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<!DOCTYPE collection SYSTEM "BioC.dtd">
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  <source>bionlp-st-2011</source>
  <date>20130823</date>
  <key>bionlp-st-2011.key</key>
  <document>
    <id>PMC-1134658-00-TIAB</id>
    <passage>
      <offset>0</offset>
      <text>BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="101" length="104"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="85" length="97"></location>
        <text>upregulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>106</offset>
      <text>Background</text>
    </passage>
    <passage>
      <offset>117</offset>
      <text>Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="166" length="174"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="286" length="291"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="433" length="438"></location>
        <text>BMP-6</text>
      </annotation>
    </passage>
    <passage>
      <offset>482</offset>
      <text>Results</text>
    </passage>
    <passage>
      <offset>490</offset>
      <text>The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM.</text>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="561" length="566"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="602" length="607"></location>
        <text>Smad1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="608" length="609"></location>
        <text>5</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="610" length="611"></location>
        <text>8</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="626" length="630"></location>
        <text>Smad</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="679" length="682"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="692" length="695"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="713" length="716"></location>
        <text>Id2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="721" length="724"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="781" length="786"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="834" length="838"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="839" length="843"></location>
        <text>CD27</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="866" length="870"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="871" length="875"></location>
        <text>CD27</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="948" length="953"></location>
        <text>CD40L</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="969" length="974"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1068" length="1073"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1139" length="1145"></location>
        <text>noggin</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1200" length="1205"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="575" length="582"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Phosphorylation</infon>
        <location offset="583" length="598"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Phosphorylation</infon>
        <location offset="631" length="646"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="651" length="659"></location>
        <text>followed</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="663" length="675"></location>
        <text>upregulation</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Gene_expression</infon>
        <location offset="725" length="735"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Regulation</infon>
        <location offset="744" length="752"></location>
        <text>affected</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Positive_regulation</infon>
        <location offset="1189" length="1199"></location>
        <text>upregulate</text>
      </annotation>
    </passage>
    <passage>
      <offset>1244</offset>
      <text>Conclusion</text>
    </passage>
    <passage>
      <offset>1255</offset>
      <text>In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.</text>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1280" length="1285"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1348" length="1353"></location>
        <text>Smad1</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1354" length="1355"></location>
        <text>5</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1356" length="1357"></location>
        <text>8</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="1389" length="1392"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Positive_regulation</infon>
        <location offset="1321" length="1328"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Phosphorylation</infon>
        <location offset="1329" length="1344"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Positive_regulation</infon>
        <location offset="1358" length="1366"></location>
        <text>followed</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Positive_regulation</infon>
        <location offset="1373" length="1385"></location>
        <text>upregulation</text>
      </annotation>
    </passage>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
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    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
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    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
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    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T28" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="E19" role="Cause"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="E21" role="Cause"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="E23" role="Cause"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T29" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E16" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1134658-01-Background</id>
    <passage>
      <offset>0</offset>
      <text>Background</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>Members of the transforming growth factor beta (TGF-beta) superfamily play central roles in controlling cellular proliferation, differentiation, migration and apoptosis [1]. These cytokines can be divided into three subgroups: TGF-beta, the activins/inhibins, and the bone morphogenetic proteins (BMPs), of which the latter constitute the largest family. BMPs are 30-38 kDa hetero- or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation [2,3]. Several studies have demonstrated an essential role of these proteins during embryogenesis, and more recently, also in adult tissues [1]. TGF-beta has been intensively studied in normal and malignant haematopoietic cells and is one of the most potent endogenous negative regulators known to date. [4]. In contrast, the effect of BMPs in the immune system has not been widely investigated. In that respect, BMP- 2, -4 and -7 have been found to control differentiation of hematopoietic stem cells [5] and early T cell development [6,7]. BMP-6 has been reported to reduce the number of cobblestone-area-forming cells of normal human haematopoietic cells [8]. Furthermore, BMP-2, -4, 6 and -7 had an antiproliferative and a proapoptotic effect on multiple myeloma cells [9-11]. In addition, by gene expression profiling, BMP-6 significantly increased the predictive value for a multi-gene signature test and was associated with a poor outcome in diffuse large B cell lymphomas (DLBCL) [12].</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="26" length="57"></location>
        <text>transforming growth factor beta</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="59" length="67"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="238" length="247"></location>
        <text>TGF-beta,</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="648" length="656"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="916" length="922"></location>
        <text>BMP- 2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="924" length="926"></location>
        <text>-4</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="931" length="933"></location>
        <text>-7</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1045" length="1050"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1179" length="1184"></location>
        <text>BMP-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1186" length="1188"></location>
        <text>-4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1190" length="1191"></location>
        <text>6</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1196" length="1198"></location>
        <text>-7</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1327" length="1332"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Positive_regulation</infon>
        <location offset="1347" length="1356"></location>
        <text>increased</text>
      </annotation>
    </passage>
    <passage>
      <offset>1498</offset>
      <text>BMP-6, like the other BMP members, signals through ligation and heterodimerzation of BMP type I [activin-like-kinase (ALK)] and type II serine-threonine kinase receptors, which subsequently propagates the signal downstream by phosphorylating Smad proteins. BMP-6 can signal through the ligation of the type I receptors Act-RIA, BMP-RIA, and BMP-RIB and the type II receptors BMP-RII, Act-RIIA and Act-RIIB, which lead to the phosphorylation of the receptor Smads (Smad-1, Smad-5, and Smad-8). The R- Smads then form complexes with the co-Smad (Smad4) and are translocated into the nucleus where they exert gene regulation [1,13].</text>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1498" length="1503"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1755" length="1760"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1817" length="1824"></location>
        <text>Act-RIA</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1826" length="1833"></location>
        <text>BMP-RIA</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1839" length="1846"></location>
        <text>BMP-RIB</text>
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      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1873" length="1880"></location>
        <text>BMP-RII</text>
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        <infon key="type">Protein</infon>
        <location offset="1882" length="1890"></location>
        <text>Act-RIIA</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1895" length="1903"></location>
        <text>Act-RIIB</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
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        <text>Smad-1</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1970" length="1976"></location>
        <text>Smad-5</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1982" length="1988"></location>
        <text>Smad-8</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="2042" length="2047"></location>
        <text>Smad4</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Binding</infon>
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        <text>ligation</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="1562" length="1579"></location>
        <text>heterodimerzation</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Binding</infon>
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        <text>ligation</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1911" length="1915"></location>
        <text>lead</text>
      </annotation>
      <annotation id="T37">
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      <text>Given the reported role of BMP-6 in B-cell malignancies and haematopoietic progenitor cells, we wanted to explore its potential role in normal human B cells. We studied the effects of BMP-6 on proliferation and apoptosis on resting and stimulated B cells. Furthermore, the expression of BMP receptors and BMP-6 induced activation of the Smad signalling pathway with subsequent regulation of the target genes Id1-Id4, were resolved. Finally, we investigated whether B cells also were capable of producing BMP-6.</text>
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  </document>
  <document>
    <id>PMC-1134658-02-Results-01</id>
    <passage>
      <offset>0</offset>
      <text>BMP-6 inhibits anti-IgM induced proliferation of human B cells</text>
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        <location offset="0" length="5"></location>
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    <passage>
      <offset>63</offset>
      <text>The effects of BMP-6 on normal and neoplastic hematopoietic cells prompted us to investigate the effects of BMP-6 on normal human B cells. All experiments in this study were performed under serum-free conditions as FCS has been shown to interfere with BMP-signalling [14](own observations). To study the effect of BMP-6 on proliferation, B-cells from healthy volunteers were stimulated with anti-IgM and/or CD40L in the presence or absence of BMP-6 for three days. We found that BMP-6 led to a 35% mean reduction of anti-IgM- induced DNA synthesis (n = 8; p &lt;/= 0.0002, Figure 1A). Similar results were obtained for B cells treated with anti-IgM and CD40L (26% mean reduction, n = 6; p &lt;/= 0.023). The BMP-6-induced inhibition of proliferation was dose-dependent in both peripheral B cells (Figure 1B) and the Burkitt lymphoma cell line Ramos (40% reduction of DNA synthesis, Figure 1C). The BMP-6 effects could be reversed by addition of the extracellular inhibitor Noggin (Figure 1D). Similarly, a combination of the soluble BMP receptors BMP-RIB-Fc and BMP-RII-Fc also neutralized the effects of BMP-6 (data not shown). Next, we wanted to test whether BMP-6 had different effect on naive and memory B cells. Naive (CD19+CD27-) and memory (CD19+CD27+) B cells were isolated from peripheral blood by cell sorting of immunobead-isolated CD19+ B cells [15], and tested for their capacity to proliferate in the presence of BMP-6. However, BMP-6 inhibited anti-IgM induced DNA synthesis in the two subpopulations to a similar extent, with a mean reduction of DNA-synthesis of 45% (n = 5; p &lt;/= 0,004) for naive B cells and 48% (n = 5; p &lt;/= 0,001) for memory B cells (Figure 1E).</text>
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  <document>
    <id>PMC-1134658-03-Results-02</id>
    <passage>
      <offset>0</offset>
      <text>BMP-6 induces cell death in human memory B cells and Ramos cells</text>
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    <passage>
      <offset>65</offset>
      <text>Next, we wanted to establish whether BMP-6 also could affect the viability of normal B cells. Cell viability was determined by propidium iodide (PI) staining after culture with or without BMP-6 for 48 hours. Interestingly, BMP-6 showed a small, but reproducible mean increase of cell death from 17 to 23% (n = 5; p &lt;/= 0,003) in anti-IgM stimulated CD27+ memory B cells. Furthermore, Ramos cells showed a mean increase in cell death from 20 to 50% (n = 3, p &lt; 0,001, figure 3) after BMP-6 treatment. In contrast, cell death of total CD19+ cells (n = 6; p &lt;/= 0,32; data not shown) or CD27-IgG- naive B cells was not significantly affected (n = 5; p &lt;/= 0,65, figure 2).</text>
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  </document>
  <document>
    <id>PMC-1134658-04-Results-03</id>
    <passage>
      <offset>0</offset>
      <text>Human B cells express BMP-6 receptors</text>
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        <location offset="22" length="27"></location>
        <text>BMP-6</text>
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    <passage>
      <offset>38</offset>
      <text>Detailed knowledge regarding expression of different BMP receptors in B cells is currently not available. To further elucidate the role of BMPs in human B cells, we performed western blot analysis for type I and type II BMP receptors. This analysis revealed that the type I receptors Act-RIA, BMP-RIB and the type II receptors BMP-RII and Act-RIIb are expressed on resting human B-cells (Figure 4). Ramos cells expressed the type I receptors Act-RIA, weakly BMP-RIB and the type II receptor BMP-RII, but more weakly than normal B cells (Figure 4). HL60 cells were used for comparison and weakly expressed Act-RIA and BMP-RII.</text>
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        <text>Act-RIA</text>
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        <location offset="331" length="338"></location>
        <text>BMP-RIB</text>
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        <text>BMP-RII</text>
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    <passage>
      <offset>665</offset>
      <text>Taken together, these data show that normal human B cells and Ramos cells express a set of BMP receptors, previously shown to bind BMP-6 [16].</text>
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  </document>
  <document>
    <id>PMC-1134658-05-Results-04</id>
    <passage>
      <offset>0</offset>
      <text>BMP-6 induces phosphorylation of Smad1/5/8</text>
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        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>BMP-6</text>
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        <location offset="33" length="38"></location>
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        <text>5</text>
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        <text>8</text>
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        <location offset="14" length="29"></location>
        <text>phosphorylation</text>
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    </passage>
    <passage>
      <offset>43</offset>
      <text>Upon ligand binding, the type II receptor transphosphorylates and activates the type I receptor. Type I receptors can signal via several pathways. We examined the effect of BMP-6 on Smad phosphorylation, as the activation of Smad is considered to be a major signalling pathway for BMPs [17]. B cells were cultured in serum-free media over night and then treated with BMP-6 for various time points. Total protein lysates were prepared, and the amounts of the phosphorylated forms of Smad1/5/8 were determined by western blot analysis. Interestingly, treatment with 500 ng/ml BMP-6 induced phosphorylation of Smad. The BMP-6 induced phosphorylation was high at the earliest time point tested (15 minutes), and remained high for at least 48 hours (Figure 5). A similar phosphorylation was observed in Ramos cells, but not in HL60 cells (Figure 6). Furthermore, we also tested whether other known downstream signalling pathways of BMP-6 could be triggered by BMP-6 in human B cells. However, we did not observe any significant changes in the level of phospho-STAT3 or phospho-p38 upon BMP-6 treatment of B cells (data not shown).</text>
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        <text>BMP-6</text>
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        <text>phosphorylation</text>
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        <text>phospho</text>
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        <text>phospho</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E25">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
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  </document>
  <document>
    <id>PMC-1134658-06-Results-05</id>
    <passage>
      <offset>0</offset>
      <text>BMP-6 induces upregulation of Id1</text>
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        <text>BMP-6</text>
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        <location offset="30" length="33"></location>
        <text>Id1</text>
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        <location offset="6" length="13"></location>
        <text>induces</text>
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        <location offset="14" length="26"></location>
        <text>upregulation</text>
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    </passage>
    <passage>
      <offset>34</offset>
      <text>Next, we wanted to explore whether the BMP-6 induced phosphorylation of Smad 1/5/8 also could induce transcriptional changes of target genes. In this regard, the inhibitors of DNA binding proteins (Ids) are considered to be some of the major target genes for Smad-signalling [17]. Thus, B cells were pre-incubated over night in X-VIVO 15, and then cultured in medium alone or in the presence of BMP-6 for various time points before preparation of total RNA. The amount of Id1-Id4 mRNA was quantified by real-time RT-PCR. Interestingly, we observed a specific four-fold upregulation of Id1 mRNA in BMP-6-treated B cells (Figure 7). The up-regulation of Id1 mRNA was characteristic of an early inducible gene, with maximal upregulation two hours after the addition of BMP-6 and returned to baseline after 24 hours. In contrast, no significant changes were observed for Id2 and Id3 mRNA, whereas Id4-transcripts were not detectable (Figure 7, data not shown).</text>
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        <text>BMP-6</text>
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        <location offset="106" length="112"></location>
        <text>Smad 1</text>
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        <text>5</text>
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        <text>8</text>
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        <text>BMP-6</text>
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        <text>Id1</text>
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        <location offset="510" length="513"></location>
        <text>Id4</text>
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        <text>Id1</text>
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        <text>BMP-6</text>
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        <text>Id1</text>
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        <text>BMP-6</text>
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        <text>Id2</text>
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        <text>Id3</text>
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        <text>Id4</text>
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        <text>induced</text>
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        <text>phosphorylation</text>
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        <text>upregulation</text>
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        <text>up-regulation</text>
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        <text>upregulation</text>
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        <text>changes</text>
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        <location offset="952" length="962"></location>
        <text>detectable</text>
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    </passage>
    <passage>
      <offset>992</offset>
      <text>Western blot analysis revealed that the BMP-6-induced upregulation of Id1 mRNA also was correlated with upregulation of Id1 protein as well. The increase in Id1 protein level was detectable after one hour and increased until 24 hours after BMP-6 addition, showing a 16-fold upregulation compared with t0 (p &lt;/= 0.020, n = 4) (Figure 8 and 9). In line with the mRNA data, no consistent change in the amounts of Id2 and Id3 protein could be observed (Figure 8 and 9). We were able to block the Id1 specific band with a blocking peptide (data not shown). Taken together, these data suggest that Id1 could be a possible target gene for mediating the effects of BMP-6 in human B cells, whereas Id2 and Id3 not seem to be involved.</text>
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      <infon key="type">Event</infon>
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    <relation id="E24">
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
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    </relation>
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      <infon key="modification type">Negation</infon>
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      <infon key="type">Event modification</infon>
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  </document>
  <document>
    <id>PMC-1134658-07-Results-06</id>
    <passage>
      <offset>0</offset>
      <text>BMP-6 production in B cells</text>
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        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>BMP-6</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="6" length="16"></location>
        <text>production</text>
      </annotation>
    </passage>
    <passage>
      <offset>28</offset>
      <text>The fact that BMP-6 has been reported to act as an autocrine stimulator in chondrocytes [18] and ovarium [19], prompted us to investigate whether normal human B cells could produce BMP-6 upon stimulation. Ramos cells, which have been described to express BMP-6 mRNA endogenously [20], and the T cell line Jurkat, served as positive and negative controls, respectively. Endogenous BMP-6 mRNA levels in normal B cells were quantified by real-time RT-PCR after stimulation with anti-IgM for different time points. Interestingly, the up-regulation of BMP-6 mRNA was characteristic of an early-to intermediate inducible gene with maximal upregulation four hours after the addition of anti-IgM. The level of BMP-6 mRNA was back to baseline after 24 hours upon stimulation (Figure 10). Furthermore, both FCS and human AB-serum induced significant upregulation of BMP-6 mRNA (Figure 11). Interestingly, in a separate study we have found that normal human T cells do not express BMP-6 mRNA after activation (Sivertsen et al, manuscript in preparation). Next, we wanted to detect BMP-6 protein in normal B-cells and tested various commercially available antibodies. However, in our hands these anti-BMP-6 antibodies did only recognize the recombinant BMP-6 protein and not the native protein.</text>
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        <location offset="209" length="214"></location>
        <text>BMP-6</text>
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        <location offset="283" length="288"></location>
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        <location offset="408" length="413"></location>
        <text>BMP-6</text>
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        <location offset="575" length="580"></location>
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        <location offset="730" length="735"></location>
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        <location offset="884" length="889"></location>
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        <location offset="998" length="1003"></location>
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        <location offset="275" length="282"></location>
        <text>express</text>
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        <text>up-regulation</text>
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        <text>upregulation</text>
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        <text>back to baseline</text>
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        <location offset="848" length="855"></location>
        <text>induced</text>
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        <text>upregulation</text>
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        <text>express</text>
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        <infon key="type">Binding</infon>
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        <text>recognize</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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  </document>
  <document>
    <id>PMC-1134658-08-Discussion</id>
    <passage>
      <offset>0</offset>
      <text>Discussion</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>Recent studies have demonstrated an important role for BMP superfamily members in hematopoietic stem cells, early thymocytes [6,7] and B-cell malignancies [8,11,12], but a role for BMPs in normal human B cells has previously not been reported. The present study demonstrated a significant antiproliferative effect of BMP-6 in peripheral blood CD19+ B cells. Additionally, BMP-6 induced cell death in CD27+ memory B cells as well as in a Burkitt lymphoma cell line (Ramos). Importantly, BMP-6 induced a rapid and marked increase in Smad-1/5/8 phosphorylation. Furthermore, the BMP-6 induced Smad phosphorylation was followed by a selective upregulation of Id1 mRNA and subsequent Id1 protein.</text>
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        <text>BMP-6</text>
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        <text>5</text>
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        <text>8</text>
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        <text>Smad</text>
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        <text>Id1</text>
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        <text>Id1</text>
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        <text>induced</text>
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        <text>increase</text>
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        <text>phosphorylation</text>
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        <text>induced</text>
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        <text>phosphorylation</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>followed</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="650" length="662"></location>
        <text>upregulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>704</offset>
      <text>In the present study, the demonstrated antiproliferative effect of BMP-6 in anti-IgM treated B cells was significant and dose-dependent. Importantly, the anti-proliferative effect of BMP-6 could be completely neutralized by the use of a natural inhibitor, Noggin. This is in line with others, showing that Noggin can function as a BMP-6 antagonist [21,22]. In addition, the combination of soluble BMP-RIB-Fc and BMP-RII-Fc fusion proteins also neutralized the anti-proliferative effect of BMP-6 in human B cells. Interestingly, as for other TGF family members, bifunctional effects have also been demonstrated for BMPs. Whereas several of the BMPs have been shown to promote proliferation in various cell types including condrocytes [23], liver [24] and granulosa cells [25], antiproliferative effects and induction of apoptosis has been reported for B and T lineage cells. Similar effects as demonstrated for BMP-6 on human B cells in the present study, were demonstrated for BMP-2, 4, 6 and -7 in human myeloma cells [9-11]. Other members of the BMP-family have also been reported to induce apoptosis, including in mouse B lineage cells [26]. Additionally, BMP-4 inhibits thymocyte proliferation [6]. Taken together, these data suggest that the role of BMPs in the regulation of proliferation and apoptosis is highly cell type dependent.</text>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="771" length="776"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="887" length="892"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="960" length="966"></location>
        <text>Noggin</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1010" length="1016"></location>
        <text>Noggin</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1035" length="1040"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1101" length="1108"></location>
        <text>BMP-RIB</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1116" length="1123"></location>
        <text>BMP-RII</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1193" length="1198"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1614" length="1619"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1681" length="1686"></location>
        <text>BMP-2</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
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        <text>4</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
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        <text>6</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1697" length="1699"></location>
        <text>-7</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1863" length="1868"></location>
        <text>BMP-4</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1041" length="1051"></location>
        <text>antagonist</text>
      </annotation>
    </passage>
    <passage>
      <offset>2045</offset>
      <text>To examine how BMP-6 exerts its functional effects in B cells, we analysed BMP receptor expression by western blot analysis. Human peripheral B cells were found to express the BMP type I receptors Act-RIA and BMP-RIB, and the type II-receptors BMP-RII and Act-RIIb, which signal after binding of several BMPs, including BMP-6 [16,13]. To further explore BMP-6 induced signalling, activation of several pathways is possible. The major signalling pathway known to date, is activation of R-Smads [13,27]. In that respect, BMPs have been shown to exert antiproliferative effects in B lineage cells via phosphorylation of R-Smad [11,28]. Furthermore, BMP-2 has been shown to induce activation of STAT3 in myeloma cells [9]. However, phosphorylation of R-Smad was not investigated in that study. BMP-2 has also been shown to induce phosphorylation of p38 [29]. Thus, phosphorylation of p38, STAT3 and Smad1/5/8 represent important BMP-signalling pathways that mediated the effects of BMPs and even cross-talk between these pathways has been reported [29,30]. In the present study, we were not able to detect BMP-6-induced changes in the phosphorylation status of STAT3 or p38 in human peripheral B cells. Instead, a rapid and marked phosphorylation of Smad1/5/8 was revealed. In a parallel study, we have found that other BMPs also induced phosphorylation of Smad1/5/8 in peripheral B cells (data not shown). We are currently pursuing microarray studies to identify the signalling pathways and target genes that are differently regulated by the various BMPs in human B cells.</text>
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        <infon key="type">Protein</infon>
        <location offset="2060" length="2065"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="2242" length="2249"></location>
        <text>Act-RIA</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="2254" length="2261"></location>
        <text>BMP-RIB</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="2289" length="2296"></location>
        <text>BMP-RII</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="2301" length="2309"></location>
        <text>Act-RIIb</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="2365" length="2370"></location>
        <text>BMP-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2399" length="2404"></location>
        <text>BMP-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2691" length="2696"></location>
        <text>BMP-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2736" length="2741"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="2835" length="2840"></location>
        <text>BMP-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2930" length="2935"></location>
        <text>STAT3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2940" length="2945"></location>
        <text>Smad1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2946" length="2947"></location>
        <text>5</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2948" length="2949"></location>
        <text>8</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="3147" length="3152"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="3202" length="3207"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="3291" length="3296"></location>
        <text>Smad1</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="3297" length="3298"></location>
        <text>5</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="3299" length="3300"></location>
        <text>8</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="3398" length="3403"></location>
        <text>Smad1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="3404" length="3405"></location>
        <text>5</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="3406" length="3407"></location>
        <text>8</text>
      </annotation>
      <annotation id="T131">
        <infon key="type">Gene_expression</infon>
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        <text>express</text>
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      <annotation id="T132">
        <infon key="type">Binding</infon>
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        <text>binding</text>
      </annotation>
      <annotation id="T133">
        <infon key="type">Positive_regulation</infon>
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        <text>induce</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="2722" length="2732"></location>
        <text>activation</text>
      </annotation>
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        <infon key="type">Phosphorylation</infon>
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        <text>phosphorylation</text>
      </annotation>
      <annotation id="T136">
        <infon key="type">Positive_regulation</infon>
        <location offset="3153" length="3160"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T137">
        <infon key="type">Regulation</infon>
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        <text>changes</text>
      </annotation>
      <annotation id="T138">
        <infon key="type">Phosphorylation</infon>
        <location offset="3176" length="3191"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T139">
        <infon key="type">Phosphorylation</infon>
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        <text>phosphorylation</text>
      </annotation>
      <annotation id="T140">
        <infon key="type">Positive_regulation</infon>
        <location offset="3371" length="3378"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T141">
        <infon key="type">Phosphorylation</infon>
        <location offset="3379" length="3394"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <passage>
      <offset>3616</offset>
      <text>Upregulation of Id1 via Smad1/5/8 phosphorylation is a known mechanism for BMP-6 signalling in other cell systems [31,32] and regulation of Id-proteins is thought to be an important mechanism for Smad-signalling [17]. In the present study, real-time RT-PCR experiments revealed a specific four-fold upregulation for Id1 in BMP-6-treated B cells, while the amount of Id2-Id4 remained unchanged. In agreement with this, western blot analysis demonstrated an upregulation of Id1 protein, while the amount of Id2 and Id3 protein levels remained unchanged. Previously, Id1 has been considered not to be expressed in later developmental stages than pro-B cells [33,34], and its constitutive expression has been reported to impair mouse B cell development [35]. Therefore, our demonstration of the time-dependent upregulation of Id1 mRNA and protein in mature normal human B cells is of particular interest. In that respect, it is noteworthy that TGF-beta signalling in early and mature B cells induces both Id2 and Id3 expression [36,37], but not Id1 (data not shown). Interestingly, these results show that various members of the TGF-beta family regulate Id proteins differently. Id2 and Id3 are considered to be the Id proteins mainly expressed in mature B cells [38]. The present study also found Id2 and Id3 protein in B cells to be more highly expressed than Id1 in resting B cells. However, BMP-6 did not induce significant changes in the protein expression of Id2 and Id3. It is believed that Id proteins block differentiation and promote proliferation in various cell types [39,33]. Id proteins act as dominant-negative inhibitors of E-proteins and Pax5 function by forming dimers with these proteins, making them unable to bind DNA. It has been proposed that the balance among E-proteins, Pax5 and Id proteins might have an important role in activated B cells [38]. In that respect, E-proteins have been implicated in both the promotion and inhibition of cell survival and growth at different points in lymphocyte development [40]. The antiproliferative and death inducing effect of BMP-6 in B cells with concomitant upregulation of Id1 protein is therefore in line with the view that Id proteins are required for the induction of growth arrest and apoptosis in B-lymphocyte progenitors by TGF-beta [40].</text>
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        <infon key="type">Protein</infon>
        <location offset="3632" length="3635"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="3640" length="3645"></location>
        <text>Smad1</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="3646" length="3647"></location>
        <text>5</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="3648" length="3649"></location>
        <text>8</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Protein</infon>
        <location offset="3691" length="3696"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Protein</infon>
        <location offset="3932" length="3935"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T55">
        <infon key="type">Protein</infon>
        <location offset="3939" length="3944"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Protein</infon>
        <location offset="3982" length="3985"></location>
        <text>Id2</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Protein</infon>
        <location offset="3986" length="3989"></location>
        <text>Id4</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Protein</infon>
        <location offset="4088" length="4091"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T59">
        <infon key="type">Protein</infon>
        <location offset="4121" length="4124"></location>
        <text>Id2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="4129" length="4132"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Protein</infon>
        <location offset="4180" length="4183"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T62">
        <infon key="type">Protein</infon>
        <location offset="4438" length="4441"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Protein</infon>
        <location offset="4556" length="4564"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
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        <text>Id2</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Protein</infon>
        <location offset="4625" length="4628"></location>
        <text>Id3</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id1</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Protein</infon>
        <location offset="4741" length="4749"></location>
        <text>TGF-beta</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id2</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id3</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id2</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id3</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id1</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>BMP-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Id2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="5085" length="5088"></location>
        <text>Id3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="5267" length="5271"></location>
        <text>Pax5</text>
      </annotation>
      <annotation id="T77">
        <infon key="type">Protein</infon>
        <location offset="5408" length="5412"></location>
        <text>Pax5</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Protein</infon>
        <location offset="5702" length="5707"></location>
        <text>BMP-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="5752" length="5755"></location>
        <text>Id1</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>TGF-beta</text>
      </annotation>
      <annotation id="T142">
        <infon key="type">Positive_regulation</infon>
        <location offset="3616" length="3628"></location>
        <text>Upregulation</text>
      </annotation>
      <annotation id="T143">
        <infon key="type">Phosphorylation</infon>
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        <text>phosphorylation</text>
      </annotation>
      <annotation id="T144">
        <infon key="type">Regulation</infon>
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        <text>mechanism</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
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        <text>upregulation</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
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        <text>amount</text>
      </annotation>
      <annotation id="T147">
        <infon key="type">Positive_regulation</infon>
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        <text>remained unchanged</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
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        <text>upregulation</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
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        <text>amount</text>
      </annotation>
      <annotation id="T150">
        <infon key="type">Positive_regulation</infon>
        <location offset="4148" length="4166"></location>
        <text>remained unchanged</text>
      </annotation>
      <annotation id="T151">
        <infon key="type">Gene_expression</infon>
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        <text>expressed</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
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        <text>expression</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
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        <text>upregulation</text>
      </annotation>
      <annotation id="T154">
        <infon key="type">Positive_regulation</infon>
        <location offset="4604" length="4611"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T155">
        <infon key="type">Gene_expression</infon>
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        <text>expression</text>
      </annotation>
      <annotation id="T156">
        <infon key="type">Gene_expression</infon>
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        <text>expressed</text>
      </annotation>
      <annotation id="T157">
        <infon key="type">Gene_expression</infon>
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        <text>expressed</text>
      </annotation>
      <annotation id="T158">
        <infon key="type">Positive_regulation</infon>
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        <text>induce</text>
      </annotation>
      <annotation id="T159">
        <infon key="type">Regulation</infon>
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        <text>changes</text>
      </annotation>
      <annotation id="T160">
        <infon key="type">Gene_expression</infon>
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        <text>expression</text>
      </annotation>
      <annotation id="T161">
        <infon key="type">Negative_regulation</infon>
        <location offset="5238" length="5248"></location>
        <text>inhibitors</text>
      </annotation>
      <annotation id="T162">
        <infon key="type">Binding</infon>
        <location offset="5284" length="5298"></location>
        <text>forming dimers</text>
      </annotation>
      <annotation id="T163">
        <infon key="type">Positive_regulation</infon>
        <location offset="5320" length="5326"></location>
        <text>making</text>
      </annotation>
      <annotation id="T164">
        <infon key="type">Binding</infon>
        <location offset="5342" length="5346"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T165">
        <infon key="type">Positive_regulation</infon>
        <location offset="5736" length="5748"></location>
        <text>upregulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>5925</offset>
      <text>Furthermore, Id proteins are known as important parts of signalling pathways involved in development, cell cycle and tumorigenesis [32]. It is well established that various members of the Id family are overexpressed in a range of human tumours and generally, Id1 appears to be the family member most widely overexpressed in a variety of human malignancies [41], including multiple myeloma [42,32]. Additionally, our findings that BMP-6 activates intracellular signalling pathways in human B cells might be of potential pathophysiological significance in lymphoma and inflammation. High BMP-6 mRNA expression in DLBCL has been shown to correlate to unfavourable outcome [12]. In this respect, it is of interest that targeted expression of Id1 to B-lymphocytes resulted in aberrant B cell development, massive apoptosis, and subsequent development of B cell lymphomas [35]. Moreover, BMP-6 has been suggested to play a role in rheumatoid arthritis (RA) [43,44] and elevated levels of Id1 and Id3 have been found in the synovia of RA-patients [45]. Altogether, these results point to an important role for Id proteins in the regulation of normal B cell homeostasis and in diseases, where B cells are involved. It will therefore be important to further elucidate the role of Id-1 in human B cells by selective over expression or inhibition of Id-1 gene expression.</text>
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        <infon key="type">Protein</infon>
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        <text>Id1</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Protein</infon>
        <location offset="6355" length="6360"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Protein</infon>
        <location offset="6511" length="6516"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Protein</infon>
        <location offset="6663" length="6666"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Protein</infon>
        <location offset="6807" length="6812"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Protein</infon>
        <location offset="6907" length="6910"></location>
        <text>Id1</text>
      </annotation>
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      <text>Given the role of BMP-6 in mature human B cells demonstrated here, identification of BMP-6 producing cells in vivo with possibility of interaction with naive and memory B cells might contribute to the understanding of mature B cell biology. High BMP-6 mRNA expression in DLBCL has been detected by gene expression profiling [12]. Furthermore, production of BMP-6 transcripts in normal activated B cells was detected in the same study. Of note, an autocrine BMP-6 loop has been reported by others in chondrocytes and in the ovarium [46,19,18]. Therefore, we wanted to explore the possibility for an autocrine BMP-6 loop in human B cells. We analysed the expression BMP-6 mRNA in peripheral blood B cells by real-time PCR, and report here the upregulation of endogenous BMP-6 transcripts after stimulation with FCS, human AB-serum and, most importantly, anti-IgM. However, our attempts to study BMP-6 protein levels were unsuccessful due to problems with unspecific binding of the anti-BMP-6 antibodies tested, and lack of specific staining in control cells known to express BMP-6 mRNA. In contrast, the recombinant protein was readily detected. In that respect, few investigators have detected BMP-6 protein in humans, especially in non-pathogenic tissue. The possibility of BMP-6 production in human B-cells is in line with a recent work that reported the production of BMP-6 in mouse B cells, infiltrating the bone marrow of mice with inflammatory arthritis [43]. In this study, a role for BMPs in the inflammatoric process of arthritis was suggested. The upregulation of the BMP-6-transcripts after IgM-crosslinking is of pathophysiologic interest [12]. A loss of TGF-beta-responsiveness has been suggested to be a critical contribution to malignant transformation [47,48] and similar oncogenic mechanisms have been postulated for BMPs. Lines of evidence suggest [49] that at early stages of carcinogenesis, BMP-6 is not a tumour promoter, but suppresses benign and malignant tumour outgrowth. These findings are in good agreement with previous findings for other TGF-beta family members, including TGF-beta1 and BMP-4 [50], indicating that cellular context of the BMP target cell might define the various observed effects. In contrast to the upregulation of BMP-6 transcript in B cells, we were not able to detect BMP-6 transcripts in human peripheral blood CD4+ or CD8+ T cells (resting or stimulated with anti-CD3 and anti-CD28; data not shown), consistent with the findings in T cell lines [20] and T cells in mice [43]. Other potential BMP-6 sources for mature B cells in vivo might be other cells of the immune system or tissue with contact to the hematopoietic system. One well recognized source for BMP-6 production is the human bone and bone marrow stroma [51,8]. Furthermore, it is noteworthy that human umbilical vein endothelial cells (HUVEC) highly express BMP-6 mRNA [52], and vascular endothelium has been reported to produce BMP-6 [53]. These studies might imply a role for BMP-6 in transendothelial migration of B cells. BMP-6 mRNA has been demonstrated in murine macrophage cell lines, but not in humans [54]. In accordance with these findings, other human cell lines of neutrophil and monocytic origin have been described to be negative for the BMP-6 transcript [20]. To our knowledge, there is currently no report about BMP-6 production of human dendritic cells.</text>
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    </relation>
    <relation id="E39">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T140" role="Trigger"></node>
      <node refid="E40" role="Theme"></node>
    </relation>
    <relation id="E41">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T140" role="Trigger"></node>
      <node refid="E42" role="Theme"></node>
    </relation>
    <relation id="E38">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T141" role="Trigger"></node>
      <node refid="T46" role="Theme"></node>
    </relation>
    <relation id="E40">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T141" role="Trigger"></node>
      <node refid="T47" role="Theme"></node>
    </relation>
    <relation id="E42">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T141" role="Trigger"></node>
      <node refid="T48" role="Theme"></node>
    </relation>
    <relation id="E43">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T142" role="Trigger"></node>
      <node refid="T49" role="Theme"></node>
      <node refid="E44" role="Cause"></node>
    </relation>
    <relation id="E45">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T142" role="Trigger"></node>
      <node refid="T49" role="Theme"></node>
      <node refid="E46" role="Cause"></node>
    </relation>
    <relation id="E47">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T142" role="Trigger"></node>
      <node refid="T49" role="Theme"></node>
      <node refid="E48" role="Cause"></node>
    </relation>
    <relation id="E44">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T143" role="Trigger"></node>
      <node refid="T50" role="Theme"></node>
    </relation>
    <relation id="E46">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T143" role="Trigger"></node>
      <node refid="T51" role="Theme"></node>
    </relation>
    <relation id="E48">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T143" role="Trigger"></node>
      <node refid="T52" role="Theme"></node>
    </relation>
    <relation id="E49">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T144" role="Trigger"></node>
      <node refid="E43" role="Theme"></node>
      <node refid="T53" role="Cause"></node>
    </relation>
    <relation id="E50">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T144" role="Trigger"></node>
      <node refid="E45" role="Theme"></node>
      <node refid="T53" role="Cause"></node>
    </relation>
    <relation id="E51">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T144" role="Trigger"></node>
      <node refid="E47" role="Theme"></node>
      <node refid="T53" role="Cause"></node>
    </relation>
    <relation id="E52">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T145" role="Trigger"></node>
      <node refid="T54" role="Theme"></node>
      <node refid="T55" role="Cause"></node>
    </relation>
    <relation id="E53">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T146" role="Trigger"></node>
      <node refid="T56" role="Theme"></node>
    </relation>
    <relation id="E54">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T146" role="Trigger"></node>
      <node refid="T57" role="Theme"></node>
    </relation>
    <relation id="E55">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T147" role="Trigger"></node>
      <node refid="E53" role="Theme"></node>
      <node refid="T55" role="Cause"></node>
    </relation>
    <relation id="E56">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T147" role="Trigger"></node>
      <node refid="E54" role="Theme"></node>
      <node refid="T55" role="Cause"></node>
    </relation>
    <relation id="E57">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T148" role="Trigger"></node>
      <node refid="T58" role="Theme"></node>
    </relation>
    <relation id="E58">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T149" role="Trigger"></node>
      <node refid="T59" role="Theme"></node>
    </relation>
    <relation id="E59">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T149" role="Trigger"></node>
      <node refid="T60" role="Theme"></node>
    </relation>
    <relation id="E60">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T150" role="Trigger"></node>
      <node refid="E58" role="Theme"></node>
    </relation>
    <relation id="E61">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T150" role="Trigger"></node>
      <node refid="E59" role="Theme"></node>
    </relation>
    <relation id="E62">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T151" role="Trigger"></node>
      <node refid="T61" role="Theme"></node>
    </relation>
    <relation id="E63">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T152" role="Trigger"></node>
      <node refid="T61" role="Theme"></node>
    </relation>
    <relation id="E64">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T153" role="Trigger"></node>
      <node refid="T62" role="Theme"></node>
    </relation>
    <relation id="E65">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T154" role="Trigger"></node>
      <node refid="E66" role="Theme"></node>
    </relation>
    <relation id="E67">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T154" role="Trigger"></node>
      <node refid="E68" role="Theme"></node>
    </relation>
    <relation id="E69">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T154" role="Trigger"></node>
      <node refid="T66" role="Theme"></node>
    </relation>
    <relation id="E66">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T155" role="Trigger"></node>
      <node refid="T64" role="Theme"></node>
    </relation>
    <relation id="E68">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T155" role="Trigger"></node>
      <node refid="T65" role="Theme"></node>
    </relation>
    <relation id="E70">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T156" role="Trigger"></node>
      <node refid="T68" role="Theme"></node>
    </relation>
    <relation id="E71">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T156" role="Trigger"></node>
      <node refid="T69" role="Theme"></node>
    </relation>
    <relation id="E72">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T157" role="Trigger"></node>
      <node refid="T72" role="Theme"></node>
    </relation>
    <relation id="E73">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T157" role="Trigger"></node>
      <node refid="T70" role="Theme"></node>
    </relation>
    <relation id="E74">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T157" role="Trigger"></node>
      <node refid="T71" role="Theme"></node>
    </relation>
    <relation id="E75">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T158" role="Trigger"></node>
      <node refid="E76" role="Theme"></node>
      <node refid="T73" role="Cause"></node>
    </relation>
    <relation id="E77">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T158" role="Trigger"></node>
      <node refid="E78" role="Theme"></node>
      <node refid="T73" role="Cause"></node>
    </relation>
    <relation id="E76">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T159" role="Trigger"></node>
      <node refid="E79" role="Theme"></node>
    </relation>
    <relation id="E78">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T159" role="Trigger"></node>
      <node refid="E80" role="Theme"></node>
    </relation>
    <relation id="E79">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T160" role="Trigger"></node>
      <node refid="T74" role="Theme"></node>
    </relation>
    <relation id="E80">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T160" role="Trigger"></node>
      <node refid="T75" role="Theme"></node>
    </relation>
    <relation id="E81">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T161" role="Trigger"></node>
      <node refid="T76" role="Theme"></node>
      <node refid="E82" role="Cause"></node>
    </relation>
    <relation id="E82">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T162" role="Trigger"></node>
      <node refid="T76" role="Theme"></node>
    </relation>
    <relation id="E83">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T163" role="Trigger"></node>
      <node refid="E84" role="Theme"></node>
      <node refid="E82" role="Cause"></node>
    </relation>
    <relation id="E84">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T164" role="Trigger"></node>
      <node refid="T76" role="Theme"></node>
    </relation>
    <relation id="E85">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T165" role="Trigger"></node>
      <node refid="T79" role="Theme"></node>
      <node refid="T78" role="Cause"></node>
    </relation>
    <relation id="E86">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T166" role="Trigger"></node>
      <node refid="T81" role="Theme"></node>
    </relation>
    <relation id="E87">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T167" role="Trigger"></node>
      <node refid="E88" role="Theme"></node>
    </relation>
    <relation id="E88">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T168" role="Trigger"></node>
      <node refid="T83" role="Theme"></node>
    </relation>
    <relation id="E89">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T169" role="Trigger"></node>
      <node refid="T84" role="Theme"></node>
    </relation>
    <relation id="E90">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T170" role="Trigger"></node>
      <node refid="E91" role="Theme"></node>
    </relation>
    <relation id="E92">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T170" role="Trigger"></node>
      <node refid="E93" role="Theme"></node>
    </relation>
    <relation id="E91">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T171" role="Trigger"></node>
      <node refid="T86" role="Theme"></node>
    </relation>
    <relation id="E93">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T171" role="Trigger"></node>
      <node refid="T87" role="Theme"></node>
    </relation>
    <relation id="E94">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T172" role="Trigger"></node>
      <node refid="E95" role="Theme"></node>
    </relation>
    <relation id="E95">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T173" role="Trigger"></node>
      <node refid="T89" role="Theme"></node>
    </relation>
    <relation id="E96">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T174" role="Trigger"></node>
      <node refid="E97" role="Theme"></node>
    </relation>
    <relation id="E97">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T175" role="Trigger"></node>
      <node refid="T89" role="Theme"></node>
    </relation>
    <relation id="E98">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T176" role="Trigger"></node>
      <node refid="T91" role="Theme"></node>
    </relation>
    <relation id="E99">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T177" role="Trigger"></node>
      <node refid="E100" role="Theme"></node>
    </relation>
    <relation id="E100">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T178" role="Trigger"></node>
      <node refid="T92" role="Theme"></node>
    </relation>
    <relation id="E101">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T179" role="Trigger"></node>
      <node refid="T93" role="Theme"></node>
    </relation>
    <relation id="E102">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T180" role="Trigger"></node>
      <node refid="T94" role="Theme"></node>
      <node refid="T94" role="Cause"></node>
    </relation>
    <relation id="E103">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T181" role="Trigger"></node>
      <node refid="T95" role="Theme"></node>
      <node refid="T95" role="Cause"></node>
    </relation>
    <relation id="E104">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T182" role="Trigger"></node>
      <node refid="T96" role="Theme"></node>
    </relation>
    <relation id="E105">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T183" role="Trigger"></node>
      <node refid="T97" role="Theme"></node>
    </relation>
    <relation id="E106">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T184" role="Trigger"></node>
      <node refid="T99" role="Theme"></node>
    </relation>
    <relation id="E107">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T185" role="Trigger"></node>
      <node refid="T100" role="Theme"></node>
    </relation>
    <relation id="E108">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T186" role="Trigger"></node>
      <node refid="T101" role="Theme"></node>
    </relation>
    <relation id="E109">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T187" role="Trigger"></node>
      <node refid="T102" role="Theme"></node>
    </relation>
    <relation id="E110">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T188" role="Trigger"></node>
      <node refid="T103" role="Theme"></node>
    </relation>
    <relation id="E111">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T189" role="Trigger"></node>
      <node refid="T109" role="Theme"></node>
    </relation>
    <relation id="E112">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T190" role="Trigger"></node>
      <node refid="T110" role="Theme"></node>
    </relation>
    <relation id="E113">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T191" role="Trigger"></node>
      <node refid="T116" role="Theme"></node>
    </relation>
    <relation id="E114">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T192" role="Trigger"></node>
      <node refid="E115" role="Theme"></node>
    </relation>
    <relation id="E115">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T193" role="Trigger"></node>
      <node refid="T117" role="Theme"></node>
    </relation>
    <relation id="E116">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T194" role="Trigger"></node>
      <node refid="T118" role="Theme"></node>
    </relation>
    <relation id="E117">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T195" role="Trigger"></node>
      <node refid="T120" role="Theme"></node>
    </relation>
    <relation id="E118">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T196" role="Trigger"></node>
      <node refid="T121" role="Theme"></node>
    </relation>
    <relation id="E119">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T197" role="Trigger"></node>
      <node refid="T122" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E31" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E55" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E56" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E60" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E61" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E62" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E62" role="Event"></node>
    </relation>
    <relation id="M8">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E69" role="Event"></node>
    </relation>
    <relation id="M9">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E70" role="Event"></node>
    </relation>
    <relation id="M10">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E71" role="Event"></node>
    </relation>
    <relation id="M11">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E75" role="Event"></node>
    </relation>
    <relation id="M12">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E77" role="Event"></node>
    </relation>
    <relation id="M13">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E84" role="Event"></node>
    </relation>
    <relation id="M14">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E86" role="Event"></node>
    </relation>
    <relation id="M15">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E103" role="Event"></node>
    </relation>
    <relation id="M16">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E104" role="Event"></node>
    </relation>
    <relation id="M17">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E107" role="Event"></node>
    </relation>
    <relation id="M18">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E108" role="Event"></node>
    </relation>
    <relation id="M19">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E112" role="Event"></node>
    </relation>
    <relation id="M20">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E117" role="Event"></node>
    </relation>
    <relation id="M21">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E118" role="Event"></node>
    </relation>
    <relation id="M22">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E119" role="Event"></node>
    </relation>
    <relation id="M23">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E119" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1134658-09-Conclusion</id>
    <passage>
      <offset>0</offset>
      <text>Conclusion</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>In conclusion, our results show that BMP-6 induces activation of intracellular Smad signalling in mature human B-cells with consecutive production of Id1 protein. Furthermore, we report that BMP-6 has an antiproliferative effect in B cells stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induces cell death in activated memory B cells and Ramos cells. Taken together, these results provide a rationale to further examine the role of BMP-6 signalling in normal B cell biology as well as in pathologic conditions like B cell malignancies and autoimmune disorders.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="48" length="53"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="161" length="164"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="202" length="207"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="321" length="326"></location>
        <text>CD40L</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="342" length="347"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="493" length="498"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Positive_regulation</infon>
        <location offset="135" length="146"></location>
        <text>consecutive</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Gene_expression</infon>
        <location offset="147" length="157"></location>
        <text>production</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1134658-10-Methods-01</id>
    <passage>
      <offset>0</offset>
      <text>Cell culture</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>If not specified, all cells were cultured in X-VIVO 15trade (BioWhittaker, Verviers, Belgium) serum-free medium at 37degreesC and 5% CO2 in air.</text>
    </passage>
    <passage>
      <offset>159</offset>
      <text>Peripheral blood was provided by the Blood Bank at Buskerud Regional Hospital with formal agreement by the patients, and approval by the regional ethics committee. Highly purified resting human B-lymphocytes (CD19+ cells) were isolated from the peripheral blood by rosetting with immunomagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) as described [55]. This procedure yields less than 0.5% T cells, 0.1% NK cells, and 0.5% monocytes as judged by indirect immunofluorescence staining.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="368" length="372"></location>
        <text>CD19</text>
      </annotation>
    </passage>
    <passage>
      <offset>649</offset>
      <text>The following cell lines from human lymphoid malignancies were maintained in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% foetal bovine serum (FCS), 100 units/ml penicillin G, and 100 units/ml of streptomycin sulphate, but serum-starved for at least four hours and cultured in X-VIVO 15trade when included in experiments: EBV-negative BL cell lines Ramos (ECACC 85030802), HL60 (JCRB0085).</text>
    </passage>
  </document>
  <document>
    <id>PMC-1134658-11-Methods-02</id>
    <passage>
      <offset>0</offset>
      <text>Growth factors/supplements</text>
    </passage>
    <passage>
      <offset>27</offset>
      <text>The following reagents were used at indicated concentrations: recombinant human (rhu) BMP-6 (1 mug/ml, if not specified otherwise), rhu BMP-RIB/ALK-6/Fc Chimera (5 mug/ml), rhu BMPR-II/Fc Chimera (5 mug/ml), and recombinant mouse Noggin (5 mug/ml) were purchased from R&amp;D Systems (Abingdon, UK); Anti-IgM F(ab)2 fragments of rabbit polyclonal antibodies to human IgM heavy chain (37.5 mug/ml) was obtained from Dako, Copenhagen, Denmark and rhu CD40 ligand (CD40L, 10 ng/ml) was a gift from Immunex Corp. (Seattle, WA).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="113" length="118"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="163" length="170"></location>
        <text>BMP-RIB</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="171" length="176"></location>
        <text>ALK-6</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="204" length="211"></location>
        <text>BMPR-II</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="257" length="263"></location>
        <text>Noggin</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="472" length="483"></location>
        <text>CD40 ligand</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="485" length="490"></location>
        <text>CD40L</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1134658-12-Methods-03</id>
    <passage>
      <offset>0</offset>
      <text>Antibodies used for flow cytometric analysis and immunoblot analysis</text>
    </passage>
    <passage>
      <offset>69</offset>
      <text>Antibodies against the human BMP-receptors Act-RIA, BMP-RIB, BMPR-II, Act-RIIA and Act-RIIb were purchased from R &amp; D Systems (Abingdon, UK). Detection of the BMP-6-protein has been tried with the following antibodies: goat polyclonal anti-BMP-6 (Santa Cruz, San Diego, CA, USA), monoclonal mouse anti-BMP-6 and polyclonal goat anti-BMP-6 from R &amp; D Systems (Abingdon, UK), and mouse monoclonal anti-BMP-6 (Chemicon International Inc, Temecula, CA, USA).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="112" length="119"></location>
        <text>Act-RIA</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="121" length="128"></location>
        <text>BMP-RIB</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="130" length="137"></location>
        <text>BMPR-II</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="139" length="147"></location>
        <text>Act-RIIA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="152" length="160"></location>
        <text>Act-RIIb</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="228" length="233"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="309" length="314"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="371" length="376"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="402" length="407"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="469" length="474"></location>
        <text>BMP-6</text>
      </annotation>
    </passage>
    <passage>
      <offset>525</offset>
      <text>Characterisation of BMP-signalling pathways was done by use of anti-phospho-Smad1, -5, -and 8 polyclonal antibody (Chemicon, Temecula, CA, USA). Expression levels of Id1-3 proteins were detected with polyclonal rabbit antibody and detection was blocked with blocking peptide from Santa Cruz Biotechnology (Santa Cruz, San Diego, CA, USA). As secondary antibodies served anti-mouse, anti-goat or anti-rabbit IgG- horseradish peroxidase (HRP) from Dakocytomation AS (Copenhagen, Denmark) for immunoblot analysis. Anti-beta-actin was from Santa-Cruz. From Becton Dickinson (San Jose, CA), we purchased anti-CD19-PE, anti-CD19-FITC. The antibodies used for cell sorting were anti-CD19 PC5 from Immunotech SA (Marseille, France) and anti-CD27 PE from Becton Dickinson, Biosciences Pharmingen (San Diego, CA, USA).</text>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="601" length="606"></location>
        <text>Smad1</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="608" length="610"></location>
        <text>-5</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="617" length="618"></location>
        <text>8</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="691" length="694"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="695" length="696"></location>
        <text>3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1041" length="1051"></location>
        <text>beta-actin</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1129" length="1133"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1143" length="1147"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1201" length="1205"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1258" length="1262"></location>
        <text>CD27</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="670" length="687"></location>
        <text>Expression levels</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="770" length="777"></location>
        <text>blocked</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1134658-13-Methods-04</id>
    <passage>
      <offset>0</offset>
      <text>Cell sorting</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>Highly purified CD19+CD27- or CD19+CD27+ cells were obtained by staining CD19+ cells with anti-CD27 PE and CD19 PC5 mAbs for 30 minutes at 4degreesC, followed by washing with PBS and sorting on FACS DiVa from Becton Dickinson.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="29" length="33"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="34" length="38"></location>
        <text>CD27</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="43" length="47"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="48" length="52"></location>
        <text>CD27</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="86" length="90"></location>
        <text>CD19</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="108" length="112"></location>
        <text>CD27</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="120" length="124"></location>
        <text>CD19</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1134658-14-Methods-05</id>
    <passage>
      <offset>0</offset>
      <text>Western-blot analysis</text>
    </passage>
    <passage>
      <offset>22</offset>
      <text>B cells from peripheral blood or cultured cell-lines were lysed in lysis buffer (glycerol 10%, beta-mercaptoethanol 5%, 0.0625 M Tris-HCL [pH 6.8], sodium dodecyl sulphate [SDS] 2.5%w/vol). Total protein (30-100 mug) from each sample was run on 10% or 12% SDS/polyacrylamide (SDS/PAGE) gels and blotted onto nitrocellulose filters (Protran; Schleicher &amp;Schuell GmbH, Dassel, Germany). Blocking, washing and incubation of the filters with primary antibodies were done according to the manufacturer's protocols at room temperature (RT). After washing with TBS/0.1% Tween-20 (TBS-T), the filters were incubated with horseradish peroxidase (HRP) coupled to relevant secondary antibodies (see above) for 60 minutes at RT. Enzyme activity was visualised by the enhanced chemiluminescence system, ECL+PLUS (Amersham, Buckinghamshire, UK). Densitometric analysis was performed by scanning hyperfilms on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). Quantification of Id1, Id2 and Id3 protein was calculated by normalizing the specific protein bands to beta-actin using Image Quant 5.5 software (Molecular Dynamics).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="999" length="1002"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="1004" length="1007"></location>
        <text>Id2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1012" length="1015"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1084" length="1094"></location>
        <text>beta-actin</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1134658-15-Methods-06</id>
    <passage>
      <offset>0</offset>
      <text>Analysis of BMP-6 messenger RNA (mRNA) expression</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="12" length="17"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Transcription</infon>
        <location offset="39" length="49"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>50</offset>
      <text>Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. Total RNA was isolated using Absolutely RNAtrade RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions. Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements. Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan(R) Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A). To measure mRNA expression of BMP6, Id1-Id4 and PGK1 PCR were carried out with TaqMan(R) universal master mix. Primers and probes were provided by Assay-on-Demand (Applied Biosystems). PCR reactions were carried out in a final volume of 25 mul (BMP-6) or 20 mul (ID1). The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA. The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems). The expression was then normalized to the expression level of PGK1. PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56]. Expression levels in B cells were then related to the expression levels in Ramos cells.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="79" length="84"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="595" length="599"></location>
        <text>BMP6</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="601" length="604"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="605" length="608"></location>
        <text>Id4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="613" length="617"></location>
        <text>PGK1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="810" length="815"></location>
        <text>BMP-6</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="985" length="989"></location>
        <text>BMP6</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1022" length="1025"></location>
        <text>Id1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1151" length="1155"></location>
        <text>PGK1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1157" length="1161"></location>
        <text>PGK1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="61" length="71"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="576" length="591"></location>
        <text>mRNA expression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="926" length="936"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="1131" length="1141"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="1203" length="1222"></location>
        <text>have low expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
    <relation id="M8">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M9">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M10">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M11">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M12">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1134658-16-Methods-07</id>
    <passage>
      <offset>0</offset>
      <text>Cell proliferation</text>
    </passage>
    <passage>
      <offset>19</offset>
      <text>For estimation of DNA synthesis, CD19+ cells (7.5 x 104 cells/0.2 ml) or Ramos cells (1 x 104 cells/0.2 ml) were cultured in triplicate in microtiter wells. The cells were pulsed with 3.7 x 104Bq [3H]thymidine (Amersham, Buckinghamshire, UK) for the last 16 h of a 72-h incubation. The cells were harvested using an automated cell harvester (Packard Instrument Company, Meriden, CT, USA) and [3H]thymidine incorporation was determined in a scintillation counter (TopCount, Packard Instrument Company Inc., Meriden, CT).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="52" length="56"></location>
        <text>CD19</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1134658-17-Methods-08</id>
    <passage>
      <offset>0</offset>
      <text>Determination of cell death</text>
    </passage>
    <passage>
      <offset>28</offset>
      <text>Cell death was measured by vital dye exclusion test by staining cells with 5 mug/ml propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml) for one minute on ice. At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer.</text>
    </passage>
  </document>
  <document>
    <id>PMC-1134658-18-Methods-09</id>
    <passage>
      <offset>0</offset>
      <text>Statistical analysis</text>
    </passage>
    <passage>
      <offset>21</offset>
      <text>The statistical significance of differences between groups was determined using the paired two-tailed Wilcoxon nonparametric test, by applying SPSS10.1 software (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered significant.</text>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-00-TIAB</id>
    <passage>
      <offset>0</offset>
      <text>Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="3"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="8" length="11"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="54" length="62"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="12" length="20"></location>
        <text>regulate</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Transcription</infon>
        <location offset="27" length="40"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>69</offset>
      <text>APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="69" length="77"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="79" length="82"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="409" length="412"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="507" length="517"></location>
        <text>Luciferase</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="710" length="713"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="874" length="884"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1377" length="1403"></location>
        <text>specificity protein (Sp) 1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1408" length="1411"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="161" length="170"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="319" length="329"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Entity</infon>
        <location offset="714" length="722"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="731" length="740"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Entity</infon>
        <location offset="1302" length="1308"></location>
        <text>GC-box</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Binding</infon>
        <location offset="1323" length="1330"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T16" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
      <node refid="T18" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T11" role="Theme2"></node>
      <node refid="T18" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-01-INTRODUCTION</id>
    <passage>
      <offset>0</offset>
      <text>INTRODUCTION</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>The recently discovered APOBEC3 family of cytidine deaminases is considered to play an important role in antiviral intrinsic immunity (1,2). In primates, the seven paralogs APOBEC3A, B, C, DE, F, G, H (A3A-H) have been described (3), and they appear to fulfill individual functions. Human APOBEC3G (A3G), the most prominent member of the APOBEC3 family has been identified as the cellular restriction factor that is responsible for inhibition of virion infectivity factor (Vif)-deleted human immunodeficiency virus-1 (HIV-1) replication in non-permissive cells (4). A3G is packaged into HIV-1Deltavif particles and causes C-to-U deaminations on the single-stranded viral DNA during reverse transcription (5-8). This leads to degradation of the uracile-containing DNA by cellular repair mechanisms or to hypermutation of the viral genome (5,6). As a result, only a marginal fraction of the A3G-containing HIV-1 particles is able to complete the replication cycle. In addition to the inhibition of HIV-1, A3G restricts replication of other lentiviruses, gammaretroviruses, deltaretroviruses, spumaviruses, long-terminal-repeat (LTR)-retrotransposons, orthohepadnaviruses and avihepadnaviruses (9-21). Interestingly, deamination seems not to be the only A3G-mediated antiviral mechanism; in the case of hepatitis B virus (HBV) and human T cell leukemia virus type 1 (HTLV-1), A3G was shown to restrict virus replication by deamination-independent mechanisms (12, 13, 19, 22-25). Another member of the APOBEC3 family, APOBEC3F (A3F), appears to have similar activities like A3G (26,27). A3F is also packaged into HIV-1Deltavif particles and induces similar C-to-U deaminations, although the proteins differ in their target sequences specificity (26,28). Furthermore, A3F proteins were detected in many tissues that express A3G and are able to form heteromultimers with A3G (26,29,30). Both proteins localize to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of non-translating mRNAs (30,31).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="186" length="194"></location>
        <text>APOBEC3A</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="196" length="197"></location>
        <text>B</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="199" length="200"></location>
        <text>C</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="202" length="204"></location>
        <text>DE</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="206" length="207"></location>
        <text>F</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="209" length="210"></location>
        <text>G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="212" length="213"></location>
        <text>H</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="302" length="310"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="312" length="315"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="459" length="484"></location>
        <text>virion infectivity factor</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="486" length="489"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="579" length="582"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="610" length="613"></location>
        <text>vif</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="902" length="905"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1016" length="1019"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1264" length="1267"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1386" length="1389"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1527" length="1535"></location>
        <text>APOBEC3F</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1537" length="1540"></location>
        <text>A3F</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1583" length="1586"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1596" length="1599"></location>
        <text>A3F</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1632" length="1635"></location>
        <text>vif</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1776" length="1779"></location>
        <text>A3F</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1832" length="1835"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1878" length="1881"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Negative_regulation</infon>
        <location offset="491" length="498"></location>
        <text>deleted</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Gene_expression</infon>
        <location offset="1824" length="1831"></location>
        <text>express</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Binding</infon>
        <location offset="1852" length="1872"></location>
        <text>form heteromultimers</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Localization</infon>
        <location offset="1908" length="1916"></location>
        <text>localize</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Entity</infon>
        <location offset="1920" length="1946"></location>
        <text>mRNA processing (P) bodies</text>
      </annotation>
    </passage>
    <passage>
      <offset>2048</offset>
      <text>A3G has been shown to be expressed in T cells, a relevant cell target for HIV-1 in vivo, but little is known about its regulation (4,29,32). There is a report describing that mitogenic stimulation of T cells upregulates A3G mRNA levels, but this was not analyzed on the transcriptional level (33). Since the A3G promoter has not been systematically analyzed so far, our aim was to clone the A3G promoter and characterize its regulation in T cells. In our study, we observed that A3G uses multiple transcriptional start sites (TSS). By generating a series of 5' deletions of the A3G promoter, we identified a 180 bp region that mediated basal transcription. In T cells, transcriptional activity of this core promoter was not inducible by mitogenic stimulation or interferon treatment, but was dependent on a GC-box which was recognized by Sp (specificity protein) 1 and Sp3 transcription factors.</text>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="2048" length="2051"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="2268" length="2271"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="2356" length="2359"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="2439" length="2442"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="2527" length="2530"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="2626" length="2629"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="2810" length="2820"></location>
        <text>interferon</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="2886" length="2912"></location>
        <text>Sp (specificity protein) 1</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="2917" length="2920"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Gene_expression</infon>
        <location offset="2073" length="2082"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Regulation</infon>
        <location offset="2167" length="2177"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Positive_regulation</infon>
        <location offset="2256" length="2267"></location>
        <text>upregulates</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Gene_expression</infon>
        <location offset="2272" length="2283"></location>
        <text>mRNA levels</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Entity</infon>
        <location offset="2443" length="2451"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Regulation</infon>
        <location offset="2473" length="2483"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Entity</infon>
        <location offset="2855" length="2861"></location>
        <text>GC-box</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Binding</infon>
        <location offset="2872" length="2882"></location>
        <text>recognized</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
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      <node refid="T9" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T10" role="Event"></node>
      <node refid="T11" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T18" role="Event"></node>
      <node refid="T19" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
      <node refid="T25" role="Theme2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
      <node refid="T39" role="ToLoc"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
      <node refid="T39" role="ToLoc"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T29" role="Theme"></node>
      <node refid="T44" role="Site"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
      <node refid="T33" role="Theme2"></node>
      <node refid="T46" role="Site"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
      <node refid="T34" role="Theme2"></node>
      <node refid="T46" role="Site"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-02-MATERIALS_AND_METHODS-01</id>
    <passage>
      <offset>0</offset>
      <text>Cell culture</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>The human T cell lines A3.01 and PM1 (NIBSC, UK) and the human myeloid cell line U937 (NIBSC, UK) were grown in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 100 U/ml penicillin-streptomycin. The human hepatic cell lines HepG2 and Huh7 (kindly provided by Dr Thomas Pietschmann, Department of Molecular Virology, University of Heidelberg) as well as HeLa cells were maintained in Dulbecco's high glucose modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine and 100 U/ml penicillin-streptomycin. Cells lines were incubated at 37degreesC with 100% humidity in 5-7% CO2 and passaged using standard cell culture techniques.</text>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-03-MATERIALS_AND_METHODS-02</id>
    <passage>
      <offset>0</offset>
      <text>Plasmids</text>
    </passage>
    <passage>
      <offset>9</offset>
      <text>For cloning of an APOBEC3G promoter-driven reporter plasmid, genomic DNA was prepared from the T cell line PM1 using the DNeasy Kit (Qiagen). The DNA sequence ranging from positions -959 to +66 relative to the identified transcription start was amplified via PCR using the primers 3Gprom1025 (5'-TGTGAACGCGTTGCTGCAGGCCATCTGGATGTATATG-3') and 3Gpromreverse (5'-ACAGCAGATCTAGGGACCTCTGATAAAGACAGG-3'). PCR reactions were performed with Pwo DNA Polymerase (Roche) using the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 58degreesC for 60 s, 72degreesC for 60 s; one cycle 72degreesC for 7 min. The amplicon was ligated into the promoterless luciferase reporter plasmid pGL3-Basic (Promega) via MluI and BglII restriction sites, which were introduced by the primers. The resulting construct contained 1025 bp of the A3G promoter and was designated pGL3-APOprom1025. Reporter plasmids containing shorter fragments of the APOBEC3G promoter were constructed using pGL3-APOprom1025 as template and the following forward primers: for plasmid pGL3-APOprom502 (containing sequence -436/+66): 3Gprom502 (5'-TGTGAACGCGTTCCATAACATGGGGACAAGA-3'); for plasmid pGL3-APOprom225 (containing sequence -159/+66): 3Gprom225 (5'-TGTGAACGCGTCGAGGGCAGGATCCGGGAGT-3'); for plasmid pGL3-APOprom180 (containing sequence -114/+66): 3Gprom180 (5'-TGTGAACGCGTTCTTGATGGTGGAGAGGAGG-3'); for plasmid pGL3-APOprom150 (containing sequence -84/+66): 3Gprom150 (5'-TGTGAACGCGTGCGGGACCACCAGGGGAGGGGCTT-3'); for plasmid pGL3-APOprom120 (containing sequence -54/+66): 3Gprom120 (5'-TGTGAACGCGTTGCTGGCTCAGCCTGGTGTG-3'); for plasmid pGL3-APOprom60 (containing sequence +7/+66): 3Gprom60 (5'-TGTGAACGCGTCCCTTTGCAATTGCCTTG-3'); each in combination with the reverse primer 3Gpromreverse (described above). PCR reactions were performed with Pfu Ultra Hotstart (Stratagene) using the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 45 s, 58degreesC for 45 s, 72degreesC for 60 s; one cycle 72degreesC for 7 min. As for pGL3-APOprom1025, MluI and BglII restriction sites were introduced via the primers and PCR products were ligated into pGL3-Basic (Promega) via these restriction sites. pGL3-APOprom180mut carries two point mutations (bold) and was generated using the primer 3GProm180mut (5'-TGTGAACGCGTTCTTGATGGTGGAGAGGAGGCTCCAGCTGTTCGGGACCACCAG-3') in combination with primer 3Gpromreverse. This PCR was performed with an annealing temperature of 65degreesC. pGL3promE1 (containing nucleotides -114/-85) and pGL3promE2 (containing nucleotides -92/-63) were constructed by annealing the following single-stranded oligonucleotides: 114_85Plus (5'- CGCGTTCTTGATGGTGGAGAGGAGGCTCCAGCTGGA-3') and 114_85Minus (5'- GATCTCCAGCTGGAGCCTCCTCTCCACCATCAAGAA-3') or 92-63Plus (5'-CGCGTCCAGCTGGGCGGGACCACCAGGGGAGGGGCA-3') and 92_63Minus (5'-GATCTGCCCCTCCCCTGGTGGTCCCGCCCAGCTGGA-3'). After annealing, the double-stranded oligonucleotides which contained the respective 30 bp of the APOBEC3G promoter and sticky ends compatible with MluI and BglII restriction sites were ligated into the pGL3-Promoter (Promega) vector. The sequences of all constructed plasmids were verified by sequence analysis. Nucleotide -219 of the cloned APOBEC3G promoter differs from the sequence in the database (GenBanktrade accession number DQ147772). An A-to-C substitution is present at this position. Numbering is relative to the major transcriptional start site we identified.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="27" length="35"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="442" length="445"></location>
        <text>Pwo</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="691" length="701"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="865" length="868"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="969" length="977"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1847" length="1850"></location>
        <text>Pfu</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="3011" length="3019"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="3256" length="3264"></location>
        <text>APOBEC3G</text>
      </annotation>
    </passage>
    <passage>
      <offset>3488</offset>
      <text>The reporter plasmids pGL3-Control and phRG-TK were purchased from Promega. pGL2-CVX contains two repeats of the IFN-responsive GAS (gamma activated sequence) elements (GATCTGGATTTAGAGTAATATGAAACTGAAAGTACTTCG) of the guanylate-binding protein (GBP) gene in front of a CMV minimal promoter and was kindly provided by Ute Pagelow and Mario Koster from the Helmholtz-Zentrum fur Infektionsforschung. Plasmid pNL4-3 (NIBSC, UK) contains the full-length HIV-1NL4-3 genome and has been described previously (34). pcDNA3.1Vif was generously provided by Nathaniel R. Landau from the Salk Institute, La Jolla. It was generated by amplifying the Vif gene from pNL4-3 and ligating it into the pcDNA3.1 vector via BamHI and XhoI restriction sites. A 3'-WPRE element was included into the XhoI site. pBS-kRSPA-TatHIV-1(NL4-3) was constructed by amplifying the two exons of Tat via PCR reaction using the molecular clone pNL4-3 as template and the following primer sets: exon1, 5UXho1HIV-Tat 1Plus (5'-GCATGCTCGAGATGGAGCCAGTAGATCCTAG-3') and HIV-Tat 1Minus (5'-TGCTTTGATAGAGAAGCTTGATG-3'); exon2, 15FHIV-Tat2Plus (5'-TTCTCTATCAAAGCAACCCACCTCCCAATCCCG-3') and 5USpe1HIV-Tat2Minus (5'-GACGTACTAGTCTATTCCTTCGGGCCTGTC-3'). XhoI and SpeI restriction sites were introduced via the primers. The sense-primer of exon2 starts with a 15-mer which is homologous to the 3' end of exon1 and necessary for fusion of both exons. PCRs were performed with Expand High Fidelity PCR System (Roche) using the following conditions: one cycle 94degreesC for 3 min; 35 cycles 94degreesC for 45 s, 55degreesC for 45 s, 68degreesC for 45 s; one cycle 68degreesC for 7 min. For fusion of both exons, the following PCR conditions were applied: one cycle 94degreesC for 3 min; 35 cycles 94degreesC for 45 s, 58degreesC for 45 s, 68degreesC for 60 s. After 10 cycles without primers, the sense-primer of exon1 and the antisense-primer of exon2 were added for the remaining cycles. The resulting amplicon was ligated into the pBS-kRSPA vector (35) via XhoI and SpeI restriction sites.</text>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="3705" length="3730"></location>
        <text>guanylate-binding protein</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="3732" length="3735"></location>
        <text>GBP</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="4124" length="4127"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="4285" length="4288"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="4348" length="4351"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="4462" length="4465"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="4520" length="4523"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="4578" length="4581"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="4643" length="4646"></location>
        <text>Tat</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T9" role="Event"></node>
      <node refid="T10" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-04-MATERIALS_AND_METHODS-03</id>
    <passage>
      <offset>0</offset>
      <text>5'-Rapid amplification of cDNA ends analysis (RACE)</text>
    </passage>
    <passage>
      <offset>52</offset>
      <text>Total RNA was isolated from A3.01 T cells using RNeasy mini kit (Qiagen). The transcriptional start sites of A3G were identified using the 5'/3' RACE Kit, 2nd Generation (Roche) according to the manufacturer's instructions. The following primers were used: RACE-APO3G1 (5'-TATCCCTTGTACACTTTGT-3') for cDNA synthesis, RACE-APO3G2 (5'-CATACTCCTGGTCACGAT-3') for the first PCR and RACE-APO3Gnest (5'-GAATACACCTGGCCTCGAA-3') for the nested PCR. Reaction products were analyzed by agarose gel electrophoresis, purified using QIAquick gel extraction kit (Qiagen), T/A-cloned into vector pCR4-TOPO (Invitrogen) and sequenced.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="161" length="164"></location>
        <text>A3G</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-05-MATERIALS_AND_METHODS-04</id>
    <passage>
      <offset>0</offset>
      <text>Luciferase assay</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="10"></location>
        <text>Luciferase</text>
      </annotation>
    </passage>
    <passage>
      <offset>17</offset>
      <text>For transient transfection of A3.01 and U937 cells, DMRIE-C transfection reagent (Life Technologies) was used (36). Cells were seeded in 6-well tissue culture plates (5 x 105 cells per well) in 1.5 ml Opti-MEM (Life Technologies) containing 0.5 microg firefly luciferase reporter plasmid and 3.5 microl DMRIE-C. After 4-5 h of incubation, 1.5 ml complete RPMI medium were added. HepG2 and Huh7 cell lines were transfected using LipofectAMINE Plus as recommended by the manufacturer (Life Technologies). Briefly, exponential growing cells (1.5 x 106) were transfected with 5 microl LipofectAMINE, 6 microl PLUS reagent and the required amount of plasmid DNA in a final volume of 1 ml Opti-MEM. Following 4 h of incubation, cells were washed in PBS and 3 ml of complete DMEM medium were added.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="277" length="287"></location>
        <text>luciferase</text>
      </annotation>
    </passage>
    <passage>
      <offset>810</offset>
      <text>For cotransfection of reporter plasmids and siRNA into HeLa cells, HiPerfect transfection reagent (Qiagen) was used according to the manufacturer's protocol for cotransfection of adherent cells with siRNA and plasmid DNA.</text>
    </passage>
    <passage>
      <offset>1033</offset>
      <text>Two days after transfection of the respective cell lines, cells were harvested in 100 microl (suspension cells) or 300 microl (adherent cells) of Passive Lysis Buffer (Promega) and luciferase assay was performed using the Dual Luciferase Assay System (Promega) according to the manufacturer's instructions. As an internal control, 50 ng (adherent cells) or 100 ng (suspension cells) of ph-RG-TK plasmid (Promega), which constitutively expresses renilla luciferase was cotransfected in every sample and firefly luciferase activities were normalized to renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown in the figures. For stimulation of cells, final concentrations of 20 ng/ml TPA (Sigma) or 30 ng/ml IFN-alpha or 30 ng/ml IFN-gamma (Tebu-Bio) were applied approximately 15 h before harvesting for luciferase assay.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1214" length="1224"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1486" length="1496"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1543" length="1553"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1592" length="1602"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1799" length="1808"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1821" length="1830"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1896" length="1906"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="1468" length="1477"></location>
        <text>expresses</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-06-MATERIALS_AND_METHODS-05</id>
    <passage>
      <offset>0</offset>
      <text>Electrophoretic mobility shift assay (EMSA)</text>
    </passage>
    <passage>
      <offset>44</offset>
      <text>For preparation of nuclear extracts, 5 x 106 A3.01 T cells were washed in cold PBS and resuspended in 500 microl buffer A (10 mM HEPES pH7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After incubation for 15 min on ice, swollen cells were pressed 10 times through a syringe with a 26G needle and centrifuged at 5000 r.p.m. for 5 min. Pellets contained the nuclei and were washed in buffer A for two times and resuspended in 50 microl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA 1 mM DTT, 1 mM PMSF). After shaking for 30 min at 4degreesC and centrifugation for 10 min at 13 000 r.p.m., supernatants were used as nuclear extracts.</text>
    </passage>
    <passage>
      <offset>714</offset>
      <text>EMSA probes were generated by annealing the following complementary oligonucleotides: APO-Sp1/3, 5'- CCAGCTGGGCGGGACCACCAGGGGAGGGGC-3' and 5'-GCCCCTCCCCTGGTGGTCCCGCCCAGCTGG-3'; APO-Sp1/3mut, 5'- CCAGCTGTTCGGGACCACCAGGGGAGGGGC-3' and 5'- GCCCCTCCCCTGGTGGTCCCGAACAGCTGG-3' according to standard procedures. Nucleotides differing from the original promoter sequence are shown in bold type. A commercially available Sp1 probe (sc-2502, referred to as Sp1cons) was purchased from Santa Cruz Biotechnology. The double-stranded oligonucleotides were 5' end-labeled using T4 polynucleotide kinase (New England Biolabs) and [gamma-32P]ATP (3000 Ci/mmol, Amersham) and purified by using Nick G50 columns (Amersham).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="804" length="807"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="895" length="898"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1126" length="1129"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1278" length="1302"></location>
        <text>T4 polynucleotide kinase</text>
      </annotation>
    </passage>
    <passage>
      <offset>1421</offset>
      <text>For EMSA, 5 microg of nuclear proteins were preincubated on ice with 2 microg of poly(dI-dC) (Roche) as an unspecific competitor and 1 microg of bovine serum albumin in band shift buffer (50 mM Tris, 150 mM KCl, 5 mM EDTA, 2.5 mM dithiothreitol, 20% Ficoll) for 15 min. 32P-labeled oligonucleotides (50 000 c.p.m.) were added in a total volume of 20 microl, incubated on ice for 20 min and loaded onto 5% native polyacrylamide gels in 0.5xTris-borate-EDTA buffer. Upon fractionation, gels were dried and exposed for autoradiography. For competition experiments, 1- or 30-fold molar excess of the unlabeled APO-Sp1/3 or APO-Sp1/3mut oligonucleotides was added to the preincubation mixture. For supershift experiments, 2 microg Sp1 antibody (sc-59x, Santa Cruz Biotechnology) or Sp3 antibody (sc-644x, Santa Cruz Biotechnology) were added to the preincubation mixture and preincubation time was extended to 30 min.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1566" length="1586"></location>
        <text>bovine serum albumin</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="2031" length="2034"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="2044" length="2047"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="2147" length="2150"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="2198" length="2201"></location>
        <text>Sp3</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-07-MATERIALS_AND_METHODS-06</id>
    <passage>
      <offset>0</offset>
      <text>Chromatin immunoprecipitation (ChIP) assay</text>
    </passage>
    <passage>
      <offset>43</offset>
      <text>A3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37degreesC. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 x 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 microl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 x 107 cells and 2 microg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 microl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 microl of the resolved DNA in the first PCR, and 1 microl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5'-ccacggtggcctccgagggtga-3' and ChIP3Gminus: 5'-ctctccaccatcaagacagac-3' (1. PCR); ChIP3G2plus: 5'-tactctccctccctgtcccca-3' and ChIP3G nested minus: 5'-aggctgatgcctccgcag-3' (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 60degreesC for 60 s, 72degreesC for 2 min; one cycle 72degreesC for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5'-taagtaccacccagagatgag-3' and ChIP3Gneg_minus: 5'-catgatcttcatggtggcacg-3' for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55degreesC.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="470" length="490"></location>
        <text>Micrococcal Nuclease</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="792" length="795"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="809" length="812"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="829" length="834"></location>
        <text>actin</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1235" length="1238"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1482" length="1496"></location>
        <text>Taq polymerase</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1739" length="1742"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1932" length="1935"></location>
        <text>A3G</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-08-MATERIALS_AND_METHODS-07</id>
    <passage>
      <offset>0</offset>
      <text>RNA interference and western blot analysis</text>
    </passage>
    <passage>
      <offset>43</offset>
      <text>Sp1 and Sp3 translation was silenced in HeLa cells using the siRNA duplexes Hs_SP1_1_HP and Hs_SP3_1_HP (Qiagen). A nonspecific siRNA (Qiagen) was used as control. HeLa cells were transfected with 150 or 300 ng siRNA per 6-well, using the HiPerfect transfection reagent (Qiagen) according to the manufacturer's protocol for reverse transfection of adherent cells in 6-well plates.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="43" length="46"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="51" length="54"></location>
        <text>Sp3</text>
      </annotation>
    </passage>
    <passage>
      <offset>425</offset>
      <text>Forty-eight hours after transfection, HeLa cells were harvested for detection of Sp1 and Sp3 proteins. Cells were washed in PBS, lysed in RIPA (25 mM Tris pH 8.0, 137 mM NaCl, 1% Glycerol, 0.5% sodium deoxycholate, 1% NP-40, 2 mM EDTA pH 8, 0.1% SDS and protease inhibitors) and lysates were cleared by centrifugation. After boiling with Laemmli's buffer, samples were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Sp1 and Sp3 proteins were detected using alpha-Sp1(Pep2) antibody (sc-59, Santa Cruz) or alpha-Sp3(D-20) antibody (sc-644, Santa Cruz) followed by incubation with alpha-rabbit-HRP (Amersham Biosciences). For detection of tubulin, alpha-tubulin (B5-1-2, Sigma) and alpha-mouse-HRP (Amersham Biosciences) antibodies were used. Signals were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="506" length="509"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="514" length="517"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="897" length="900"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="905" length="908"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="944" length="947"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="992" length="995"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1118" length="1125"></location>
        <text>tubulin</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1133" length="1140"></location>
        <text>tubulin</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="493" length="502"></location>
        <text>detection</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="923" length="931"></location>
        <text>detected</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-09-RESULTS-01</id>
    <passage>
      <offset>0</offset>
      <text>Characterization of the transcriptional start sites of APOBEC3G by 5'-RACE</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="55" length="63"></location>
        <text>APOBEC3G</text>
      </annotation>
    </passage>
    <passage>
      <offset>75</offset>
      <text>To identify the transcriptional start sites (TSS) of APOBEC3G (A3G) in A3.01 T cells, we performed 5'-rapid amplification of cDNA ends analysis (RACE) with A3G-specific primers (see Figure 1). Agarose gel electrophoresis resolved the nested PCR products into three bands of different electrophoretic mobility with a dominant middle band (Figure 2). For each band, the DNA was cloned and sequence analysis of six or seven individual transformants was performed. We observed that the transcriptional start sites of the A3G gene were located between 58 and 361 nt upstream of the ATG start codon (Figure 1). Although TSS were variable and most sites were only detected once among the 19 clones analyzed, one TSS was identified in six individual clones. This TSS was located 66 nt upstream of the start of the published A3G mRNA sequence (GenBanktrade accession number NM021822) and we defined this position as the major transcriptional start site of the A3G gene.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="128" length="136"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="138" length="141"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="231" length="234"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="592" length="595"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="891" length="894"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1026" length="1029"></location>
        <text>A3G</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-10-RESULTS-02</id>
    <passage>
      <offset>0</offset>
      <text>The core promoter of A3G is located within the region -114/+66 relative to the TSS</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="21" length="24"></location>
        <text>A3G</text>
      </annotation>
    </passage>
    <passage>
      <offset>83</offset>
      <text>For characterization of the A3G promoter, we cloned the 1025 bp located at position -959/+66 relative to the identified transcription start into the promoterless pGL3-Basic luciferase reporter plasmid and designated the plasmid pGL3-Basic-APOprom1025. Similarly, pGL3-APOprom502 (containing sequence -436/+66), pGL3-APOprom225 (containing sequence -159/+66) and further 5' deletion reporter constructs containing 180, 150, 120 or 60 bp upstream of position +65 were generated (Figure 3A). In order to analyze transcriptional activity, the luciferase reporter plasmids were transiently transfected into A3.01 T cells. Luciferase assays revealed a approximately20-fold increased transcriptional activity of the 1025 bp sequence as compared to the empty vector, indicating that we had identified an active A3G promoter sequence (Figure 3B). This transcription rate was not significantly altered by the 5' deletions leading to the 502, 225 and 180 bp fragments (Figure 3B). In contrast, a drop in luciferase activity was observed in the case of the 150 bp fragment. This construct only retained 28% of the transcriptional activity of the 180 bp promoter in A3.01 T cells and activity of the 120 bp fragment was further reduced. Comparable reductions relative to the activity of 180 bp fragment were observed in the myeloid cell line U937 and the hepatic cell lines HepG2 and Huh7 (Figure 3C), indicating that the core promoter of A3G is located within the region -114/+66 relative to the TSS.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="111" length="114"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="256" length="266"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="622" length="632"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="700" length="710"></location>
        <text>Luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="886" length="889"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1076" length="1086"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1509" length="1512"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Positive_regulation</infon>
        <location offset="750" length="759"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Transcription</infon>
        <location offset="760" length="784"></location>
        <text>transcriptional activity</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Entity</infon>
        <location offset="792" length="808"></location>
        <text>1025 bp sequence</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="967" length="974"></location>
        <text>altered</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Negative_regulation</infon>
        <location offset="1068" length="1072"></location>
        <text>drop</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
      <node refid="T11" role="CSite"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-11-RESULTS-03</id>
    <passage>
      <offset>0</offset>
      <text>The A3G promoter is not inducible in T cells</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="4" length="7"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Entity</infon>
        <location offset="8" length="16"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Positive_regulation</infon>
        <location offset="24" length="33"></location>
        <text>inducible</text>
      </annotation>
    </passage>
    <passage>
      <offset>45</offset>
      <text>According to the current knowledge, APOBEC3G plays a role in the innate defence against pathogens like HIV-1. The latter has evolved a mechanism to counteract A3G activity by expressing the regulatory protein Vif (4). Vif induces proteasomal degradation of A3G and additionally inhibits A3G activity by further mechanisms (8, 37-43). To investigate whether the overexpression of HIV-1 proteins also influences A3G promoter activity, we either expressed HIV-1 Vif or the HIV-1 Tat protein, the latter has been shown to activate different viral and cellular promoters (44,45). In addition, increasing amounts of the plasmid pNL4-3, containing the full-length genome of HIV-1, were transfected. These constructs or the empty vector pcDNA3.1 were cotransfected with the 1025 bp construct into A3.01 T cells. Luciferase assays showed that the transcriptional activity of the A3G promoter was not significantly altered in the presence of Vif, Tat or HIV-1NL4-3 (Figure 4A), suggesting that HIV-1 is not modulating A3G expression on the transcriptional level.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="81" length="89"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="204" length="207"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="254" length="257"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="263" length="266"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="302" length="305"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="332" length="335"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="455" length="458"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="504" length="507"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="521" length="524"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="849" length="859"></location>
        <text>Luciferase</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="915" length="918"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="977" length="980"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="982" length="985"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1053" length="1056"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Negative_regulation</infon>
        <location offset="193" length="203"></location>
        <text>counteract</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Gene_expression</infon>
        <location offset="220" length="230"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="267" length="274"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein_catabolism</infon>
        <location offset="287" length="298"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Negative_regulation</infon>
        <location offset="323" length="331"></location>
        <text>inhibits</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Gene_expression</infon>
        <location offset="488" length="497"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Regulation</infon>
        <location offset="1042" length="1052"></location>
        <text>modulating</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Gene_expression</infon>
        <location offset="1057" length="1067"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>1099</offset>
      <text>It has been shown that the amount of A3G mRNA in T cells is increased in response to mitogenic stimulation with phorbol ester (46-48). In addition, interferons have been described to upregulate A3G expression in hepatocytes and macrophages (46,48). To investigate whether these stimuli interfere with A3G promoter activity in T cells, we transfected A3.01 T cells with the 1025 bp promoter or the empty vector pGL3-Basic (vector) and treated the cells with phorbol ester (TPA). The luciferase assay showed that the approximately15-fold increased transcriptional activity of the 1025 bp promoter relative to the empty vector was not further enhanced by TPA treatment (Figure 4B), although the functional activity of TPA was confirmed by induction of the SV40 promoter-containing reporter plasmid pGL3-Control (data not shown). Similarly, treatment of A3.01 T cells transfected with the A3G promoter deletion constructs with IFN-alpha or IFN-gamma showed no effect (Figure 4C). Interestingly, a control plasmid (pGL2-CVX) containing twoIFN-responsive GAS ( gamma activated sequence) elements upstream of the luciferase reporter gene was only induced by IFN-alpha in these cells (Figure 4C). Since two reports describe A3G upregulation by interferons in hepatocytes (47,48), we additionally performed the experiment in the hepatic cell line HepG2. In line with these publications, we observed an induction of the A3G promoter by approximately 2-fold after IFN-alpha or IFN-gamma stimulation (Figure 4D) with IFN-gamma being slightly more potent. For both interferon types, induction of A3G promoter activity was observed for all deletion constructs except for the 60 bp fragment, indicating that the responsible region is located within the 60 nt present in the 120 bp, but not in the 60 bp fragment.</text>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1136" length="1139"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1293" length="1296"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1400" length="1403"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1581" length="1591"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1984" length="1987"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="2022" length="2031"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="2035" length="2044"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="2205" length="2215"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="2250" length="2259"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="2315" length="2318"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="2509" length="2512"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="2552" length="2561"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="2565" length="2574"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="2604" length="2613"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="2682" length="2685"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Positive_regulation</infon>
        <location offset="1159" length="1168"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Positive_regulation</infon>
        <location offset="1282" length="1292"></location>
        <text>upregulate</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Gene_expression</infon>
        <location offset="1297" length="1307"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Positive_regulation</infon>
        <location offset="1635" length="1644"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Transcription</infon>
        <location offset="1645" length="1660"></location>
        <text>transcriptional</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Positive_regulation</infon>
        <location offset="1661" length="1669"></location>
        <text>activity</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Positive_regulation</infon>
        <location offset="1739" length="1747"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Positive_regulation</infon>
        <location offset="2319" length="2331"></location>
        <text>upregulation</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Positive_regulation</infon>
        <location offset="2492" length="2501"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Entity</infon>
        <location offset="2513" length="2521"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T31" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="E3" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
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    <relation id="E5">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="T25" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
      <node refid="T50" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E17" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-12-RESULTS-04</id>
    <passage>
      <offset>0</offset>
      <text>A GC-box located at position -87/-78 of the A3G promoter is important for transcriptional activity</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="44" length="47"></location>
        <text>A3G</text>
      </annotation>
    </passage>
    <passage>
      <offset>99</offset>
      <text>The reporter studies shown in Figure 3 had demonstrated a drop in luciferase activity after deletion of the 30 nt at the 5' end of the 180 bp core promoter. We therefore inspected the 30 bp sequence deleted in the 150 bp fragment and identified a GC-box at position -87/-78 (see Figure 1). The sequence TGGGCGGGAC, which is interrupted in the 150 bp fragment, represents a variant of the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif recognized by Sp1 and Sp3 transcription factors. To analyze whether this putative Sp1/Sp3-binding site mediates transcriptional activity of the 180 bp core promoter, we introduced two point mutations which changed the sequence from TGGGCGGGAC to TGTTCGGGAC (mutations shown in bold). This resulted in a 71% reduction of the transcriptional activity compared to the unmodified 180 bp promoter (Figure 5A) and this value was only marginally higher than the luciferase activity of the 150 bp fragment, indicating that the identified motif is essential for basal activity of the A3G core promoter. To further examine the transcriptional potency of the 30 nt present in the 180 bp promoter, we cloned the region -114/-85 (containing all nucleotides which are deleted in the 150 bp fragment, designated E1) or the region -92/-63 (containing the putative Sp1/Sp3 motif, designated E2) into the vector pGL3-Promoter (see Figure 1). This vector contains a luciferase reporter gene under the control of an SV40 promoter without enhancer sequences, and putative transcriptionally active sequences can be cloned upstream of the SV40 promoter. Luciferase assays showed that the E1 element increased SV40 promoter activity only by approximately2-fold (Figure 5B). In contrast, the 30 nt of E2 enhanced the transcriptional activity of the SV40 promoter by approximately4.3-fold, indicating that the intact GC-box present in the E2 element was responsible for the strongly enhanced transcriptional activity of the SV40 promoter.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="165" length="175"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="544" length="547"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="552" length="555"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="985" length="995"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1105" length="1108"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1477" length="1487"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1661" length="1671"></location>
        <text>Luciferase</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Entity</infon>
        <location offset="483" length="529"></location>
        <text>the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Binding</infon>
        <location offset="530" length="540"></location>
        <text>recognized</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Regulation</infon>
        <location offset="1502" length="1519"></location>
        <text>under the control</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Entity</infon>
        <location offset="1797" length="1802"></location>
        <text>30 nt</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="1809" length="1817"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="1822" length="1846"></location>
        <text>transcriptional activity</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="1958" length="1969"></location>
        <text>responsible</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="1987" length="1995"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="1996" length="2020"></location>
        <text>transcriptional activity</text>
      </annotation>
    </passage>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
      <node refid="T9" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T4" role="Theme2"></node>
      <node refid="T9" role="Site"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
      <node refid="T12" role="CSite"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-13-RESULTS-05</id>
    <passage>
      <offset>0</offset>
      <text>Sp1 and Sp3 transcription factors bind to the GC-box at position -87/-78 of the A3G promoter</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="3"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="8" length="11"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="80" length="83"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="34" length="38"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Entity</infon>
        <location offset="46" length="52"></location>
        <text>GC-box</text>
      </annotation>
    </passage>
    <passage>
      <offset>93</offset>
      <text>To investigate whether the GC-box represents a binding site for the transcription factors Sp1 and Sp3, we performed EMSA analyses with nuclear extracts isolated from A3.01 T cells. As probes, we radioactively labeled the unmodified E2 sequence (nucleotides -92/-63 of the A3G promoter, probe designated APO-Sp1/3) or the same region carrying the two point mutations described above (probe designated APO-Sp1/3mut). As control, we used a commercially available Sp1 consensus oligonucleotide (Sp1cons), which is known to be recognized by Sp1 transcription factors. Four DNA-protein complexes were observed in the presence of the APO-Sp1/3 probe (Figure 6A). The upper two complexes were specific since they disappeared in the presence of a 30-fold molar excess of unlabeled APO-Sp1/3 probe (Figure 6A, lane 7). Further confirmation of the specificity of these DNA-protein complexes was demonstrated by the inability of the unlabeled APO-Sp1/3mut probe to abolish binding (Figure 6A, lanes 12 and 13). As expected, the two specific complexes were also present in the case of the Sp1 cons control probe (Figure 6A, lanes 2 and 3). In contrast, none of these specific complexes was observed when the mutated probe was used (Figure 6A, lanes 10 and 11), confirming that protein binding was dependent on the identified GC-box. In order to characterize the complexes, we performed supershift experiments using Sp1- and Sp3-specific antibodies. The upper of the complexes that appeared in combination with the Sp1cons or APO-Sp1/3 probes, shifted in the presence of the Sp1 antibody (Figure 6B, lanes 3 and 7), whereas the lower complex shifted in the presence of the Sp3 antibody (Figure 6B, lanes 4 and 8). Taken together, the EMSA demonstrated that the GC-box located on the A3G core promoter serves as a binding site for Sp1 and Sp3.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="183" length="186"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="191" length="194"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="365" length="368"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="629" length="632"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1495" length="1498"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1504" length="1507"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1654" length="1657"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1752" length="1755"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1862" length="1865"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1909" length="1912"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1917" length="1920"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Entity</infon>
        <location offset="120" length="126"></location>
        <text>GC-box</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Binding</infon>
        <location offset="127" length="152"></location>
        <text>represents a binding site</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Binding</infon>
        <location offset="615" length="625"></location>
        <text>recognized</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Entity</infon>
        <location offset="1840" length="1846"></location>
        <text>GC-box</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Binding</infon>
        <location offset="1880" length="1904"></location>
        <text>serves as a binding site</text>
      </annotation>
    </passage>
    <passage>
      <offset>1923</offset>
      <text>To show binding of Sp1 and Sp3 factors also in the context of the endogenous A3G promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Sp1 and Sp3 antibodies, but not an actin antibody, immunoprecipitated the A3G promoter in A3.01 T cells (Figure 6C, upper panel). In contrast, no PCR signal was received with a primer pair recognizing a region in the A3G gene approximately4000 bp downstream of the Sp1/Sp3-binding site (Figure 6C, lower panel). Only the positive controls showed a DNA band, with an A3G expression plasmid or the sheared and cross-linked input DNA used as template. This demonstrates the binding of Sp1 and Sp3 transcription factors to the endogenous A3G promoter.</text>
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        <infon key="type">Protein</infon>
        <location offset="1942" length="1945"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1950" length="1953"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="2000" length="2003"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="2074" length="2077"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="2082" length="2085"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="2109" length="2114"></location>
        <text>actin</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="2148" length="2151"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="2291" length="2294"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="2440" length="2443"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="2556" length="2559"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="2564" length="2567"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="2608" length="2611"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="1931" length="1938"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Entity</infon>
        <location offset="2004" length="2012"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Gene_expression</infon>
        <location offset="2444" length="2454"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Binding</infon>
        <location offset="2545" length="2552"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Entity</infon>
        <location offset="2612" length="2620"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
      <node refid="T28" role="Site2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
      <node refid="T28" role="Site2"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T4" role="Theme2"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T5" role="Theme2"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T13" role="Theme2"></node>
      <node refid="T32" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T14" role="Theme2"></node>
      <node refid="T32" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T17" role="Theme2"></node>
      <node refid="T35" role="Site2"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
      <node refid="T17" role="Theme2"></node>
      <node refid="T35" role="Site2"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
      <node refid="T26" role="Theme2"></node>
      <node refid="T38" role="Site2"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T25" role="Theme"></node>
      <node refid="T26" role="Theme2"></node>
      <node refid="T38" role="Site2"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-14-RESULTS-06</id>
    <passage>
      <offset>0</offset>
      <text>Silencing of Sp1 and Sp3 reduces A3G promoter activity</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="13" length="16"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="21" length="24"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="33" length="36"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Negative_regulation</infon>
        <location offset="0" length="9"></location>
        <text>Silencing</text>
      </annotation>
    </passage>
    <passage>
      <offset>55</offset>
      <text>To confirm the role of Sp1 and Sp3 in regulation of A3G promoter activity, we silenced their translation via RNA interference. Functionality of the siRNAs directed against Sp1 or Sp3 was confirmed by western blot analysis: protein levels of Sp1 as well as the long and short isoforms of Sp3 were strongly reduced in the presence of 150 or 300 ng specific siRNA (Figure 7A). An unspecific control siRNA had no influence (Figure 7A). We then cotransfected the luciferase reporter plasmid containing the 180 bp A3G promoter together with 100 ng siRNA using an optimized protocol for the cotransfection of plasmid plus siRNA. This resulted in a 31-43% reduction of luciferase activity in the presence of Sp1- or Sp3-specific siRNA compared to the control siRNA. In contrast, no influence on transcriptional activity of the 150 bp fragment, which does not contain the Sp1/Sp3-binding motif, was observed. Thus, both Sp1 and Sp3 factors are mediating transcriptional activity of the A3G promoter.</text>
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        <infon key="type">Protein</infon>
        <location offset="78" length="81"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="86" length="89"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="107" length="110"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="227" length="230"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="234" length="237"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="296" length="299"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="342" length="345"></location>
        <text>Sp3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="513" length="575"></location>
        <text>luciferase reporter plasmid containing the 180 bp A3G promoter</text>
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        <infon key="type">Protein</infon>
        <location offset="755" length="758"></location>
        <text>Sp1</text>
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        <infon key="type">Protein</infon>
        <location offset="763" length="766"></location>
        <text>Sp3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="966" length="969"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="974" length="977"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1032" length="1035"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="278" length="292"></location>
        <text>protein levels</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="360" length="367"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="495" length="508"></location>
        <text>cotransfected</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Negative_regulation</infon>
        <location offset="703" length="712"></location>
        <text>reduction</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="776" length="781"></location>
        <text>siRNA</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="E9" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-15-DISCUSSION</id>
    <passage>
      <offset>0</offset>
      <text>DISCUSSION</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>Shortly after the discovery of APOBEC3G, it became clear that this human gene plays an important role in antiretroviral defense (4). Originally identified as a restriction factor of HIV-1 infection, expression of APOBEC3G was found in human peripheral blood lymphocytes and macrophages, which represent the main target cells for HIV-1 (32). In addition, the protein was detected in lung, liver, spleen, testis and ovary, but little is known about its transcriptional regulation (29,32,47). To address this question, we cloned the human APOBEC3G promoter and analyzed its regulation in T cells. Applying 5'-RACE, we identified multiple start sites for transcription of the A3G gene. This observation is consistent with the fact that the gene appears to lack canonical CCAAT and TATA boxes (32), a condition often associated with multiple transcriptional start sites (49-51). However, one single start site was detected in 6 of 19 clones and was designated as +1. This TSS is located 66 bp upstream of the start of the published mRNA sequence (GenBanktrade NM021822).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="42" length="50"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="224" length="232"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="547" length="555"></location>
        <text>APOBEC3G</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="683" length="686"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Gene_expression</infon>
        <location offset="210" length="220"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Gene_expression</infon>
        <location offset="381" length="389"></location>
        <text>detected</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Entity</infon>
        <location offset="556" length="564"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Regulation</infon>
        <location offset="582" length="592"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Transcription</infon>
        <location offset="662" length="675"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>1078</offset>
      <text>We cloned a 1025 bp promoter, which ranges from position -959/+66 relative to the identified TSS. Luciferase reporter assays showed that this promoter was transcriptionally active in A3.01 T cells. Treatment with the phorbol ester TPA did not further enhance transcriptional activity, although upregulation of A3G mRNA levels in T cells by TPA has been described (33). Rose et al. used actinomycin D to block further transcription and did not find evidence for enhanced mRNA stability. Therefore, the authors suggested that an enhanced transcription rate could be responsible for the increased amount of A3G mRNA after TPA treatment. However, the promoter analysis we performed indicates a different mechanism that is independent of transcriptional regulation. In addition, we observed that the A3G promoter was not inducible by IFN-alpha or IFN-gamma in A3.01 T cells, whereas in the hepatic cell line HepG2, a moderate induction was measured. It has been described previously that A3G gene expression is upregulated by interferons in hepatocytes and macrophages (46-48, 52). In this context, two interferon-responsive elements have been identified at the positions -6/+9 and +1/+15 relative to the TSS (48). Consistent with this data, we observed an interferon induction in hepatocytes for the fragments ranging between 1025 and 120 bp length. The activity of the 60 bp fragment that does not contain these motifs was not affected. Thus, the induction we observed was most likely mediated by the described interferon-responsive elements. However, according to our results, these motifs can enhance transcription in hepatic cells, but not in T cells. This is surprising, since IFN-alpha-inducible signaling cascades are present in A3.01 T cells: we showed that the control plasmid harboring interferon-responsive GAS elements was markedly induced by IFN-alpha treatment. In contrast, the GAS element was not responsive to IFN-gamma treatment in these cells, suggesting that our T cell line was not able to mediate IFN-gamma-induced signals. So far, enhanced A3G promoter activity by interferons is clearly described in hepatocytes and macrophages, whereas the situation in T cells is still unclear. A recent study found no influence of IFN-alpha or IFN-gamma on A3G expression in resting primary blood lymphocytes (52). Another publication describes an enhanced expression of A3G after IFN-alpha treatment in resting primary CD4 T cells, but not in activated T cells (53). This observation is not contradictory to our results since T cell lines are mitotically active and therefore rather in an activated than in a resting state. In conclusion, the current data suggests that regulation of the A3G promoter activity by interferons is dependent on the cell type and possibly also from the cellular activation status.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1176" length="1186"></location>
        <text>Luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1388" length="1391"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1682" length="1685"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1873" length="1876"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1907" length="1916"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1920" length="1929"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="2061" length="2064"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="2756" length="2765"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="2929" length="2938"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="3001" length="3010"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="3093" length="3102"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="3137" length="3140"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="3315" length="3324"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="3328" length="3337"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="3341" length="3344"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="3455" length="3458"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="3465" length="3474"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="3504" length="3507"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="3773" length="3776"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Positive_regulation</infon>
        <location offset="1329" length="1336"></location>
        <text>enhance</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Transcription</infon>
        <location offset="1337" length="1352"></location>
        <text>transcriptional</text>
      </annotation>
      <annotation id="T87">
        <infon key="type">Positive_regulation</infon>
        <location offset="1372" length="1384"></location>
        <text>upregulation</text>
      </annotation>
      <annotation id="T88">
        <infon key="type">Negative_regulation</infon>
        <location offset="1481" length="1486"></location>
        <text>block</text>
      </annotation>
      <annotation id="T89">
        <infon key="type">Transcription</infon>
        <location offset="1495" length="1508"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T90">
        <infon key="type">Positive_regulation</infon>
        <location offset="1605" length="1613"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T91">
        <infon key="type">Transcription</infon>
        <location offset="1614" length="1627"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T92">
        <infon key="type">Positive_regulation</infon>
        <location offset="1662" length="1671"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T93">
        <infon key="type">Entity</infon>
        <location offset="1877" length="1885"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T94">
        <infon key="type">Positive_regulation</infon>
        <location offset="1894" length="1903"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T95">
        <infon key="type">Gene_expression</infon>
        <location offset="2070" length="2080"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T96">
        <infon key="type">Positive_regulation</infon>
        <location offset="2084" length="2095"></location>
        <text>upregulated</text>
      </annotation>
      <annotation id="T97">
        <infon key="type">Entity</infon>
        <location offset="2444" length="2458"></location>
        <text>60 bp fragment</text>
      </annotation>
      <annotation id="T98">
        <infon key="type">Regulation</infon>
        <location offset="2502" length="2510"></location>
        <text>affected</text>
      </annotation>
      <annotation id="T99">
        <infon key="type">Entity</infon>
        <location offset="2572" length="2616"></location>
        <text>the described interferon-responsive elements</text>
      </annotation>
      <annotation id="T100">
        <infon key="type">Positive_regulation</infon>
        <location offset="2670" length="2677"></location>
        <text>enhance</text>
      </annotation>
      <annotation id="T101">
        <infon key="type">Transcription</infon>
        <location offset="2678" length="2691"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T102">
        <infon key="type">Regulation</infon>
        <location offset="3302" length="3311"></location>
        <text>influence</text>
      </annotation>
      <annotation id="T103">
        <infon key="type">Gene_expression</infon>
        <location offset="3345" length="3355"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T104">
        <infon key="type">Positive_regulation</infon>
        <location offset="3432" length="3440"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T105">
        <infon key="type">Gene_expression</infon>
        <location offset="3441" length="3451"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>3896</offset>
      <text>We additionally analyzed the influence of HIV-1 proteins on A3G promoter activity. The HIV-1 Vif protein is neutralizing the antiviral function of A3G by a multitude of ways. Targeting the A3G protein for proteasomal degradation is considered to be the main mode of action (37-41), but it becomes more and more clear that other mechanisms are also involved. There is evidence that Vif also promotes exclusion of A3G from the virus particles and an influence on the A3G translation process is discussed (8,42,43). The question whether Vif alters transcription controlled by the A3G promoter has not been analyzed so far. Our analysis indicates that transcription from the A3G promoter is unaffected by Vif or other HIV-1 proteins. Taken together, in T cell lines, the A3G promoter appears constitutively active.</text>
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        <infon key="type">Protein</infon>
        <location offset="3956" length="3959"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="3989" length="3992"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="4043" length="4046"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="4085" length="4088"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="4277" length="4280"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="4308" length="4311"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="4361" length="4364"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="4430" length="4433"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="4473" length="4476"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="4567" length="4570"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="4597" length="4600"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="4663" length="4666"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T106">
        <infon key="type">Positive_regulation</infon>
        <location offset="4071" length="4080"></location>
        <text>Targeting</text>
      </annotation>
      <annotation id="T107">
        <infon key="type">Protein_catabolism</infon>
        <location offset="4101" length="4124"></location>
        <text>proteasomal degradation</text>
      </annotation>
      <annotation id="T108">
        <infon key="type">Regulation</infon>
        <location offset="4434" length="4440"></location>
        <text>alters</text>
      </annotation>
      <annotation id="T109">
        <infon key="type">Transcription</infon>
        <location offset="4441" length="4454"></location>
        <text>transcription</text>
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      <annotation id="T110">
        <infon key="type">Regulation</infon>
        <location offset="4455" length="4465"></location>
        <text>controlled</text>
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      <annotation id="T111">
        <infon key="type">Entity</infon>
        <location offset="4477" length="4485"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T112">
        <infon key="type">Transcription</infon>
        <location offset="4544" length="4557"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T113">
        <infon key="type">Positive_regulation</infon>
        <location offset="4558" length="4562"></location>
        <text>from</text>
      </annotation>
      <annotation id="T114">
        <infon key="type">Entity</infon>
        <location offset="4571" length="4579"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T115">
        <infon key="type">Regulation</infon>
        <location offset="4583" length="4593"></location>
        <text>unaffected</text>
      </annotation>
      <annotation id="T116">
        <infon key="type">Entity</infon>
        <location offset="4667" length="4675"></location>
        <text>promoter</text>
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      <annotation id="T117">
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      <offset>4708</offset>
      <text>By generating a series of 5' deletions, we showed that the core promoter is located within the region -114/+66 relative to the TSS. This 180 bp promoter was transcriptionally active in the lymphoid, myeloid and hepatic cell lines we tested, indicating that ubiquitous transcription factors are involved in regulation of A3G gene transcription. A GC-box with the sequence TGGGCGGGAC was identified at position -87/-78 of the A3G promoter. This GC-box is essential for basal promoter activity, since changing the motif to TGTTCGGGAC by introducing two point mutations strongly reduced A3G promoter activity. The transcriptional potency of the GC-box was further demonstrated by cloning it upstream of an SV40 promoter. This resulted in a 4-fold enhanced transcription rate. The hypothesis that a general transcription factor is involved in regulation of A3G transcription was confirmed by EMSA. Supershift analysis showed that the transcription factors Sp1 and Sp3 which are ubiquitously expressed in mammalian cells, bind specifically to the identified motif. In addition, the binding of Sp1 and Sp3 to the endogenous A3G promoter was demonstrated by a chromatin immunoprecipitation assay. This observation is consistent with the results of the 5'-RACE, since Sp1 is known to play an important role for RNA polymerase II to bind to the transcription initiation site in TATA-boxless promoters and is associated with multiple transcription initiation sites (54-56). We also tested whether overexpression of Sp1 or Sp3 proteins had an influence on A3G promoter activity. Luciferase assays revealed that A3G promoter activities remained unchanged (data not shown), most probably due to the high basal expression level of the endogenous Sp1 and/or Sp3 proteins. The Sp family of transcription factors is involved in transcriptional regulation of many housekeeping, tissue-specific, viral and inducible genes (57). Whereas Sp1 typically acts as an activator, Sp3 can serve as a repressor or activator (57). However, in the context of the A3G promoter, both transcription factors serve as activators, as shown by siRNA-mediated silencing of the single factors. Although siRNAs directed against Sp1 or Sp3 significantly reduced transcription controlled by the 180 bp promoter, transcriptional activity was not reduced to the level of the 150 bp fragment. This can be explained by the remaining low amounts of Sp1/Sp3 proteins which can be seen in the western blot analysis. Alternatively, other transcription factors could be involved in A3G regulation.</text>
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        <text>A3G</text>
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        <text>Luciferase</text>
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        <text>A3G</text>
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        <text>regulation</text>
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        <text>transcription</text>
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        <text>regulation</text>
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        <text>transcription</text>
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        <text>expressed</text>
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        <location offset="5724" length="5728"></location>
        <text>bind</text>
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        <text>binding</text>
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        <text>promoter</text>
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        <text>play an important role</text>
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        <text>bind</text>
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        <text>overexpression</text>
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        <text>expression</text>
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        <text>serve as activators,</text>
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        <location offset="6828" length="6837"></location>
        <text>silencing</text>
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        <location offset="6919" length="6926"></location>
        <text>reduced</text>
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        <text>transcription</text>
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        <location offset="6941" length="6951"></location>
        <text>controlled</text>
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        <text>180 bp promoter</text>
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        <text>transcriptional</text>
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        <text>reduced</text>
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        <text>regulation</text>
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    <passage>
      <offset>7254</offset>
      <text>Our results also provide information about the regulation of APOBEC3F (A3F), another member of the human APOBEC3 family. A3F is expressed in many human tissues that also express A3G and both proteins have been shown to form heteromultimers, which are most likely generated through binding to an RNA intermediate (26,29,30). A3F only differs in 16 nt from the 560 nt upstream of the TSS of A3G (GenBanktrade DQ146365 and DQ147772). The 180-bp core promoter region, which we identified for A3G has 100% identity with the A3F sequence. Therefore, Sp1/Sp3 transcription factors are most likely also mediating basal transcription of the A3F gene.</text>
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        <text>A3F</text>
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        <text>A3F</text>
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        <text>A3G</text>
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        <text>A3F</text>
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        <location offset="7643" length="7646"></location>
        <text>A3G</text>
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        <text>A3G</text>
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        <location offset="7773" length="7776"></location>
        <text>A3F</text>
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        <text>Sp1</text>
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        <location offset="7802" length="7805"></location>
        <text>Sp3</text>
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        <location offset="7886" length="7889"></location>
        <text>A3F</text>
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        <location offset="7301" length="7311"></location>
        <text>regulation</text>
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        <text>expressed</text>
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        <text>express</text>
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        <text>form heteromultimers</text>
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        <text>mediating</text>
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        <location offset="7865" length="7878"></location>
        <text>transcription</text>
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    <passage>
      <offset>7897</offset>
      <text>Our study revealed that the human A3G gene is controlled by a promoter with multiple transcriptional start sites. In A3.01 T cells, the A3G promoter appears constitutively active and is not inducible by TPA, type I or II interferons or by HIV-1 proteins. The core promoter is located within a region of 180 bp at position -114/+66 relative to the TSS. A GC-box is crucial to the function of the core promoter and represents a binding site for Sp1 and Sp3 transcription factors. Our results can serve as a basis for future studies aimed at understanding how A3G and A3F expression is controlled in different tissues and how these restriction factors can be used to develop novel therapeutic strategies against HIV-1 infection.</text>
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        <text>A3G</text>
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        <text>crucial</text>
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        <text>binding</text>
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        <text>expression</text>
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      <node refid="T18" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T103" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T104" role="Trigger"></node>
      <node refid="E26" role="Theme"></node>
      <node refid="T21" role="Cause"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T105" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
      <node refid="E28" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T107" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T108" role="Trigger"></node>
      <node refid="E30" role="Theme"></node>
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    </relation>
    <relation id="E31">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T109" role="Trigger"></node>
      <node refid="T32" role="Theme"></node>
    </relation>
    <relation id="E30">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T110" role="Trigger"></node>
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      <node refid="T32" role="Cause"></node>
      <node refid="T111" role="CSite"></node>
    </relation>
    <relation id="E32">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T112" role="Trigger"></node>
      <node refid="T33" role="Theme"></node>
    </relation>
    <relation id="E33">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T113" role="Trigger"></node>
      <node refid="E32" role="Theme"></node>
      <node refid="T33" role="Cause"></node>
      <node refid="T114" role="CSite"></node>
    </relation>
    <relation id="E34">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T115" role="Trigger"></node>
      <node refid="E32" role="Theme"></node>
      <node refid="T34" role="Cause"></node>
    </relation>
    <relation id="E35">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T117" role="Trigger"></node>
      <node refid="T35" role="Theme"></node>
      <node refid="T116" role="Site"></node>
    </relation>
    <relation id="E36">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T118" role="Trigger"></node>
      <node refid="E37" role="Theme"></node>
    </relation>
    <relation id="E37">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T119" role="Trigger"></node>
      <node refid="T36" role="Theme"></node>
    </relation>
    <relation id="E38">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T120" role="Trigger"></node>
      <node refid="E39" role="Theme"></node>
    </relation>
    <relation id="E39">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T121" role="Trigger"></node>
      <node refid="T40" role="Theme"></node>
    </relation>
    <relation id="E40">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T122" role="Trigger"></node>
      <node refid="T41" role="Theme"></node>
    </relation>
    <relation id="E41">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T122" role="Trigger"></node>
      <node refid="T42" role="Theme"></node>
    </relation>
    <relation id="E42">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T123" role="Trigger"></node>
      <node refid="T41" role="Theme"></node>
    </relation>
    <relation id="E43">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T123" role="Trigger"></node>
      <node refid="T42" role="Theme"></node>
    </relation>
    <relation id="E44">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T124" role="Trigger"></node>
      <node refid="T43" role="Theme"></node>
      <node refid="T45" role="Theme2"></node>
      <node refid="T125" role="Site2"></node>
    </relation>
    <relation id="E45">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T124" role="Trigger"></node>
      <node refid="T44" role="Theme"></node>
      <node refid="T45" role="Theme2"></node>
      <node refid="T125" role="Site2"></node>
    </relation>
    <relation id="E46">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T126" role="Trigger"></node>
      <node refid="E47" role="Theme"></node>
      <node refid="T46" role="Cause"></node>
    </relation>
    <relation id="E47">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T127" role="Trigger"></node>
      <node refid="T47" role="Theme"></node>
    </relation>
    <relation id="E48">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="T48" role="Theme"></node>
    </relation>
    <relation id="E49">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="T49" role="Theme"></node>
    </relation>
    <relation id="E50">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T129" role="Trigger"></node>
      <node refid="T53" role="Theme"></node>
    </relation>
    <relation id="E51">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T129" role="Trigger"></node>
      <node refid="T54" role="Theme"></node>
    </relation>
    <relation id="E52">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T131" role="Trigger"></node>
      <node refid="T57" role="Theme"></node>
      <node refid="T55" role="Cause"></node>
      <node refid="T130" role="Site"></node>
    </relation>
    <relation id="E53">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T131" role="Trigger"></node>
      <node refid="T57" role="Theme"></node>
      <node refid="T56" role="Cause"></node>
      <node refid="T130" role="Site"></node>
    </relation>
    <relation id="E54">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T132" role="Trigger"></node>
      <node refid="T55" role="Theme"></node>
    </relation>
    <relation id="E55">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T132" role="Trigger"></node>
      <node refid="T56" role="Theme"></node>
    </relation>
    <relation id="E56">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T133" role="Trigger"></node>
      <node refid="E57" role="Theme"></node>
    </relation>
    <relation id="E57">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T134" role="Trigger"></node>
      <node refid="T57" role="Theme"></node>
    </relation>
    <relation id="E58">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T135" role="Trigger"></node>
      <node refid="E57" role="Theme"></node>
      <node refid="T57" role="Cause"></node>
      <node refid="T136" role="CSite"></node>
    </relation>
    <relation id="E59">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T137" role="Trigger"></node>
      <node refid="T57" role="Theme"></node>
    </relation>
    <relation id="E60">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T138" role="Trigger"></node>
      <node refid="E59" role="Theme"></node>
    </relation>
    <relation id="E61">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T139" role="Trigger"></node>
      <node refid="T62" role="Theme"></node>
    </relation>
    <relation id="E62">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T140" role="Trigger"></node>
      <node refid="T63" role="Theme"></node>
    </relation>
    <relation id="E63">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T141" role="Trigger"></node>
      <node refid="T65" role="Theme"></node>
    </relation>
    <relation id="E64">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T142" role="Trigger"></node>
      <node refid="T66" role="Theme"></node>
    </relation>
    <relation id="E65">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T143" role="Trigger"></node>
      <node refid="T65" role="Theme"></node>
      <node refid="T66" role="Theme2"></node>
    </relation>
    <relation id="E66">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T144" role="Trigger"></node>
      <node refid="E67" role="Theme"></node>
      <node refid="T71" role="Cause"></node>
    </relation>
    <relation id="E68">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T144" role="Trigger"></node>
      <node refid="E67" role="Theme"></node>
      <node refid="T72" role="Cause"></node>
    </relation>
    <relation id="E67">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T145" role="Trigger"></node>
      <node refid="T73" role="Theme"></node>
    </relation>
    <relation id="E69">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T146" role="Trigger"></node>
      <node refid="T74" role="Theme"></node>
      <node refid="T74" role="Cause"></node>
      <node refid="T147" role="CSite"></node>
    </relation>
    <relation id="E70">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T149" role="Trigger"></node>
      <node refid="T75" role="Theme"></node>
      <node refid="T148" role="Site"></node>
    </relation>
    <relation id="E71">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T150" role="Trigger"></node>
      <node refid="T75" role="Theme"></node>
      <node refid="T148" role="Site"></node>
    </relation>
    <relation id="E72">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T152" role="Trigger"></node>
      <node refid="E73" role="Theme"></node>
      <node refid="T75" role="Cause"></node>
      <node refid="T151" role="CSite"></node>
    </relation>
    <relation id="E74">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T152" role="Trigger"></node>
      <node refid="E75" role="Theme"></node>
      <node refid="T75" role="Cause"></node>
      <node refid="T151" role="CSite"></node>
    </relation>
    <relation id="E73">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T154" role="Trigger"></node>
      <node refid="T75" role="Theme"></node>
      <node refid="T76" role="Theme2"></node>
      <node refid="T153" role="Site"></node>
    </relation>
    <relation id="E75">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T154" role="Trigger"></node>
      <node refid="T75" role="Theme"></node>
      <node refid="T77" role="Theme2"></node>
      <node refid="T153" role="Site"></node>
    </relation>
    <relation id="E76">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T155" role="Trigger"></node>
      <node refid="T78" role="Theme"></node>
    </relation>
    <relation id="E77">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T155" role="Trigger"></node>
      <node refid="T79" role="Theme"></node>
    </relation>
    <relation id="E78">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T156" role="Trigger"></node>
      <node refid="E76" role="Theme"></node>
    </relation>
    <relation id="E79">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T156" role="Trigger"></node>
      <node refid="E77" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E19" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E20" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E22" role="Event"></node>
    </relation>
    <relation id="M8">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E24" role="Event"></node>
    </relation>
    <relation id="M9">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E25" role="Event"></node>
    </relation>
    <relation id="M10">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E29" role="Event"></node>
    </relation>
    <relation id="M11">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E34" role="Event"></node>
    </relation>
    <relation id="M12">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E35" role="Event"></node>
    </relation>
    <relation id="M13">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E60" role="Event"></node>
    </relation>
    <relation id="M14">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E66" role="Event"></node>
    </relation>
    <relation id="M15">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E68" role="Event"></node>
    </relation>
    <relation id="M16">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E71" role="Event"></node>
    </relation>
    <relation id="M17">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E78" role="Event"></node>
    </relation>
    <relation id="M18">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E79" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-16-caption-01</id>
    <passage>
      <offset>0</offset>
      <text>Sequence of the A3G promoter and the downstream region. The first 1000 bp upstream of the major TSS are shown in lower case, the first 800 bp of the transcribed sequence are shown in upper case. Introns are removed, but their positions are indicated. Arrows refer to transcriptional start sites and their observed frequency is given by the numbers above. The primer binding sites for 5'-RACE analysis and cloning of the luciferase reporter constructs are underlined and the names of the primers are annotated. Gray and black arrowheads define the regions designated E1 and E2 which were cloned into vector pGL3-Promoter and used as EMSA probes. The ATG start codon and the identified Sp1/Sp3 transcription factor binding site are shown in bold.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="16" length="19"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="267" length="294"></location>
        <text>transcriptional start sites</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="420" length="430"></location>
        <text>luciferase</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-17-caption-02</id>
    <passage>
      <offset>0</offset>
      <text>5'-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5'-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="24" length="27"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="81" length="84"></location>
        <text>A3G</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-1920263-18-caption-03</id>
    <passage>
      <offset>0</offset>
      <text>Luciferase activities of A3G promoter constructs in different cell lines. (A) A3G promoter 5' deletion constructs of different sizes were cloned into pGL3-Basic luciferase reporter plasmids. Numbering is relative to the major TSS. A putative Sp1/Sp3 consensus site (gray square) is depicted. (B) A3.01 T cells were transiently transfected with the A3G promoter deletion constructs. Numbers on the x-axis refer to the length of the A3G promoter fragments in bp. (C) A3G promoter plasmids were transfected into U937, HepG2 and Huh7 cell lines. Numbers in the legends refer to the length of the A3G promoter fragments in bp. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="10"></location>
        <text>Luciferase</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="25" length="28"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="78" length="81"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="161" length="171"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="348" length="351"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="431" length="434"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="465" length="468"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="592" length="595"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="659" length="669"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="685" length="695"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="746" length="756"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="11" length="21"></location>
        <text>activities</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Entity</infon>
        <location offset="29" length="37"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="726" length="737"></location>
        <text>coexpressed</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
      <node refid="T13" role="CSite"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-19-caption-04</id>
    <passage>
      <offset>0</offset>
      <text>A3G promoter activities after coexpression of HIV-1 proteins or treatment with TPA or interferons. (A) A3.01 T cells were cotransfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter and 1 microg of Vif expression plasmid, 1 microg Tat expression plasmid or increasing amounts of HIV-1NL4-3 (0.1, 0.5 and 1 microg). After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (B) A3.01 T cells were transiently transfected with pGL3-Basic reporter plasmid containing the 1025 bp A3G promoter or with empty vector. Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 20 ng/ml TPA. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities and the values for the empty vectors (untreated and TPA-stimulated) were set as 1. (C) A3.01 T cells were transiently transfected with the A3G promoter constructs or with the interferon-responsive reporter plasmid pGL2-CVX (GAS). Fifteen hour before harvesting for luciferase assay, a subset of the cell culture was stimulated with 30 ng/ml IFN-alpha or IFN-gamma. Forty-eight hour after transfection, luciferase assay was performed. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. (D) HepG2 cells were used for transfection. The experiment was performed as described in (C). Mean values (+/-SD) of representative experiments performed in triplicate are shown.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="3"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="192" length="195"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="221" length="224"></location>
        <text>Vif</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="254" length="257"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="375" length="385"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="401" length="411"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="462" length="472"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="588" length="591"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="658" length="668"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="776" length="786"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="816" length="826"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="877" length="887"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1038" length="1041"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1074" length="1084"></location>
        <text>interferon</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1164" length="1174"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1240" length="1249"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1253" length="1262"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1301" length="1311"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1341" length="1351"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1402" length="1412"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="442" length="453"></location>
        <text>coexpressed</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="857" length="868"></location>
        <text>coexpressed</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1382" length="1393"></location>
        <text>coexpressed</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-20-caption-05</id>
    <passage>
      <offset>0</offset>
      <text>A GC-box mediates transcriptional activity of the 180 bp core promoter. (A) A3.01 T cells were transfected with reporter plasmid pGL3-Basic containing 180, 150 or 120 bp of the A3G promoter. The two G-to-T substitutions introduced into the GC-box of the 180 bp fragment (180mut) are specified. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown. (B) pGL3-Promoter reporter plasmids containing the regions E1 or E2 (see Figure 1) upstream of the SV40 promoter were transfected into A3.01 T cells. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="177" length="180"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="331" length="341"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="357" length="367"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="418" length="428"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="685" length="695"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="757" length="767"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="398" length="409"></location>
        <text>coexpressed</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Gene_expression</infon>
        <location offset="737" length="748"></location>
        <text>coexpressed</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-21-caption-06</id>
    <passage>
      <offset>0</offset>
      <text>Sp1 and Sp3 bind to the GC-box present in the A3G promoter. (A) Nuclear extracts of A3.01 T cells were incubated with a 32P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein-DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). (B) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody-protein-oligo complexes (lanes 3, 4, 7, 8, 11, 12). (C) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="3"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="8" length="11"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="46" length="49"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="143" length="146"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="269" length="272"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="287" length="290"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="291" length="292"></location>
        <text>3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="301" length="304"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="305" length="306"></location>
        <text>3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="486" length="489"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="490" length="491"></location>
        <text>3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="533" length="536"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="537" length="538"></location>
        <text>3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="588" length="591"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="597" length="600"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="880" length="883"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="885" length="888"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="892" length="897"></location>
        <text>actin</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="933" length="936"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="967" length="970"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Binding</infon>
        <location offset="12" length="16"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Entity</infon>
        <location offset="24" length="30"></location>
        <text>GC-box</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
      <node refid="T22" role="Site2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
      <node refid="T22" role="Site2"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1920263-22-caption-07</id>
    <passage>
      <offset>0</offset>
      <text>Silencing of Sp1 and Sp3 reduces A3G promoter activity. (A) HeLa cells were transfected with 150 and 300 ng of unspecific, Sp1-specific or Sp3-specific siRNA. After 48 h, cells were harvested and Sp1 and Sp3 proteins were detected by western blot analysis. As loading control, protein levels of tubulin are shown. (B) HeLa cells were cotransfected with reporter plasmid pGL3-Basic containing 180 or 150 bp of the A3G promoter and siRNA. Hundred nanogram of unspecific siRNA (control), Sp1-specific siRNA (Sp1), Sp3-specific siRNA (Sp3) or a mixture of 50 ng Sp1-specific plus 50 ng Sp3-specific siRNA (Sp1+Sp3) were used. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (+/-SD) of a representative experiment performed in triplicate are shown.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="13" length="16"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="21" length="24"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="33" length="36"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="123" length="126"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="139" length="142"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="196" length="199"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="204" length="207"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="295" length="302"></location>
        <text>tubulin</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="413" length="416"></location>
        <text>A3G</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="485" length="488"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="505" length="508"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="511" length="514"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="531" length="534"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="558" length="561"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="582" length="585"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="602" length="605"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="606" length="609"></location>
        <text>Sp3</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="630" length="640"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="702" length="712"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Negative_regulation</infon>
        <location offset="0" length="9"></location>
        <text>Silencing</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="682" length="693"></location>
        <text>coexpressed</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1942070-00-TIAB</id>
    <passage>
      <offset>0</offset>
      <text>Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFkappaB activity in vertebrate B-cells</text>
    </passage>
    <passage>
      <offset>141</offset>
      <text>Abstract</text>
    </passage>
    <passage>
      <offset>150</offset>
      <text>To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569-1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFkappaB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="702" length="707"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Positive_regulation</infon>
        <location offset="681" length="689"></location>
        <text>required</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Regulation</infon>
        <location offset="693" length="701"></location>
        <text>regulate</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Phosphorylation</infon>
        <location offset="708" length="723"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T2" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T3" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T4" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1942070-01-Introduction</id>
    <passage>
      <offset>0</offset>
      <text>Introduction</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCgamma and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3-6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8-12].</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="90" length="94"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="96" length="100"></location>
        <text>PKD2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="105" length="109"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="753" length="757"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Binding</infon>
        <location offset="718" length="725"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Positive_regulation</infon>
        <location offset="738" length="749"></location>
        <text>contributes</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="758" length="768"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <passage>
      <offset>853</offset>
      <text>PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13-16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFkappaB transcription factor and in regulating cell survival during oxidative stress [17,21-23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26-28].</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1381" length="1385"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1399" length="1404"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1608" length="1612"></location>
        <text>MEF2</text>
      </annotation>
    </passage>
    <passage>
      <offset>1682</offset>
      <text>To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFkappaB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.</text>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="2180" length="2185"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="2162" length="2175"></location>
        <text>indispensable</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Phosphorylation</infon>
        <location offset="2186" length="2201"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1942070-02-Materials_and_methods-01</id>
    <passage>
      <offset>0</offset>
      <text>Cell culture, transient transfections and cell stimulation</text>
    </passage>
    <passage>
      <offset>59</offset>
      <text>The generation, culture and activation of PKD1-/-, PKD3-/- and PKD1/3-/- knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29].</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="101" length="105"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="110" length="114"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="122" length="126"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="127" length="128"></location>
        <text>3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Negative_regulation</infon>
        <location offset="132" length="140"></location>
        <text>knockout</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1942070-03-Materials_and_methods-02</id>
    <passage>
      <offset>0</offset>
      <text>sIgM staining</text>
    </passage>
    <passage>
      <offset>14</offset>
      <text>DT40 B cells (2x106 cells per point) were resuspended in 200mul buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry.</text>
    </passage>
    <passage>
      <offset>296</offset>
      <text>All results shown are representative of at two to four independent experiments unless otherwise indicated.</text>
    </passage>
  </document>
  <document>
    <id>PMC-1942070-04-Results-01</id>
    <passage>
      <offset>0</offset>
      <text>Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="8" length="13"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Negative_regulation</infon>
        <location offset="0" length="4"></location>
        <text>Loss</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Phosphorylation</infon>
        <location offset="14" length="29"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <passage>
      <offset>87</offset>
      <text>DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3-/- cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="126" length="130"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="135" length="139"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="245" length="249"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="253" length="257"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="340" length="344"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="355" length="359"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="395" length="399"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="509" length="513"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="528" length="532"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="533" length="534"></location>
        <text>3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="585" length="589"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Gene_expression</infon>
        <location offset="100" length="107"></location>
        <text>express</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Negative_regulation</infon>
        <location offset="219" length="223"></location>
        <text>lack</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Gene_expression</infon>
        <location offset="224" length="234"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Gene_expression</infon>
        <location offset="378" length="387"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Gene_expression</infon>
        <location offset="514" length="524"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Negative_regulation</infon>
        <location offset="535" length="550"></location>
        <text>double knockout</text>
      </annotation>
    </passage>
    <passage>
      <offset>738</offset>
      <text>Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3-/- B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.</text>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="893" length="897"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="898" length="899"></location>
        <text>3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1005" length="1010"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1043" length="1047"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1153" length="1158"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1186" length="1190"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1255" length="1260"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1324" length="1328"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1332" length="1336"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1444" length="1449"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1557" length="1562"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1582" length="1586"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1590" length="1594"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1694" length="1699"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1749" length="1753"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1758" length="1762"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="1832" length="1836"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="1923" length="1928"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="2002" length="2006"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="2105" length="2110"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="2145" length="2149"></location>
        <text>PKD3</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="2161" length="2165"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="2179" length="2184"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="2256" length="2261"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Negative_regulation</infon>
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        <text>defective</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Phosphorylation</infon>
        <location offset="1134" length="1149"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Entity</infon>
        <location offset="1162" length="1171"></location>
        <text>serine 82</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Regulation</infon>
        <location offset="1241" length="1251"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Phosphorylation</infon>
        <location offset="1261" length="1276"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Negative_regulation</infon>
        <location offset="1309" length="1316"></location>
        <text>lacking</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Positive_regulation</infon>
        <location offset="1420" length="1429"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Phosphorylation</infon>
        <location offset="1450" length="1465"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Entity</infon>
        <location offset="1469" length="1472"></location>
        <text>S82</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Positive_regulation</infon>
        <location offset="1548" length="1556"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T55">
        <infon key="type">Phosphorylation</infon>
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        <text>phosphorylation</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Negative_regulation</infon>
        <location offset="1602" length="1610"></location>
        <text>knockout</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Positive_regulation</infon>
        <location offset="1667" length="1674"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Phosphorylation</infon>
        <location offset="1675" length="1690"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T59">
        <infon key="type">Entity</infon>
        <location offset="1703" length="1706"></location>
        <text>S82</text>
      </annotation>
      <annotation id="T60">
        <infon key="type">Negative_regulation</infon>
        <location offset="1711" length="1720"></location>
        <text>abolished</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Negative_regulation</infon>
        <location offset="1737" length="1743"></location>
        <text>lacked</text>
      </annotation>
      <annotation id="T62">
        <infon key="type">Positive_regulation</infon>
        <location offset="1801" length="1808"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Gene_expression</infon>
        <location offset="1809" length="1819"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Positive_regulation</infon>
        <location offset="1880" length="1901"></location>
        <text>sufficient to restore</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Regulation</infon>
        <location offset="1909" length="1919"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Phosphorylation</infon>
        <location offset="1929" length="1944"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Gene_expression</infon>
        <location offset="1969" length="1979"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Negative_regulation</infon>
        <location offset="1992" length="2001"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Positive_regulation</infon>
        <location offset="2067" length="2074"></location>
        <text>restore</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Positive_regulation</infon>
        <location offset="2097" length="2104"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Phosphorylation</infon>
        <location offset="2111" length="2126"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Regulation</infon>
        <location offset="2170" length="2178"></location>
        <text>regulate</text>
      </annotation>
      <annotation id="T73">
        <infon key="type">Phosphorylation</infon>
        <location offset="2185" length="2200"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
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    </relation>
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      <infon key="type">Event</infon>
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    <relation id="E34">
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="T30" role="Theme"></node>
    </relation>
    <relation id="E36">
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      <infon key="type">Event</infon>
      <node refid="T67" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
    </relation>
    <relation id="E37">
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      <infon key="type">Event</infon>
      <node refid="T68" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
    </relation>
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      <infon key="type">Event</infon>
      <node refid="T69" role="Trigger"></node>
      <node refid="E39" role="Theme"></node>
      <node refid="E36" role="Cause"></node>
    </relation>
    <relation id="E39">
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      <infon key="type">Event</infon>
      <node refid="T70" role="Trigger"></node>
      <node refid="E40" role="Theme"></node>
    </relation>
    <relation id="E40">
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      <infon key="type">Event</infon>
      <node refid="T71" role="Trigger"></node>
      <node refid="T32" role="Theme"></node>
    </relation>
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      <infon key="type">Event</infon>
      <node refid="T72" role="Trigger"></node>
      <node refid="E42" role="Theme"></node>
      <node refid="T33" role="Cause"></node>
    </relation>
    <relation id="E43">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T72" role="Trigger"></node>
      <node refid="E42" role="Theme"></node>
      <node refid="T34" role="Cause"></node>
    </relation>
    <relation id="E42">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T73" role="Trigger"></node>
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    </relation>
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      <infon key="modification type">Speculation</infon>
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    </relation>
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      <infon key="modification type">Speculation</infon>
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    </relation>
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  </document>
  <document>
    <id>PMC-1942070-05-Results-02</id>
    <passage>
      <offset>0</offset>
      <text>Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases</text>
    </passage>
    <passage>
      <offset>93</offset>
      <text>PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3-/-: Flag-PKD3+ve) or absence (PKD1/3-/-) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3-/- cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3-/- B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3-/- cells was slightly slower than that of wild type DT40 cells (12.7+/-2.8h versus 10.2+/-0.4h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3-/- cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.</text>
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        <text>3</text>
      </annotation>
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        <text>re-expression</text>
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        <text>re-expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>1259</offset>
      <text>PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30-32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.</text>
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        <text>loss</text>
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        <text>expression</text>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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  <document>
    <id>PMC-1942070-06-Results-03</id>
    <passage>
      <offset>0</offset>
      <text>Antigen receptor regulated signalling pathways in PKD-null DT40 B cells</text>
    </passage>
    <passage>
      <offset>72</offset>
      <text>To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3-/- (and in PKD1/3-/-:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3-/- cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3-/- B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33-35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3-/--Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3-/- (and PKD1/3-/--Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway.</text>
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        <text>3</text>
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        <text>3</text>
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        <text>PKD1</text>
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        <text>3</text>
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        <text>3</text>
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        <text>3</text>
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      </annotation>
      <annotation id="T45">
        <infon key="type">Positive_regulation</infon>
        <location offset="635" length="642"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Positive_regulation</infon>
        <location offset="643" length="653"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Phosphorylation</infon>
        <location offset="719" length="734"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Negative_regulation</infon>
        <location offset="777" length="787"></location>
        <text>detectable</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Negative_regulation</infon>
        <location offset="798" length="802"></location>
        <text>null</text>
      </annotation>
    </passage>
    <passage>
      <offset>1762</offset>
      <text>To search for other potential PKD targets that may show defective regulation in PKD1/3-/- DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3-/- cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3-/- DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera.</text>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1842" length="1846"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1847" length="1848"></location>
        <text>3</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="2155" length="2159"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="2160" length="2161"></location>
        <text>3</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="2260" length="2264"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="2265" length="2266"></location>
        <text>3</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="2444" length="2448"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="2449" length="2450"></location>
        <text>3</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="2685" length="2693"></location>
        <text>MAPKAPK2</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Negative_regulation</infon>
        <location offset="2436" length="2440"></location>
        <text>loss</text>
      </annotation>
    </passage>
    <passage>
      <offset>2864</offset>
      <text>BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFkappaB transcription factor. Thus, PKD-mediated activation of NFkappaB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFkappaB activation [38]. In B cells, NFkappaB is known to be regulated via DAG and PKCbeta [39,40] but whether PKDs are key intermediaries for NFkappaB regulation has not been explored.</text>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="3318" length="3334"></location>
        <text>Bcr-Abl oncogene</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="3393" length="3397"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Protein</infon>
        <location offset="3414" length="3418"></location>
        <text>HPK1</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Protein</infon>
        <location offset="3512" length="3519"></location>
        <text>PKCbeta</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Positive_regulation</infon>
        <location offset="3366" length="3392"></location>
        <text>expression of an activated</text>
      </annotation>
    </passage>
    <passage>
      <offset>3616</offset>
      <text>The data (Fig. 4A) show that NFkappaB transcriptional activity was strongly induced in both wild-type and PKD1/3-/- DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFkappaB transcriptional activity was abolished in PKCbeta-/- DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCbeta-/- cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFkappaB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFkappaB.</text>
      <annotation id="T40">
        <infon key="type">Protein</infon>
        <location offset="3722" length="3726"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Protein</infon>
        <location offset="3727" length="3728"></location>
        <text>3</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="3895" length="3902"></location>
        <text>PKCbeta</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="4011" length="4015"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="4045" length="4052"></location>
        <text>PKCbeta</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Phosphorylation</infon>
        <location offset="3988" length="4007"></location>
        <text>autophosphorylation</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Entity</infon>
        <location offset="4019" length="4023"></location>
        <text>S916</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Positive_regulation</infon>
        <location offset="4029" length="4037"></location>
        <text>observed</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="E9" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E9" role="Cause"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="E9" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T33" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T34" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T51" role="Trigger"></node>
      <node refid="T37" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T52" role="Trigger"></node>
      <node refid="T43" role="Theme"></node>
      <node refid="T53" role="Site"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T54" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-1942070-07-Discussion</id>
    <passage>
      <offset>0</offset>
      <text>Discussion</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27. It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFkappaB transcription factors [41]. Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression. We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFkappaB regulation in lymphocytes. We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42]. However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFkappaB transcription factors. These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation, survival and NFkappaB activation [20,23]. Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages. Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="201" length="206"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="1040" length="1045"></location>
        <text>HSP27</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1113" length="1121"></location>
        <text>MAPKAPK2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1507" length="1511"></location>
        <text>PKD1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1512" length="1513"></location>
        <text>2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Entity</infon>
        <location offset="28" length="34"></location>
        <text>serine</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Regulation</infon>
        <location offset="65" length="73"></location>
        <text>regulate</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Phosphorylation</infon>
        <location offset="182" length="197"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Regulation</infon>
        <location offset="1006" length="1016"></location>
        <text>regulating</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Phosphorylation</infon>
        <location offset="1021" length="1036"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Entity</infon>
        <location offset="1049" length="1058"></location>
        <text>serine 82</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="1094" length="1100"></location>
        <text>target</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T6" role="Site"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T11" role="Site"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T11" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-00-TIAB</id>
    <passage>
      <offset>0</offset>
      <text>GATA3-Driven Th2 Responses Inhibit TGF-beta1-Induced FOXP3 Expression and the Formation of Regulatory T Cells</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="35" length="44"></location>
        <text>TGF-beta1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="53" length="58"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Negative_regulation</infon>
        <location offset="27" length="34"></location>
        <text>Inhibit</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="45" length="52"></location>
        <text>Induced</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Gene_expression</infon>
        <location offset="59" length="69"></location>
        <text>Expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>111</offset>
      <text>Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="340" length="345"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="660" length="673"></location>
        <text>interleukin 4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="675" length="679"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="728" length="733"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="842" length="848"></location>
        <text>GATA-3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="896" length="933"></location>
        <text>transforming growth factor (TGF)-beta</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="943" length="948"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1009" length="1014"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1022" length="1027"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1200" length="1204"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1306" length="1310"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1314" length="1319"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Regulation</infon>
        <location offset="482" length="492"></location>
        <text>regulating</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="497" length="507"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Negative_regulation</infon>
        <location offset="719" length="727"></location>
        <text>inhibits</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Gene_expression</infon>
        <location offset="827" length="841"></location>
        <text>overexpressing</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Negative_regulation</infon>
        <location offset="883" length="892"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="934" length="942"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Positive_regulation</infon>
        <location offset="949" length="958"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Positive_regulation</infon>
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        <text>mediated</text>
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      <annotation id="T39">
        <infon key="type">Binding</infon>
        <location offset="998" length="1005"></location>
        <text>binding</text>
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      <annotation id="T40">
        <infon key="type">Entity</infon>
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        <text>promoter</text>
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        <infon key="type">Regulation</infon>
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        <text>target</text>
      </annotation>
    </passage>
    <passage>
      <offset>1450</offset>
      <text>Author Summary</text>
    </passage>
    <passage>
      <offset>1465</offset>
      <text>Specific immune responses against foreign or autologous antigens are driven by specialized epitope-specific T cells, whose numbers expand upon recognition of antigen found on professional antigen-presenting cells. The subsequent maturation process involves the differentiation of certain T cell phenotypes such as pro-inflammatory cells (Th1, Th2, Th17) or regulatory T (Treg) cells, which serve to keep the immune response in check. The current study focuses on the role of two key transcription factors-FOXP3 and GATA3-in controlling the commitment of these cells. We demonstrate that the Th2 cytokine IL-4 inhibits the induction of FOXP3 and thus inhibits the generation of inducible Treg cells. We show that IL-4-induced GATA3 mediates FOXP3 inhibition by directly binding to a GATA element in the FOXP3 promoter. We hypothesize that therapeutic agents aimed at neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and thus promotion of tolerance in allergies and other Th2-dominated diseases.</text>
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        <infon key="type">Protein</infon>
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        <infon key="type">Protein</infon>
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        <infon key="type">Protein</infon>
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        <text>IL-4</text>
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        <infon key="type">Protein</infon>
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        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
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        <text>IL-4</text>
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        <infon key="type">Protein</infon>
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        <text>GATA3</text>
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        <infon key="type">Protein</infon>
        <location offset="2205" length="2210"></location>
        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
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        <text>FOXP3</text>
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        <text>induction</text>
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        <text>induced</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>mediates</text>
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        <infon key="type">Negative_regulation</infon>
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        <text>inhibition</text>
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        <infon key="type">Binding</infon>
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        <text>binding</text>
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        <infon key="type">Entity</infon>
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        <text>GATA element</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="2331" length="2343"></location>
        <text>neutralizing</text>
      </annotation>
    </passage>
    <passage>
      <offset>2500</offset>
      <text>It is shown that Th2 responses prevent the generation of inducible Tregs. This is mediated by IL-4 induction of GATA3, which binds directly to and represses the FOXP3 promoter. This mechanism is likely to be relevant in the induction of immunotolerance, particularly in allergic diseases.</text>
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        <text>IL-4</text>
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        <infon key="type">Protein</infon>
        <location offset="2612" length="2617"></location>
        <text>GATA3</text>
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        <infon key="type">Protein</infon>
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        <text>FOXP3</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>induction</text>
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        <infon key="type">Binding</infon>
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        <text>binds</text>
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        <infon key="type">Negative_regulation</infon>
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        <text>represses</text>
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        <infon key="type">Entity</infon>
        <location offset="2667" length="2675"></location>
        <text>promoter</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    </relation>
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      <node refid="E5" role="Theme"></node>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <node refid="T9" role="Cause"></node>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <node refid="E8" role="Theme"></node>
      <node refid="E12" role="Cause"></node>
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    <relation id="E12">
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      <infon key="type">Event</infon>
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      <node refid="T11" role="Theme"></node>
      <node refid="T12" role="Theme2"></node>
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    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T19" role="Theme"></node>
    </relation>
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      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
      <node refid="T20" role="Cause"></node>
    </relation>
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      <infon key="type">Event</infon>
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    <relation id="E19">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T23" role="Theme2"></node>
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    </relation>
    <relation id="E21">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
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    <relation id="E23">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <node refid="T53" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-01-Introduction</id>
    <passage>
      <offset>0</offset>
      <text>Introduction</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently. Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively. A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations. The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa. Specifically interleukin 4 (IL-4)-induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2]. GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3]. Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5]. A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4-dominated responses can break antigen-specific immune tolerance [7]. Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="660" length="673"></location>
        <text>interleukin 4</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="675" length="679"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="689" length="694"></location>
        <text>STAT6</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="699" length="704"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="784" length="789"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="877" length="881"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="883" length="887"></location>
        <text>IL-5</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="892" length="897"></location>
        <text>IL-13</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1012" length="1017"></location>
        <text>T-bet</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1037" length="1042"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1241" length="1246"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1341" length="1345"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1461" length="1466"></location>
        <text>GATA3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1508" length="1513"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="681" length="688"></location>
        <text>induced</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="864" length="873"></location>
        <text>induction</text>
      </annotation>
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        <infon key="type">Regulation</infon>
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        <text>modulates</text>
      </annotation>
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        <text>Overexpression of a dominant negative form</text>
      </annotation>
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        <location offset="1514" length="1522"></location>
        <text>blockade</text>
      </annotation>
    </passage>
    <passage>
      <offset>1596</offset>
      <text>The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens. Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery. These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines. Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions. These induced Treg (iTreg) cells were reported to express FOXP3 [10]. The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14]. TGF-beta has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-betaRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell-dependent multiorgan inflammatory disease [16]. Although the effect of TGF-beta on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.</text>
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        <text>FOXP3</text>
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        <text>TGF-beta</text>
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        <text>express</text>
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        <text>express</text>
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        <text>express</text>
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        <text>induce</text>
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        <text>expression</text>
      </annotation>
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        <text>lacking</text>
      </annotation>
    </passage>
    <passage>
      <offset>2967</offset>
      <text>The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively. We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo. We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.</text>
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        <text>T-bet</text>
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        <text>GATA3</text>
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        <text>FOXP3</text>
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        <text>GATA3</text>
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        <text>inhibits</text>
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        <text>induction</text>
      </annotation>
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        <text>limits</text>
      </annotation>
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        <text>expression</text>
      </annotation>
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  </document>
  <document>
    <id>PMC-2222968-02-Results-01</id>
    <passage>
      <offset>0</offset>
      <text>FOXP3 Induction in T Cell Subsets</text>
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    <passage>
      <offset>34</offset>
      <text>It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation. However, it is not known if all CD4+ T cells have the same capacity to express FOXP3. To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1). The CD4+CD25-, CD45RA+, CD45RO+, and CD4+CD45RO+CD25- were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-beta. Th1 cells showed only a 10-fold increase. In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression. The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).</text>
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        <text>up-regulate</text>
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    <passage>
      <offset>1097</offset>
      <text>Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-beta signaling and thus prevent FOXP3 induction. However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data). These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B). The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A). Only iTreg cells expressed FOXP3 in resting conditions. Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression. Repeated exposure to TGF-beta did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells. Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4-producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B). Furthermore TGF-beta-mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B). Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS). IL-4-expressing cells were most abundant among CD45RO+CD25- cells, which did not co-express FOXP3 (Figure 3A, left panel). In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel). As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background. The IL-4+ cells were confined to the FOXP3- cells, as shown for the CD45RO+CD25-, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B). In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C). Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap. Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.</text>
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  </document>
  <document>
    <id>PMC-2222968-03-Results-02</id>
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      <offset>0</offset>
      <text>FOXP3 and GATA3 Kinetics in Differentiating Cells</text>
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      <offset>50</offset>
      <text>The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs. Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-gamma, and anti IL-12), Th2, and iTreg phenotypes. After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18]. Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel). Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B). Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process. The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B). On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B). Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.</text>
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  </document>
  <document>
    <id>PMC-2222968-04-Results-03</id>
    <passage>
      <offset>0</offset>
      <text>IL-4 Inhibits TGF-beta-Mediated iTreg Commitment</text>
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        <text>IL-4</text>
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      <offset>49</offset>
      <text>IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage. We therefore asked whether IL-4 is able to inhibit TGF-beta induction of FOXP3 during the priming of naive T cells. Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta and/or IL-4 and harvested after 5 d. IL-4 efficiently repressed the TGF-beta-mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B). Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3]. However, at low concentration, IL-4 was able to marginally induce FOXP3 expression. Of note, GATA3 was also induced in the presence of TGF-beta at high IL-4 concentration (Figure 5A and 5B). The IL-4-mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-beta, which demonstrates that IL-4 as well as TGF-beta signaling were functional under these conditions (Figure 5C). Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D). Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo. A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25. We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19]. In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2). Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25- cells, but a real decrease of CD4+CD25+ T cells. In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo. To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity. In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C). Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).</text>
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  <document>
    <id>PMC-2222968-05-Results-04</id>
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      <offset>0</offset>
      <text>The Role of TGF-beta in T Cell Differentiation</text>
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      <text>Although TGF-beta-reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-beta-mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A). It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3- cells and thus decrease the relative percentage of FOXP3+ cells. However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3-. In fact both populations showed similarly enhanced proliferation (Figure 6A). Furthermore the TGF-beta-mediated induction of FOXP3 expression was not caused by overgrowth of a CD25-FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells. CD25 was down-regulated in TGF-beta-treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-beta and IL-4.</text>
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    <passage>
      <offset>1129</offset>
      <text>The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B). In line with the previous findings, the IL-4-producing cells and the FOXP3 expressing cells are nonoverlapping populations. Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression. The expression kinetic of naive T cells exposed to IL-4 and TGF-beta demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4-8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.</text>
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    <passage>
      <offset>1870</offset>
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  </document>
  <document>
    <id>PMC-2222968-06-Results-05</id>
    <passage>
      <offset>0</offset>
      <text>GATA3 Is a Negative Regulator of FOXP3 Expression</text>
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        <location offset="0" length="5"></location>
        <text>GATA3</text>
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        <text>FOXP3</text>
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    <passage>
      <offset>50</offset>
      <text>FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3. Besides GATA3's well-known positive effect on gene regulation, GATA3's repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells. To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3. After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-beta. TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel). TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-DeltaDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A). In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3). The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data). These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22]. To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25- cells were isolated, activated with OVA in the presence or absence of TGF-beta, and Foxp3 expression was analyzed after 4 d. The naive CD4+CD25- cells were Foxp3- (unpublished data). As described for the human cells, TGF-beta dramatically up-regulated Foxp3 in the DO11.10 littermate control mice. In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-beta and OVA (Figure 7B). All mice produced similar amounts of TGF-beta; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-beta signaling.</text>
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  </document>
  <document>
    <id>PMC-2222968-07-Results-06</id>
    <passage>
      <offset>0</offset>
      <text>GATA3 Represses the FOXP3 Promoter</text>
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      <offset>35</offset>
      <text>To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered. The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24]. This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation. The functional relevance of this site was studied using a FOXP3-promoter construct [24]. We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity. The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A). Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B). To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site. This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3-) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C). Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay. HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site. After the pull-down, GATA3 binding was detected by Western blot. Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach. Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E). These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter. To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F). Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.</text>
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  </document>
  <document>
    <id>PMC-2222968-08-Discussion</id>
    <passage>
      <offset>0</offset>
      <text>Discussion</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells. After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.</text>
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    <passage>
      <offset>287</offset>
      <text>In accordance with other studies, we found that CD4+CD25- cells were able to up-regulate FOXP3 [12,27]. Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile. In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-beta to induce iTreg cells [10,28-34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3. This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation. In this competitive process TGF-beta appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38]. In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-beta showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data). Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42-45]. Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.</text>
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    </passage>
    <passage>
      <offset>1916</offset>
      <text>The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-beta-mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype. To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4. This has been shown to increase the effect of the cytokine in vivo [19]. Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment. Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells. But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression. IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17-producing T cells (Th17) [46,47]. Similar effects have been recently described for IL-6, which, combined with TGF-beta, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49]. Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.</text>
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        <location offset="2214" length="2218"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="2302" length="2306"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Protein</infon>
        <location offset="2307" length="2311"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Protein</infon>
        <location offset="2391" length="2395"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Protein</infon>
        <location offset="2401" length="2406"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Protein</infon>
        <location offset="2461" length="2465"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="2667" length="2671"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="2713" length="2717"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="2735" length="2740"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="2841" length="2846"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="2859" length="2863"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Protein</infon>
        <location offset="2959" length="2964"></location>
        <text>IL-17</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Protein</infon>
        <location offset="3048" length="3052"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Protein</infon>
        <location offset="3075" length="3083"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="3294" length="3298"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="3303" length="3307"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T140">
        <infon key="type">Negative_regulation</infon>
        <location offset="1983" length="1990"></location>
        <text>inhibit</text>
      </annotation>
      <annotation id="T141">
        <infon key="type">Positive_regulation</infon>
        <location offset="2015" length="2024"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T142">
        <infon key="type">Gene_expression</infon>
        <location offset="2659" length="2666"></location>
        <text>express</text>
      </annotation>
      <annotation id="T143">
        <infon key="type">Negative_regulation</infon>
        <location offset="2727" length="2734"></location>
        <text>inhibit</text>
      </annotation>
      <annotation id="T144">
        <infon key="type">Gene_expression</infon>
        <location offset="2741" length="2751"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T145">
        <infon key="type">Positive_regulation</infon>
        <location offset="2806" length="2816"></location>
        <text>contribute</text>
      </annotation>
      <annotation id="T146">
        <infon key="type">Negative_regulation</infon>
        <location offset="2833" length="2837"></location>
        <text>drop</text>
      </annotation>
      <annotation id="T147">
        <infon key="type">Gene_expression</infon>
        <location offset="2847" length="2857"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T148">
        <infon key="type">Gene_expression</infon>
        <location offset="2965" length="2974"></location>
        <text>producing</text>
      </annotation>
    </passage>
    <passage>
      <offset>3310</offset>
      <text>IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25- [50]. In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression. Importantly, these concentrations were not sufficient to induce GATA3 expression. IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].</text>
      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="3310" length="3314"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Protein</infon>
        <location offset="3369" length="3374"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Protein</infon>
        <location offset="3394" length="3397"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T55">
        <infon key="type">Protein</infon>
        <location offset="3398" length="3402"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Protein</infon>
        <location offset="3454" length="3458"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Protein</infon>
        <location offset="3499" length="3503"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Protein</infon>
        <location offset="3543" length="3548"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T59">
        <infon key="type">Protein</infon>
        <location offset="3625" length="3630"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T60">
        <infon key="type">Protein</infon>
        <location offset="3643" length="3647"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Protein</infon>
        <location offset="3713" length="3718"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T149">
        <infon key="type">Positive_regulation</infon>
        <location offset="3534" length="3542"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T150">
        <infon key="type">Gene_expression</infon>
        <location offset="3549" length="3559"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T151">
        <infon key="type">Positive_regulation</infon>
        <location offset="3618" length="3624"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T152">
        <infon key="type">Gene_expression</infon>
        <location offset="3631" length="3641"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T153">
        <infon key="type">Regulation</infon>
        <location offset="3704" length="3712"></location>
        <text>regulate</text>
      </annotation>
      <annotation id="T154">
        <infon key="type">Gene_expression</infon>
        <location offset="3719" length="3729"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>3765</offset>
      <text>Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4-dependent inhibition of FOXP3 could be mediated by GATA3. In fact, GATA3-inducing IL-4 concentrations repressed TGF-beta-mediated FOXP3 expression, whereas IL-4 as well as TGF-beta signaling were intact. This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation. Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3. Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells. This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice). In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-beta. Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%-25% lower frequency compared to wild-type DO11.10 mice. Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin. Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52]. In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55]. Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.</text>
      <annotation id="T62">
        <infon key="type">Protein</infon>
        <location offset="3771" length="3776"></location>
        <text>IL-13</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Protein</infon>
        <location offset="3805" length="3810"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
        <location offset="3831" length="3836"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Protein</infon>
        <location offset="3863" length="3867"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Protein</infon>
        <location offset="3892" length="3897"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Protein</infon>
        <location offset="3919" length="3924"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Protein</infon>
        <location offset="3935" length="3940"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Protein</infon>
        <location offset="3950" length="3954"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Protein</infon>
        <location offset="3980" length="3988"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Protein</infon>
        <location offset="3998" length="4003"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Protein</infon>
        <location offset="4024" length="4028"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T73">
        <infon key="type">Protein</infon>
        <location offset="4040" length="4048"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T74">
        <infon key="type">Protein</infon>
        <location offset="4126" length="4131"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T75">
        <infon key="type">Protein</infon>
        <location offset="4136" length="4141"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T76">
        <infon key="type">Protein</infon>
        <location offset="4285" length="4290"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T77">
        <infon key="type">Protein</infon>
        <location offset="4337" length="4342"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Protein</infon>
        <location offset="4368" length="4373"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T79">
        <infon key="type">Protein</infon>
        <location offset="4403" length="4408"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Protein</infon>
        <location offset="4487" length="4492"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Protein</infon>
        <location offset="4553" length="4558"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Protein</infon>
        <location offset="4579" length="4582"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Protein</infon>
        <location offset="4583" length="4588"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Protein</infon>
        <location offset="4652" length="4657"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Protein</infon>
        <location offset="4713" length="4718"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Protein</infon>
        <location offset="4776" length="4784"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T87">
        <infon key="type">Protein</infon>
        <location offset="4810" length="4813"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T88">
        <infon key="type">Protein</infon>
        <location offset="4814" length="4819"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T89">
        <infon key="type">Protein</infon>
        <location offset="4844" length="4849"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T90">
        <infon key="type">Protein</infon>
        <location offset="4949" length="4954"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T91">
        <infon key="type">Protein</infon>
        <location offset="5189" length="5194"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T92">
        <infon key="type">Protein</infon>
        <location offset="5288" length="5293"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T93">
        <infon key="type">Protein</infon>
        <location offset="5317" length="5322"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T94">
        <infon key="type">Protein</infon>
        <location offset="5396" length="5414"></location>
        <text>Repressor of GATA3</text>
      </annotation>
      <annotation id="T95">
        <infon key="type">Protein</infon>
        <location offset="5416" length="5419"></location>
        <text>ROG</text>
      </annotation>
      <annotation id="T96">
        <infon key="type">Protein</infon>
        <location offset="5459" length="5462"></location>
        <text>CD8</text>
      </annotation>
      <annotation id="T97">
        <infon key="type">Protein</infon>
        <location offset="5610" length="5615"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T155">
        <infon key="type">Negative_regulation</infon>
        <location offset="3798" length="3804"></location>
        <text>reduce</text>
      </annotation>
      <annotation id="T156">
        <infon key="type">Positive_regulation</infon>
        <location offset="3824" length="3830"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T157">
        <infon key="type">Negative_regulation</infon>
        <location offset="3878" length="3888"></location>
        <text>inhibition</text>
      </annotation>
      <annotation id="T158">
        <infon key="type">Positive_regulation</infon>
        <location offset="3907" length="3915"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T159">
        <infon key="type">Positive_regulation</infon>
        <location offset="3941" length="3949"></location>
        <text>inducing</text>
      </annotation>
      <annotation id="T160">
        <infon key="type">Negative_regulation</infon>
        <location offset="3970" length="3979"></location>
        <text>repressed</text>
      </annotation>
      <annotation id="T161">
        <infon key="type">Positive_regulation</infon>
        <location offset="3989" length="3997"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T162">
        <infon key="type">Gene_expression</infon>
        <location offset="4004" length="4014"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T163">
        <infon key="type">Positive_regulation</infon>
        <location offset="4280" length="4284"></location>
        <text>high</text>
      </annotation>
      <annotation id="T164">
        <infon key="type">Gene_expression</infon>
        <location offset="4291" length="4301"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T165">
        <infon key="type">Negative_regulation</infon>
        <location offset="4326" length="4333"></location>
        <text>absence</text>
      </annotation>
      <annotation id="T166">
        <infon key="type">Negative_regulation</infon>
        <location offset="4393" length="4402"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T167">
        <infon key="type">Positive_regulation</infon>
        <location offset="4409" length="4418"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T168">
        <infon key="type">Gene_expression</infon>
        <location offset="4542" length="4552"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T169">
        <infon key="type">Positive_regulation</infon>
        <location offset="4634" length="4648"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T170">
        <infon key="type">Negative_regulation</infon>
        <location offset="4696" length="4702"></location>
        <text>failed</text>
      </annotation>
      <annotation id="T171">
        <infon key="type">Positive_regulation</infon>
        <location offset="4706" length="4712"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T172">
        <infon key="type">Gene_expression</infon>
        <location offset="4719" length="4729"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T173">
        <infon key="type">Positive_regulation</infon>
        <location offset="5195" length="5209"></location>
        <text>overexpression</text>
      </annotation>
    </passage>
    <passage>
      <offset>5674</offset>
      <text>Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region. A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24]. Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3. This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments. Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells. It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57-59]. Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.</text>
      <annotation id="T98">
        <infon key="type">Protein</infon>
        <location offset="5702" length="5707"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T99">
        <infon key="type">Protein</infon>
        <location offset="5718" length="5723"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T100">
        <infon key="type">Protein</infon>
        <location offset="5762" length="5767"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T101">
        <infon key="type">Protein</infon>
        <location offset="5920" length="5925"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T102">
        <infon key="type">Protein</infon>
        <location offset="6129" length="6134"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T103">
        <infon key="type">Protein</infon>
        <location offset="6206" length="6211"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T104">
        <infon key="type">Protein</infon>
        <location offset="6255" length="6260"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T105">
        <infon key="type">Protein</infon>
        <location offset="6345" length="6350"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T106">
        <infon key="type">Protein</infon>
        <location offset="6409" length="6414"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T107">
        <infon key="type">Protein</infon>
        <location offset="6618" length="6623"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T108">
        <infon key="type">Protein</infon>
        <location offset="6677" length="6682"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T174">
        <infon key="type">Negative_regulation</infon>
        <location offset="5708" length="5717"></location>
        <text>repressed</text>
      </annotation>
      <annotation id="T175">
        <infon key="type">Gene_expression</infon>
        <location offset="5724" length="5734"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T176">
        <infon key="type">Binding</infon>
        <location offset="5747" length="5754"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T177">
        <infon key="type">Entity</infon>
        <location offset="5768" length="5783"></location>
        <text>promoter region</text>
      </annotation>
      <annotation id="T178">
        <infon key="type">Entity</infon>
        <location offset="5787" length="5808"></location>
        <text>palindromic GATA-site</text>
      </annotation>
      <annotation id="T179">
        <infon key="type">Gene_expression</infon>
        <location offset="6121" length="6128"></location>
        <text>express</text>
      </annotation>
      <annotation id="T180">
        <infon key="type">Gene_expression</infon>
        <location offset="6198" length="6205"></location>
        <text>express</text>
      </annotation>
      <annotation id="T181">
        <infon key="type">Binding</infon>
        <location offset="6246" length="6251"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T182">
        <infon key="type">Binding</infon>
        <location offset="6351" length="6356"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T183">
        <infon key="type">Gene_expression</infon>
        <location offset="6624" length="6634"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T184">
        <infon key="type">Negative_regulation</infon>
        <location offset="6635" length="6638"></location>
        <text>low</text>
      </annotation>
    </passage>
    <passage>
      <offset>6708</offset>
      <text>The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).</text>
      <annotation id="T109">
        <infon key="type">Protein</infon>
        <location offset="6744" length="6749"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T110">
        <infon key="type">Protein</infon>
        <location offset="6761" length="6766"></location>
        <text>FOXP3</text>
      </annotation>
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      <offset>6999</offset>
      <text>In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4. GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter. We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression. These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.</text>
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    <relation id="E64">
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      <infon key="type">Event</infon>
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    <relation id="E73">
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      <infon key="type">Event</infon>
      <node refid="T186" role="Trigger"></node>
      <node refid="E74" role="Theme"></node>
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    <relation id="E75">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T186" role="Trigger"></node>
      <node refid="E76" role="Theme"></node>
      <node refid="T111" role="Cause"></node>
    </relation>
    <relation id="E74">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T187" role="Trigger"></node>
      <node refid="T110" role="Theme"></node>
      <node refid="T112" role="Theme2"></node>
      <node refid="T188" role="Site"></node>
    </relation>
    <relation id="E76">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T187" role="Trigger"></node>
      <node refid="T110" role="Theme"></node>
      <node refid="T113" role="Theme2"></node>
      <node refid="T188" role="Site"></node>
    </relation>
    <relation id="E77">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T189" role="Trigger"></node>
      <node refid="T114" role="Theme"></node>
      <node refid="T115" role="Cause"></node>
    </relation>
    <relation id="E78">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T190" role="Trigger"></node>
      <node refid="E79" role="Theme"></node>
      <node refid="T116" role="Cause"></node>
    </relation>
    <relation id="E79">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T191" role="Trigger"></node>
      <node refid="T117" role="Theme"></node>
    </relation>
    <relation id="E80">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T192" role="Trigger"></node>
      <node refid="T116" role="Theme"></node>
      <node refid="T118" role="Theme2"></node>
      <node refid="T194" role="Site2"></node>
    </relation>
    <relation id="E81">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T193" role="Trigger"></node>
      <node refid="T118" role="Theme"></node>
      <node refid="T116" role="Cause"></node>
      <node refid="T194" role="Site"></node>
    </relation>
    <relation id="E82">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T195" role="Trigger"></node>
      <node refid="E83" role="Theme"></node>
      <node refid="T119" role="Cause"></node>
    </relation>
    <relation id="E83">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T196" role="Trigger"></node>
      <node refid="T120" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E30" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E32" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E34" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E40" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E42" role="Event"></node>
    </relation>
    <relation id="M8">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E44" role="Event"></node>
    </relation>
    <relation id="M9">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E45" role="Event"></node>
    </relation>
    <relation id="M10">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E47" role="Event"></node>
    </relation>
    <relation id="M11">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E67" role="Event"></node>
    </relation>
    <relation id="M12">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E73" role="Event"></node>
    </relation>
    <relation id="M13">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E75" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-09-Materials_and_Methods-01</id>
    <passage>
      <offset>0</offset>
      <text>Mice.</text>
    </passage>
    <passage>
      <offset>6</offset>
      <text>Normal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Transgenic DO11.10 mice, expressing a T cell receptor for OVA323-339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice. Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8-12 wk. Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="226" length="232"></location>
        <text>GATA-3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="254" length="257"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="280" length="283"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="284" length="289"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="318" length="321"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="322" length="327"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="215" length="225"></location>
        <text>expressing</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-10-Materials_and_Methods-02</id>
    <passage>
      <offset>0</offset>
      <text>Isolation of CD4+ T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="13" length="16"></location>
        <text>CD4</text>
      </annotation>
    </passage>
    <passage>
      <offset>27</offset>
      <text>CD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60]. The purity of CD4+ T cells was initially tested by FACS and was &gt;/= 95%. Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive. The clones were characterized by high IL-4 secretion.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="27" length="30"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="108" length="111"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="197" length="200"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="402" length="406"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Positive_regulation</infon>
        <location offset="397" length="401"></location>
        <text>high</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="407" length="416"></location>
        <text>secretion</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-11-Materials_and_Methods-03</id>
    <passage>
      <offset>0</offset>
      <text>Quantitative real-time PCR.</text>
    </passage>
    <passage>
      <offset>28</offset>
      <text>The PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer 5'-GAA ACA GCA CAT TCC CAG AGT TC-3'; FOXP3 reverse primer 5'-ATG GCC CAG CGG ATG AG-3'; EF-1alpha forward primer and reverse primer as described [61]; GATA3 forward primer 5'-GCG GGC TCT ATC ACA AAA TGA-3' and rwd 5'-GCT CTC CTG GCT GCA GAC AGC-3'. The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="190" length="195"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="249" length="254"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="300" length="309"></location>
        <text>EF-1alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="363" length="368"></location>
        <text>GATA3</text>
      </annotation>
    </passage>
    <passage>
      <offset>652</offset>
      <text>Quantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers: Ubiquitin C, 5'- AGG TCA AAC AGG AAG ACA GAC GTA-3' and 5'-TCACACCCAAGAACAAGCACA-3'; Smad-7, 5'-GAA ACC GGG GGA ACG AAT TAT-3' and 5'-CGC GAG TCT TCT CCT CCC A-3'; TGF-ss1, 5'-TGA CGT CAC TGG AGT TGT ACG G-3' and 5'-GGT TCA TGT CAT GGA TGG TGC-3'. Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification. PCR conditions were 10 min at 95 degreesC, followed by 40 cycles of 15 s at 95 degreesC and 60 degreesC for 1 min using an 7300 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative DeltaDeltaCT method.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="783" length="794"></location>
        <text>Ubiquitin C</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="868" length="874"></location>
        <text>Smad-7</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="947" length="954"></location>
        <text>TGF-ss1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1393" length="1404"></location>
        <text>ubiquitin C</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-12-Materials_and_Methods-04</id>
    <passage>
      <offset>0</offset>
      <text>Inducible murine Treg culture.</text>
    </passage>
    <passage>
      <offset>31</offset>
      <text>Naive CD4+ T cells (CD4+, CD62L+, and CD25-) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences). 5 x 105 T cells were co-cultured with 2.5 x 104 bone marrow-derived dendritic cells [62] and 0.01 mug/ml OVA323-339 peptide (Ansynth) in the presence or absence of 20-ng/mul rhTGF-ss1 (Peprotech) in 48-well plates. After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="37" length="40"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="51" length="54"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="57" length="62"></location>
        <text>CD62L</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="69" length="73"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="339" length="346"></location>
        <text>TGF-ss1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="441" length="446"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="447" length="457"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-13-Materials_and_Methods-05</id>
    <passage>
      <offset>0</offset>
      <text>In vitro T cell differentiation.</text>
    </passage>
    <passage>
      <offset>33</offset>
      <text>CD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 mug/ml, Okt3, IgG1) and anti-CD28 (2 mug/ml) in Th1 conditions: 25 ng/ml IL-12, 5 mug/ml anti-IL-4 (R&amp;D systems); in Th2 conditions: 25 ng/ml IL-4, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12 (R&amp;D systems); or in Treg conditions: 10 ng/ml TGF-beta, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12, 5 mug/ml anti-IL-4. Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="33" length="36"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="38" length="44"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="67" length="73"></location>
        <text>CD45RO</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="194" length="197"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="230" length="234"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="274" length="279"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="295" length="299"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="343" length="347"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="363" length="372"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="388" length="393"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="441" length="449"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="465" length="474"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="490" length="495"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="511" length="515"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="572" length="576"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="74" length="83"></location>
        <text>depletion</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-14-Materials_and_Methods-06</id>
    <passage>
      <offset>0</offset>
      <text>Cloning of the FOXP3 promoter and construction of mutant constructs.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="15" length="20"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
    <passage>
      <offset>69</offset>
      <text>The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 -511/+176 [24]. Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions. The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="73" length="78"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="165" length="170"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="220" length="225"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-15-Materials_and_Methods-07</id>
    <passage>
      <offset>0</offset>
      <text>Transfections and reporter gene assays.</text>
    </passage>
    <passage>
      <offset>40</offset>
      <text>T cells were rested in serum-free AIM-V medium (Life Technologies) overnight. 3.5 mug of the FOXP3 promoter luciferase reporter vector and 0.5 mug phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 muL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega Biotech) according to the manufacturer's instructions. Data were normalized by the activity of renilla luciferase.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="133" length="138"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="148" length="158"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="217" length="220"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="447" length="457"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="492" length="502"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="628" length="638"></location>
        <text>luciferase</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-16-Materials_and_Methods-08</id>
    <passage>
      <offset>0</offset>
      <text>RNA isolation and cDNA synthesis.</text>
    </passage>
    <passage>
      <offset>34</offset>
      <text>RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of human samples was performed with TaqMan reverse-transcription reagents (Applied Biosystems) with random hexamers according to the manufacturer's protocol.</text>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-17-Materials_and_Methods-09</id>
    <passage>
      <offset>0</offset>
      <text>Recombinant TAT proteins.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="12" length="15"></location>
        <text>TAT</text>
      </annotation>
    </passage>
    <passage>
      <offset>26</offset>
      <text>The cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63]. Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography. Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="45" length="50"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="76" length="81"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="175" length="178"></location>
        <text>TAT</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="349" length="352"></location>
        <text>TAT</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Negative_regulation</infon>
        <location offset="83" length="90"></location>
        <text>lacking</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Entity</infon>
        <location offset="95" length="111"></location>
        <text>two zinc fingers</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T7" role="Site"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-18-Materials_and_Methods-10</id>
    <passage>
      <offset>0</offset>
      <text>TAT-GATA3 transduction.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="3"></location>
        <text>TAT</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="4" length="9"></location>
        <text>GATA3</text>
      </annotation>
    </passage>
    <passage>
      <offset>24</offset>
      <text>CD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h. After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-beta (10 ng/ml). Each day, the TAT proteins were freshly added to the medium. FOXP3 expression was measured after 5 d by intracellular staining.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="24" length="27"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="28" length="34"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="143" length="148"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="238" length="241"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="251" length="255"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="260" length="268"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="295" length="298"></location>
        <text>TAT</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="342" length="347"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="348" length="358"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-19-Materials_and_Methods-11</id>
    <passage>
      <offset>0</offset>
      <text>Intracellular cytokine staining.</text>
    </passage>
    <passage>
      <offset>33</offset>
      <text>T cells were stimulated with 2 x 10-7 M PMA and 1 mug/ml of ionomycin (Sigma Chemicals) for 4 h. The following mAb was used: anti-IL-4-PE (8D4-8, BD). Matched isotype controls were used at the same protein concentration as the respective antibodies. Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="163" length="167"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-20-Materials_and_Methods-12</id>
    <passage>
      <offset>0</offset>
      <text>Flow cytometry.</text>
    </passage>
    <passage>
      <offset>16</offset>
      <text>For analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4degreesC in staining buffer with the following antibodies: anti-CD152-PE (CTLA-4; BD), anti-PD-1 (eBiosciences), anti-GITR (R &amp; D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcgammaRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4",6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="32" length="37"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="129" length="133"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="273" length="278"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="509" length="514"></location>
        <text>CD152</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="537" length="541"></location>
        <text>PD-1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="563" length="567"></location>
        <text>GITR</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="590" length="594"></location>
        <text>CD69</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="619" length="624"></location>
        <text>CD103</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="648" length="653"></location>
        <text>CD62L</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="911" length="914"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="921" length="924"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="935" length="939"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1078" length="1081"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1121" length="1124"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1125" length="1130"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1169" length="1173"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1185" length="1190"></location>
        <text>CD62L</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1204" length="1207"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1317" length="1322"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1407" length="1412"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1482" length="1485"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="38" length="48"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1323" length="1333"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-21-Materials_and_Methods-13</id>
    <passage>
      <offset>0</offset>
      <text>Western blotting.</text>
    </passage>
    <passage>
      <offset>18</offset>
      <text>For FOXP3 analysis on the protein level, 1 x 106 CD4+CD25- cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 degreesC. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3-31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="22" length="27"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="67" length="70"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="71" length="75"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="406" length="411"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="702" length="707"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="749" length="754"></location>
        <text>T-bet</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="794" length="799"></location>
        <text>GAPDH</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="828" length="833"></location>
        <text>SMAD2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="856" length="861"></location>
        <text>STAT6</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="878" length="883"></location>
        <text>STAT6</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-22-Materials_and_Methods-14</id>
    <passage>
      <offset>0</offset>
      <text>Administration of cytokines and antibodies in vivo.</text>
    </passage>
    <passage>
      <offset>52</offset>
      <text>Age- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 mug rmIL-4, 50 mug anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 mug rmIL-4 plus 50 mug anti-IL-4 mAb (11B11 or MAB404) for 7 d. Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression. The anti-mouse IL-4 mAb MAB404 was obtained from R&amp;D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="184" length="188"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="238" length="242"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="260" length="264"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="372" length="375"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="377" length="380"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="386" length="390"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="418" length="422"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="487" length="491"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Gene_expression</infon>
        <location offset="391" length="401"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-23-Materials_and_Methods-15</id>
    <passage>
      <offset>0</offset>
      <text>ChIP.</text>
    </passage>
    <passage>
      <offset>6</offset>
      <text>ChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3-31; Santa Cruz Biotechnology) for 2 h. Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 degreesC for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction. The amount of DNA was determined by conventional PCR. The PCR addressed for the FOXP3 promoter region -246 to -511 and was performed using the following primers: 5'-gtgccctttacgagt catctg-3' and 5'-gtgccctttacgagtcatctg-3'. The PCR products were visualized using an ethidium bromide gel.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="427" length="432"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="812" length="817"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-24-Materials_and_Methods-16</id>
    <passage>
      <offset>0</offset>
      <text>Pull-down assay.</text>
    </passage>
    <passage>
      <offset>17</offset>
      <text>CD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37degreesC. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 mug of poly(dI-dC) (Sigma) and 70 mul of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 degreesC. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 degreesC for 10 min, and the eluants on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology). Accumulated signals were analyzed using AIDA software (Raytest).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="17" length="20"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="920" length="925"></location>
        <text>GATA3</text>
      </annotation>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-25-Supporting_Information</id>
    <passage>
      <offset>0</offset>
      <text>Supporting Information</text>
    </passage>
    <passage>
      <offset>23</offset>
      <text>Phenotype of In Vitro Differentiated T Cells</text>
    </passage>
    <passage>
      <offset>68</offset>
      <text>After two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A). In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="163" length="166"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="167" length="171"></location>
        <text>CD28</text>
      </annotation>
    </passage>
    <passage>
      <offset>388</offset>
      <text>(1.0 MB AI).</text>
    </passage>
    <passage>
      <offset>402</offset>
      <text>Click here for additional data file.</text>
    </passage>
    <passage>
      <offset>440</offset>
      <text>In Vivo Treatment of Mice with IL-4 Antibody-Cytokine Complexes</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="471" length="475"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>504</offset>
      <text>B6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404). Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression. Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A-F). Numbers indicate percentages of CD4+ CD25high CD3+ cells. Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A-F) are shown as mean +/- SD. The data are representative of three independent experiments.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="607" length="611"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="627" length="631"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="642" length="646"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="689" length="693"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="704" length="708"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="783" length="786"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="788" length="791"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="797" length="801"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="823" length="827"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="835" length="838"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="853" length="856"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="858" length="861"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="915" length="918"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="920" length="924"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="929" length="932"></location>
        <text>CD3</text>
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        <infon key="type">Protein</infon>
        <location offset="966" length="969"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="971" length="975"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Gene_expression</infon>
        <location offset="802" length="812"></location>
        <text>expression</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="839" length="849"></location>
        <text>expression</text>
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    <passage>
      <offset>1104</offset>
      <text>(369 KB AI).</text>
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    <passage>
      <offset>1118</offset>
      <text>Click here for additional data file.</text>
    </passage>
    <passage>
      <offset>1156</offset>
      <text>Effect of IL-4 on Already Existing Natural or Inducible Treg Cells</text>
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        <infon key="type">Protein</infon>
        <location offset="1166" length="1170"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>1223</offset>
      <text>(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis. The results shown represent the mean +/- SD of three independent experiments.</text>
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        <infon key="type">Protein</infon>
        <location offset="1227" length="1230"></location>
        <text>CD4</text>
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      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1231" length="1235"></location>
        <text>CD25</text>
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      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1305" length="1308"></location>
        <text>CD3</text>
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        <location offset="1309" length="1313"></location>
        <text>CD28</text>
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        <location offset="1319" length="1323"></location>
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        <location offset="1369" length="1373"></location>
        <text>IL-4</text>
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    <passage>
      <offset>1507</offset>
      <text>(B) iTreg cells were induced in vitro. FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-beta, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml).</text>
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        <infon key="type">Protein</infon>
        <location offset="1546" length="1551"></location>
        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
        <location offset="1665" length="1668"></location>
        <text>CD3</text>
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        <infon key="type">Protein</infon>
        <location offset="1669" length="1673"></location>
        <text>CD28</text>
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        <infon key="type">Protein</infon>
        <location offset="1691" length="1699"></location>
        <text>TGF-beta</text>
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        <infon key="type">Protein</infon>
        <location offset="1706" length="1710"></location>
        <text>IL-2</text>
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        <infon key="type">Protein</infon>
        <location offset="1780" length="1784"></location>
        <text>IL-4</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="1552" length="1562"></location>
        <text>espression</text>
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    <passage>
      <offset>1799</offset>
      <text>(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells. CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration. After vigorous washing, their suppressive capacity on responder CD4+CD25- was tested. IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells. 1 x 104 CD4+CD25+ nTreg cells were added to 5 x 104 CD4+CD25- and 5 x 104 irradiated PBMCs. The results are representative of three independent experiments.</text>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="1837" length="1840"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="1841" length="1845"></location>
        <text>CD25</text>
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      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="1882" length="1885"></location>
        <text>CD4</text>
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      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="1886" length="1890"></location>
        <text>CD25</text>
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      <annotation id="T38">
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        <location offset="1905" length="1908"></location>
        <text>CD4</text>
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        <infon key="type">Protein</infon>
        <location offset="1909" length="1913"></location>
        <text>CD25</text>
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        <infon key="type">Protein</infon>
        <location offset="2000" length="2004"></location>
        <text>IL-4</text>
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        <infon key="type">Protein</infon>
        <location offset="2084" length="2087"></location>
        <text>CD4</text>
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      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="2088" length="2092"></location>
        <text>CD25</text>
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        <infon key="type">Protein</infon>
        <location offset="2106" length="2110"></location>
        <text>IL-4</text>
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        <location offset="2179" length="2182"></location>
        <text>CD4</text>
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        <infon key="type">Protein</infon>
        <location offset="2183" length="2187"></location>
        <text>CD25</text>
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        <infon key="type">Protein</infon>
        <location offset="2207" length="2210"></location>
        <text>CD4</text>
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        <location offset="2211" length="2215"></location>
        <text>CD25</text>
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      <annotation id="T48">
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        <location offset="2251" length="2254"></location>
        <text>CD4</text>
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        <location offset="2255" length="2259"></location>
        <text>CD25</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>increasing</text>
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    <passage>
      <offset>2357</offset>
      <text>(269 KB AI).</text>
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    <passage>
      <offset>2371</offset>
      <text>Click here for additional data file.</text>
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    <passage>
      <offset>2409</offset>
      <text>Schematic Structure of the FOXP3 Gene and Location of the GATA3 Sites</text>
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        <location offset="2436" length="2441"></location>
        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
        <location offset="2467" length="2472"></location>
        <text>GATA3</text>
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    <passage>
      <offset>2479</offset>
      <text>The scheme shows the location of the 11 exons spaced by a large intron (6000 bp) from the 5'untranslated region (UTR). Human, murine, and rat sequences are aligned and transcription start site (TSS) is indicated with an arrow.</text>
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    <passage>
      <offset>2707</offset>
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    <passage>
      <offset>2721</offset>
      <text>Click here for additional data file.</text>
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      <infon key="type">Event</infon>
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      <node refid="T9" role="Theme"></node>
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      <infon key="type">Event</infon>
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      <node refid="T10" role="Theme"></node>
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      <node refid="T12" role="Theme"></node>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <node refid="T28" role="Theme"></node>
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    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T55" role="Trigger"></node>
      <node refid="T40" role="Theme"></node>
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    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="type">Event modification</infon>
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      <infon key="type">Event modification</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
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  </document>
  <document>
    <id>PMC-2222968-26-caption-01</id>
    <passage>
      <offset>0</offset>
      <text>Th2 Cells Cannot Induce FOXP3 Expression</text>
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        <infon key="type">Protein</infon>
        <location offset="24" length="29"></location>
        <text>FOXP3</text>
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      <annotation id="T10">
        <infon key="type">Positive_regulation</infon>
        <location offset="17" length="23"></location>
        <text>Induce</text>
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        <location offset="30" length="40"></location>
        <text>Expression</text>
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    </passage>
    <passage>
      <offset>41</offset>
      <text>(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-beta as indicated on the x-axis of (B). Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR. Bars show the mean +/- SD of 4 independent experiments.</text>
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        <infon key="type">Protein</infon>
        <location offset="96" length="99"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="100" length="104"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="121" length="129"></location>
        <text>TGF-beta</text>
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      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="203" length="208"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
    <passage>
      <offset>304</offset>
      <text>(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-beta, or anti-IL-4 as indicated. The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1). Bars show the mean +/- SD of four independent experiments.</text>
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        <infon key="type">Protein</infon>
        <location offset="382" length="385"></location>
        <text>CD3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="386" length="390"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="392" length="400"></location>
        <text>TGF-beta</text>
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        <infon key="type">Protein</infon>
        <location offset="410" length="414"></location>
        <text>IL-4</text>
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    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
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      <infon key="type">Event</infon>
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      <node refid="T1" role="Theme"></node>
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      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
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  </document>
  <document>
    <id>PMC-2222968-27-caption-02</id>
    <passage>
      <offset>0</offset>
      <text>Th2 Cells Do Not Express FOXP3</text>
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        <location offset="25" length="30"></location>
        <text>FOXP3</text>
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        <location offset="17" length="24"></location>
        <text>Express</text>
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    <passage>
      <offset>31</offset>
      <text>(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-beta. FOXP3 expression was measured after 5 d in culture. The dot blots are representative of three independent experiments.</text>
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        <location offset="66" length="71"></location>
        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
        <location offset="202" length="210"></location>
        <text>TGF-beta</text>
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        <infon key="type">Protein</infon>
        <location offset="212" length="217"></location>
        <text>FOXP3</text>
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        <text>expression</text>
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        <location offset="218" length="228"></location>
        <text>expression</text>
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    <passage>
      <offset>332</offset>
      <text>(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed. Data are representative of three independent experiments.</text>
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    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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  </document>
  <document>
    <id>PMC-2222968-28-caption-03</id>
    <passage>
      <offset>0</offset>
      <text>Th2- or IL-4-Producing Cells Lack FOXP3</text>
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        <infon key="type">Protein</infon>
        <location offset="8" length="12"></location>
        <text>IL-4</text>
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      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="34" length="39"></location>
        <text>FOXP3</text>
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      <annotation id="T19">
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        <location offset="13" length="22"></location>
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        <location offset="29" length="33"></location>
        <text>Lack</text>
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    </passage>
    <passage>
      <offset>40</offset>
      <text>(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation. CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25- (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO-CD25- subsets (central panel). A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B). The dotted gray line indicates the IC background level. The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4. Data are representative of three independent experiments.</text>
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        <infon key="type">Protein</infon>
        <location offset="75" length="80"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="85" length="89"></location>
        <text>IL-4</text>
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        <infon key="type">Protein</infon>
        <location offset="138" length="141"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="178" length="184"></location>
        <text>CD45RO</text>
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      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="189" length="193"></location>
        <text>CD25</text>
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      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="268" length="274"></location>
        <text>CD45RO</text>
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        <infon key="type">Protein</infon>
        <location offset="275" length="279"></location>
        <text>CD25</text>
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        <infon key="type">Protein</infon>
        <location offset="302" length="308"></location>
        <text>CD45RO</text>
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        <infon key="type">Protein</infon>
        <location offset="309" length="313"></location>
        <text>CD25</text>
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        <infon key="type">Protein</infon>
        <location offset="338" length="344"></location>
        <text>CD45RO</text>
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        <infon key="type">Protein</infon>
        <location offset="345" length="349"></location>
        <text>CD25</text>
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        <infon key="type">Protein</infon>
        <location offset="619" length="624"></location>
        <text>CRTH2</text>
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        <infon key="type">Protein</infon>
        <location offset="637" length="641"></location>
        <text>IL-4</text>
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        <infon key="type">Protein</infon>
        <location offset="679" length="685"></location>
        <text>CD45RO</text>
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        <location offset="704" length="709"></location>
        <text>FOXP3</text>
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        <location offset="714" length="718"></location>
        <text>IL-4</text>
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        <text>expression</text>
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        <text>expression</text>
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        <text>secreting</text>
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      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="692" length="699"></location>
        <text>stained</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-29-caption-04</id>
    <passage>
      <offset>0</offset>
      <text>FOXP3 Induction During the Differentiation Process</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="6" length="15"></location>
        <text>Induction</text>
      </annotation>
    </passage>
    <passage>
      <offset>51</offset>
      <text>(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta (5 ng/ml) or IL-4 (25 ng/ml). The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression. Bars show the mean +/- SD of three independent experiments.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="61" length="64"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="65" length="71"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="118" length="121"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="122" length="126"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="146" length="154"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="168" length="172"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="281" length="286"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="291" length="296"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="297" length="307"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>370</offset>
      <text>(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure. Data are representative of three independent experiments.</text>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="388" length="393"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="398" length="403"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="440" length="443"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="444" length="450"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="404" length="412"></location>
        <text>staining</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-30-caption-05</id>
    <passage>
      <offset>0</offset>
      <text>Effect of IL-4 on FOXP3 Induction</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="10" length="14"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="18" length="23"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Regulation</infon>
        <location offset="0" length="6"></location>
        <text>Effect</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="24" length="33"></location>
        <text>Induction</text>
      </annotation>
    </passage>
    <passage>
      <offset>34</offset>
      <text>(A) A statistical analysis was performed with six donors on day 5 (TGF-beta (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors. Statistical analysis was performed using the Dunnett test. Statistical significance is indicated by asterisks (*p &lt;/= 0.05, **p &lt;/= 0.01, Dunnett).</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="101" length="109"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="141" length="145"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>375</offset>
      <text>(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-beta (5 ng/ml) with an increasing concentration of IL-4, as indicated. Cells were harvested for mRNA quantification after 5 d.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="379" length="382"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="383" length="389"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="459" length="467"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="514" length="518"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="486" length="496"></location>
        <text>increasing</text>
      </annotation>
    </passage>
    <passage>
      <offset>591</offset>
      <text>(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-beta (10ng/ml), and IL-4 (100 ng/ml) as indicated. After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6. Total STAT6 and GAPDH served as internal control.</text>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="595" length="598"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="599" length="605"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="660" length="663"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="664" length="668"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="670" length="678"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="694" length="698"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="811" length="816"></location>
        <text>SMAD2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="821" length="826"></location>
        <text>STAT6</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="834" length="839"></location>
        <text>STAT6</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="844" length="849"></location>
        <text>GAPDH</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Phosphorylation</infon>
        <location offset="796" length="810"></location>
        <text>phosphorylated</text>
      </annotation>
    </passage>
    <passage>
      <offset>879</offset>
      <text>(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B. Data are representative of three independent experiments.</text>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="897" length="902"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="907" length="912"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="950" length="953"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="954" length="960"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="973" length="977"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Gene_expression</infon>
        <location offset="913" length="921"></location>
        <text>staining</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-31-caption-06</id>
    <passage>
      <offset>0</offset>
      <text>IL-4 Inhibits TGF-beta-Mediated iTreg Commitment</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="4"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="14" length="22"></location>
        <text>TGF-beta</text>
      </annotation>
    </passage>
    <passage>
      <offset>49</offset>
      <text>CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-beta, and IL-4, as indicated. After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B). Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (**p &lt;/= 0.01, ***p &lt;/= 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-beta. Data are representative of three independent experiments.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="62" length="65"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="66" length="72"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="117" length="120"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="121" length="125"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="127" length="135"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="141" length="145"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="446" length="451"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="456" length="461"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="513" length="516"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="517" length="523"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="541" length="544"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="545" length="547"></location>
        <text>28</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="549" length="553"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="558" length="566"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="462" length="470"></location>
        <text>staining</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-32-caption-07</id>
    <passage>
      <offset>0</offset>
      <text>GATA3 Acts as a Negative Regulator of FOXP3 Expression</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="38" length="43"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
    <passage>
      <offset>55</offset>
      <text>(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells. GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel). Data are representative of four independent experiments.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="71" length="74"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="75" length="79"></location>
        <text>CDRA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="136" length="145"></location>
        <text>TAT-GATA3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="185" length="190"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="230" length="233"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="234" length="238"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="264" length="267"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="325" length="330"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="173" length="181"></location>
        <text>presence</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="331" length="341"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>449</offset>
      <text>(B) CD4+CD25- T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-beta for 96 h. Surface CD4 and intracellular FOXP3 were measured by FACS. These data are representative of three independent experiments.</text>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="453" length="456"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="457" length="461"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="510" length="513"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="514" length="519"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="550" length="558"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="577" length="580"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="599" length="604"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
    <passage>
      <offset>693</offset>
      <text>(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-beta expression. Bars show the mean +/- SD of three independent experiments.</text>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="785" length="790"></location>
        <text>SMAD7</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="795" length="803"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="804" length="814"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-33-caption-08</id>
    <passage>
      <offset>0</offset>
      <text>GATA3 Represses the Human FOXP3 Promoter</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="26" length="31"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="6" length="15"></location>
        <text>Represses</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Entity</infon>
        <location offset="32" length="40"></location>
        <text>Promoter</text>
      </annotation>
    </passage>
    <passage>
      <offset>41</offset>
      <text>(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="82" length="85"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="179" length="184"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="214" length="224"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="314" length="324"></location>
        <text>luciferase</text>
      </annotation>
    </passage>
    <passage>
      <offset>397</offset>
      <text>(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector. Bars show the mean +/- SD of three independent experiments.</text>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="407" length="410"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="445" length="450"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="495" length="500"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="501" length="511"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>600</offset>
      <text>(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.</text>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="647" length="650"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="696" length="701"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="714" length="719"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="860" length="870"></location>
        <text>luciferase</text>
      </annotation>
    </passage>
    <passage>
      <offset>944</offset>
      <text>(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides. The oligonucleotides-transcription factor complexes were separated on a SDS-PAGE gel. The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb. Total nuclear extracts were also run as controls. Data are representative of three different experiments.</text>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1011" length="1016"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1237" length="1242"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1313" length="1318"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="1226" length="1233"></location>
        <text>amounts</text>
      </annotation>
    </passage>
    <passage>
      <offset>1431</offset>
      <text>(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter. The "input" represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments.</text>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1480" length="1485"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1501" length="1506"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="1486" length="1493"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Entity</infon>
        <location offset="1507" length="1515"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
      <node refid="T20" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T18" role="Theme2"></node>
      <node refid="T24" role="Site2"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-34-caption-09</id>
    <passage>
      <offset>0</offset>
      <text>Phenotype of In Vitro Differentiated T Cells</text>
    </passage>
    <passage>
      <offset>45</offset>
      <text>After two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A). In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B).</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="140" length="143"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="144" length="148"></location>
        <text>CD28</text>
      </annotation>
    </passage>
    <passage>
      <offset>365</offset>
      <text>(1.0 MB AI).</text>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-35-caption-10</id>
    <passage>
      <offset>0</offset>
      <text>Click here for additional data file.</text>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-36-caption-11</id>
    <passage>
      <offset>0</offset>
      <text>In Vivo Treatment of Mice with IL-4 Antibody-Cytokine Complexes</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="31" length="35"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>64</offset>
      <text>B6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404). Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression. Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A-F). Numbers indicate percentages of CD4+ CD25high CD3+ cells. Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A-F) are shown as mean +/- SD. The data are representative of three independent experiments.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="167" length="171"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="187" length="191"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="202" length="206"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="264" length="268"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="343" length="346"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="348" length="351"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="357" length="361"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="383" length="387"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="395" length="398"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="413" length="416"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="418" length="421"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="475" length="478"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="480" length="484"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="489" length="492"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="526" length="529"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="531" length="535"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="362" length="372"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="399" length="409"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>664</offset>
      <text>(369 KB AI).</text>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2222968-37-caption-12</id>
    <passage>
      <offset>0</offset>
      <text>Click here for additional data file.</text>
    </passage>
  </document>
  <document>
    <id>PMC-2222968-38-caption-13</id>
    <passage>
      <offset>0</offset>
      <text>Effect of IL-4 on Already Existing Natural or Inducible Treg Cells</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="10" length="14"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>67</offset>
      <text>(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis. The results shown represent the mean +/- SD of three independent experiments.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="71" length="74"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="75" length="79"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="149" length="152"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="153" length="157"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="163" length="167"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="213" length="217"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>351</offset>
      <text>(B) iTreg cells were induced in vitro. FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-beta, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml).</text>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="390" length="395"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="509" length="512"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="513" length="517"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="535" length="543"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="550" length="554"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="624" length="628"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Gene_expression</infon>
        <location offset="396" length="406"></location>
        <text>espression</text>
      </annotation>
    </passage>
    <passage>
      <offset>643</offset>
      <text>(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells. CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration. After vigorous washing, their suppressive capacity on responder CD4+CD25- was tested. IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells. 1 x 104 CD4+CD25+ nTreg cells were added to 5 x 104 CD4+CD25- and 5 x 104 irradiated PBMCs. The results are representative of three independent experiments.</text>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="681" length="684"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="685" length="689"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="726" length="729"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="730" length="734"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="749" length="752"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="753" length="757"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="844" length="848"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="928" length="931"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="932" length="936"></location>
        <text>CD25</text>
      </annotation>
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      <offset>0</offset>
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    <id>PMC-2806624-00-TIAB</id>
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      <offset>0</offset>
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      <offset>120</offset>
      <text>Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-beta (TGF-beta) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-beta (CBFbeta) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2-/- mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4+ CD25high CD127- T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-beta mRNA compared with CD4+CD25- cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-beta-induced Foxp3 expression in iT reg cell differentiation and function.</text>
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  </document>
  <document>
    <id>PMC-2806624-01-INTRODUCTION</id>
    <passage>
      <offset>0</offset>
      <text>INTRODUCTION</text>
    </passage>
    <passage>
      <offset>13</offset>
      <text>Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003; Akdis, 2006). Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003; Williams and Rudensky, 2007). A spontaneous mutation of the X-linked Foxp3 gene in scurfy mice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006).</text>
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        <infon key="type">Protein</infon>
        <location offset="76" length="91"></location>
        <text>forkhead box P3</text>
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      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="93" length="98"></location>
        <text>FOXP3</text>
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      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="107" length="112"></location>
        <text>Foxp3</text>
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      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="302" length="307"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="562" length="567"></location>
        <text>Foxp3</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="40" length="50"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Gene_expression</infon>
        <location offset="395" length="405"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>824</offset>
      <text>Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood. It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-kappaB) to regulate the transcription of different target genes (Schubert et al., 2001; Bettelli et al., 2005; Wu et al., 2006). The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming. GATA3 binds to the FOXP3 promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007). It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+ T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007). Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+ phenotype (Fontenot et al., 2003; Hori et al., 2003). The crucial role of TGF-beta in their generation has been demonstrated.</text>
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        <infon key="type">Protein</infon>
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        <text>Foxp3</text>
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      <annotation id="T7">
        <infon key="type">Protein</infon>
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        <text>Foxp3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
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        <text>IL-4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
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        <text>FOXP3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1423" length="1428"></location>
        <text>GATA3</text>
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      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1442" length="1447"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1477" length="1482"></location>
        <text>FOXP3</text>
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      <annotation id="T13">
        <infon key="type">Protein</infon>
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        <text>T-bet</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>GATA3</text>
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        <infon key="type">Protein</infon>
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        <text>Foxp3</text>
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        <infon key="type">Protein</infon>
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        <text>STAT1</text>
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        <infon key="type">Protein</infon>
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        <text>STAT4</text>
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      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1727" length="1732"></location>
        <text>STAT6</text>
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      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1877" length="1882"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="2035" length="2040"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="2116" length="2124"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Regulation</infon>
        <location offset="931" length="941"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Negative_regulation</infon>
        <location offset="1374" length="1382"></location>
        <text>inhibits</text>
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      <annotation id="T59">
        <infon key="type">Gene_expression</infon>
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        <text>expression</text>
      </annotation>
      <annotation id="T60">
        <infon key="type">Binding</infon>
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        <text>binds</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Entity</infon>
        <location offset="1448" length="1456"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T62">
        <infon key="type">Negative_regulation</infon>
        <location offset="1465" length="1472"></location>
        <text>repress</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Positive_regulation</infon>
        <location offset="1483" length="1499"></location>
        <text>trans-activation</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Negative_regulation</infon>
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        <text>oppose</text>
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      <annotation id="T65">
        <infon key="type">Positive_regulation</infon>
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        <text>induction</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Positive_regulation</infon>
        <location offset="1733" length="1742"></location>
        <text>dependent</text>
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      <annotation id="T67">
        <infon key="type">Gene_expression</infon>
        <location offset="1883" length="1893"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>2169</offset>
      <text>The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-alpha [CBFalpha], and polyoma enhancer-binding protein-2alpha [PEBP2alpha]) contains three members, RUNX1 (AML1/CBFA2/PEBP2alphaB), RUNX2 (AML3/CBFA1/ PEBP2alphaA), and RUNX3 (AML2/CBFA3/PEBP2alphaC). They are essential transcriptional regulators of different developmental pathways. RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997). RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996; Inoue et al., 2002; Levanon et al., 2002). RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002; Egawa et al., 2007). RUNX1 is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), and RUNX3 methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005).</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX</text>
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        <infon key="type">Protein</infon>
        <location offset="2376" length="2381"></location>
        <text>RUNX1</text>
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      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="2383" length="2387"></location>
        <text>AML1</text>
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        <text>CBFA2</text>
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      <annotation id="T26">
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        <text>PEBP2alphaB</text>
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        <text>RUNX2</text>
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        <text>AML3</text>
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        <infon key="type">Protein</infon>
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        <text>CBFA1</text>
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        <infon key="type">Protein</infon>
        <location offset="2427" length="2438"></location>
        <text>PEBP2alphaA</text>
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        <infon key="type">Protein</infon>
        <location offset="2445" length="2450"></location>
        <text>RUNX3</text>
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      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="2560" length="2565"></location>
        <text>RUNX2</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX1</text>
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      <annotation id="T34">
        <infon key="type">Protein</infon>
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        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
        <location offset="2858" length="2863"></location>
        <text>RUNX1</text>
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      <annotation id="T36">
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        <location offset="2868" length="2873"></location>
        <text>RUNX3</text>
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      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="3001" length="3006"></location>
        <text>RUNX1</text>
      </annotation>
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        <location offset="3103" length="3108"></location>
        <text>RUNX3</text>
      </annotation>
    </passage>
    <passage>
      <offset>3206</offset>
      <text>RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999). The Runt domain-containing protein constitutes the alpha-chain partner of the heterodimeric CBF complex. RUNX proteins heterodimerize with the non-DNA-binding partner, CBFbeta, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993). The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements. The effects of the RUNX-CBFbeta complex regulation are clearly cell lineage and stage specific. They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005).</text>
      <annotation id="T39">
        <infon key="type">Protein</infon>
        <location offset="3206" length="3210"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Protein</infon>
        <location offset="3391" length="3394"></location>
        <text>CBF</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Protein</infon>
        <location offset="3467" length="3474"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="3562" length="3571"></location>
        <text>ubiquitin</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="3619" length="3622"></location>
        <text>CBF</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="3745" length="3749"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="3750" length="3757"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Binding</infon>
        <location offset="3418" length="3432"></location>
        <text>heterodimerize</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Binding</infon>
        <location offset="3683" length="3690"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <passage>
      <offset>3988</offset>
      <text>Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-beta, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells. This study demonstrates that RUNX1 and RUNX3 induced by TGF-beta are involved in the development and suppressive function of Foxp3+ iT reg cells.</text>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="4018" length="4022"></location>
        <text>RUNX</text>
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        <infon key="type">Protein</infon>
        <location offset="4097" length="4105"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Protein</infon>
        <location offset="4137" length="4142"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="4147" length="4152"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="4174" length="4179"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="4275" length="4280"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="4285" length="4290"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Protein</infon>
        <location offset="4302" length="4310"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Protein</infon>
        <location offset="4371" length="4376"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Binding</infon>
        <location offset="4080" length="4091"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Regulation</infon>
        <location offset="4127" length="4133"></location>
        <text>impact</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Gene_expression</infon>
        <location offset="4160" length="4170"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T73">
        <infon key="type">Positive_regulation</infon>
        <location offset="4291" length="4298"></location>
        <text>induced</text>
      </annotation>
    </passage>
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      <infon key="type">Equiv</infon>
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    <relation id="*">
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    <relation id="*">
      <infon key="type">Equiv</infon>
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    <relation id="E1">
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    <relation id="E2">
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    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E5">
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T61" role="Site2"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E6" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T18" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
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      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="T18" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T67" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T68" role="Trigger"></node>
      <node refid="T41" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T69" role="Trigger"></node>
      <node refid="T43" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T70" role="Trigger"></node>
      <node refid="T46" role="Theme"></node>
      <node refid="T47" role="Theme2"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T71" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="T48" role="Cause"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T71" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="T49" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T72" role="Trigger"></node>
      <node refid="T50" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T73" role="Trigger"></node>
      <node refid="T51" role="Theme"></node>
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    </relation>
    <relation id="E26">
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      <infon key="type">Event</infon>
      <node refid="T73" role="Trigger"></node>
      <node refid="T52" role="Theme"></node>
      <node refid="T53" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E22" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E24" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2806624-02-RESULTS-01</id>
    <passage>
      <offset>0</offset>
      <text>The role of RUNX1 and RUNX3 transcription factors in TGF-beta-mediated iT reg cell generation</text>
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        <infon key="type">Protein</infon>
        <location offset="12" length="17"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="22" length="27"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="53" length="61"></location>
        <text>TGF-beta</text>
      </annotation>
    </passage>
    <passage>
      <offset>94</offset>
      <text>To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells. Stimulation with anti-CD2/3/28 mAbs or TGF-beta alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A). In contrast, the combination of both TGF-beta and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells. This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX</text>
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        <infon key="type">Protein</infon>
        <location offset="202" length="205"></location>
        <text>CD4</text>
      </annotation>
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        <text>CD2</text>
      </annotation>
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        <location offset="328" length="329"></location>
        <text>3</text>
      </annotation>
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        <text>28</text>
      </annotation>
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        <text>TGF-beta</text>
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        <location offset="395" length="400"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="405" length="410"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="465" length="473"></location>
        <text>TGF-beta</text>
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        <location offset="483" length="486"></location>
        <text>CD2</text>
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        <text>3</text>
      </annotation>
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        <text>28</text>
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        <location offset="505" length="510"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="515" length="520"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="539" length="544"></location>
        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
        <location offset="572" length="575"></location>
        <text>CD4</text>
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        <infon key="type">Protein</infon>
        <location offset="672" length="677"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="682" length="687"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="378" length="391"></location>
        <text>up-regulation</text>
      </annotation>
      <annotation id="T92">
        <infon key="type">Transcription</infon>
        <location offset="411" length="415"></location>
        <text>mRNA</text>
      </annotation>
      <annotation id="T93">
        <infon key="type">Positive_regulation</infon>
        <location offset="497" length="504"></location>
        <text>induced</text>
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      <annotation id="T94">
        <infon key="type">Transcription</infon>
        <location offset="521" length="526"></location>
        <text>mRNAs</text>
      </annotation>
      <annotation id="T95">
        <infon key="type">Transcription</infon>
        <location offset="545" length="549"></location>
        <text>mRNA</text>
      </annotation>
      <annotation id="T96">
        <infon key="type">Positive_regulation</infon>
        <location offset="655" length="668"></location>
        <text>up-regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>793</offset>
      <text>To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B). Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-beta-mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA. The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).</text>
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        <infon key="type">Protein</infon>
        <location offset="818" length="823"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="828" length="833"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="877" length="880"></location>
        <text>CD4</text>
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        <location offset="966" length="971"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="975" length="980"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1010" length="1018"></location>
        <text>TGF-beta</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1041" length="1046"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1061" length="1064"></location>
        <text>CD4</text>
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        <infon key="type">Protein</infon>
        <location offset="1149" length="1154"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1190" length="1195"></location>
        <text>RUNX1</text>
      </annotation>
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        <location offset="1200" length="1205"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1233" length="1236"></location>
        <text>CD4</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="834" length="844"></location>
        <text>expression</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="849" length="861"></location>
        <text>knocked down</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="952" length="962"></location>
        <text>Deficiency</text>
      </annotation>
      <annotation id="T100">
        <infon key="type">Negative_regulation</infon>
        <location offset="1002" length="1009"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T101">
        <infon key="type">Positive_regulation</infon>
        <location offset="1019" length="1027"></location>
        <text>mediated</text>
      </annotation>
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        <infon key="type">Transcription</infon>
        <location offset="1028" length="1037"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T103">
        <infon key="type">Transcription</infon>
        <location offset="1155" length="1159"></location>
        <text>mRNA</text>
      </annotation>
      <annotation id="T104">
        <infon key="type">Negative_regulation</infon>
        <location offset="1172" length="1179"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T105">
        <infon key="type">Negative_regulation</infon>
        <location offset="1211" length="1223"></location>
        <text>knocked down</text>
      </annotation>
    </passage>
    <passage>
      <offset>1304</offset>
      <text>The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated. Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed. We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells. On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).</text>
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        <infon key="type">Protein</infon>
        <location offset="1321" length="1326"></location>
        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX3</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="1463" length="1466"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="1689" length="1694"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Protein</infon>
        <location offset="1720" length="1725"></location>
        <text>T-bet</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1754" length="1759"></location>
        <text>RORC2</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
        <location offset="1809" length="1814"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="1879" length="1884"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="1921" length="1926"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="1932" length="1937"></location>
        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Regulation</infon>
        <location offset="1679" length="1685"></location>
        <text>change</text>
      </annotation>
      <annotation id="T107">
        <infon key="type">Transcription</infon>
        <location offset="1695" length="1705"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T108">
        <infon key="type">Transcription</infon>
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        <text>expression</text>
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      <annotation id="T109">
        <infon key="type">Transcription</infon>
        <location offset="1760" length="1775"></location>
        <text>mRNA expression</text>
      </annotation>
      <annotation id="T110">
        <infon key="type">Negative_regulation</infon>
        <location offset="1820" length="1832"></location>
        <text>knocked down</text>
      </annotation>
      <annotation id="T111">
        <infon key="type">Transcription</infon>
        <location offset="1885" length="1889"></location>
        <text>mRNA</text>
      </annotation>
      <annotation id="T112">
        <infon key="type">Negative_regulation</infon>
        <location offset="1908" length="1917"></location>
        <text>decreased</text>
      </annotation>
      <annotation id="T113">
        <infon key="type">Negative_regulation</infon>
        <location offset="1938" length="1947"></location>
        <text>deficient</text>
      </annotation>
    </passage>
    <passage>
      <offset>2001</offset>
      <text>The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry. FOXP3 was only slightly reduced after RUNX1 silencing. Transfection of siRNA for RUNX3 had a stronger effect. The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D). Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2). The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3. In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-gamma in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-beta did not show any significant difference (Fig. S3).</text>
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        <infon key="type">Protein</infon>
        <location offset="2015" length="2019"></location>
        <text>RUNX</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2071" length="2076"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2090" length="2093"></location>
        <text>CD4</text>
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        <infon key="type">Protein</infon>
        <location offset="2161" length="2166"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2199" length="2204"></location>
        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
        <location offset="2242" length="2247"></location>
        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
        <location offset="2289" length="2294"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2323" length="2328"></location>
        <text>RUNX1</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
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        <text>CD4</text>
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        <infon key="type">Protein</infon>
        <location offset="2482" length="2487"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Protein</infon>
        <location offset="2492" length="2497"></location>
        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
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        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
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        <text>IL-4</text>
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        <infon key="type">Protein</infon>
        <location offset="2628" length="2632"></location>
        <text>IL-5</text>
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        <infon key="type">Protein</infon>
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        <text>IL-10</text>
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        <infon key="type">Protein</infon>
        <location offset="2641" length="2646"></location>
        <text>IL-13</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
        <location offset="2652" length="2661"></location>
        <text>IFN-gamma</text>
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        <infon key="type">Protein</infon>
        <location offset="2694" length="2699"></location>
        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
        <location offset="2728" length="2731"></location>
        <text>CD4</text>
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        <infon key="type">Protein</infon>
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        <text>CD2</text>
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        <infon key="type">Protein</infon>
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        <text>3</text>
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        <infon key="type">Protein</infon>
        <location offset="2787" length="2789"></location>
        <text>28</text>
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        <infon key="type">Protein</infon>
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        <text>TGF-beta</text>
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        <infon key="type">Regulation</infon>
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        <text>effect</text>
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        <infon key="type">Negative_regulation</infon>
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        <text>silencing</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="2037" length="2047"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T117">
        <infon key="type">Negative_regulation</infon>
        <location offset="2185" length="2192"></location>
        <text>reduced</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="2205" length="2214"></location>
        <text>silencing</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="2216" length="2237"></location>
        <text>Transfection of siRNA</text>
      </annotation>
      <annotation id="T120">
        <infon key="type">Negative_regulation</infon>
        <location offset="2254" length="2269"></location>
        <text>stronger effect</text>
      </annotation>
      <annotation id="T121">
        <infon key="type">Negative_regulation</infon>
        <location offset="2295" length="2304"></location>
        <text>reduction</text>
      </annotation>
      <annotation id="T122">
        <infon key="type">Negative_regulation</infon>
        <location offset="2344" length="2352"></location>
        <text>silenced</text>
      </annotation>
      <annotation id="T123">
        <infon key="type">Negative_regulation</infon>
        <location offset="2498" length="2507"></location>
        <text>knockdown</text>
      </annotation>
      <annotation id="T124">
        <infon key="type">Regulation</infon>
        <location offset="2558" length="2567"></location>
        <text>functions</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="2575" length="2584"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T126">
        <infon key="type">Gene_expression</infon>
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        <text>levels</text>
      </annotation>
      <annotation id="T127">
        <infon key="type">Negative_regulation</infon>
        <location offset="2710" length="2727"></location>
        <text>siRNA-transfected</text>
      </annotation>
      <annotation id="T128">
        <infon key="type">Regulation</infon>
        <location offset="2815" length="2846"></location>
        <text>show any significant difference</text>
      </annotation>
    </passage>
    <passage>
      <offset>2859</offset>
      <text>To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127- CD25high T reg cells and compared them with CD4+ CD127+ CD25- T cells. Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25- cells. As expected, IL-10, TGF-beta, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A). There was no difference in RUNX1 mRNA expression between these two cell subsets. We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006). Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).</text>
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        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
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        <text>RUNX3</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>CD4</text>
      </annotation>
      <annotation id="T75">
        <infon key="type">Protein</infon>
        <location offset="2979" length="2984"></location>
        <text>CD127</text>
      </annotation>
      <annotation id="T76">
        <infon key="type">Protein</infon>
        <location offset="2986" length="2990"></location>
        <text>CD25</text>
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      <annotation id="T77">
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        <location offset="3030" length="3033"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Protein</infon>
        <location offset="3035" length="3040"></location>
        <text>CD127</text>
      </annotation>
      <annotation id="T79">
        <infon key="type">Protein</infon>
        <location offset="3042" length="3046"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Protein</infon>
        <location offset="3122" length="3127"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Protein</infon>
        <location offset="3147" length="3150"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Protein</infon>
        <location offset="3151" length="3155"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Protein</infon>
        <location offset="3177" length="3182"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Protein</infon>
        <location offset="3184" length="3192"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Protein</infon>
        <location offset="3198" length="3203"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Protein</infon>
        <location offset="3295" length="3300"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T87">
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        <location offset="3427" length="3432"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="3503" length="3508"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T89">
        <infon key="type">Protein</infon>
        <location offset="3513" length="3518"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T90">
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        <location offset="3626" length="3631"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T129">
        <infon key="type">Gene_expression</infon>
        <location offset="2905" length="2914"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T130">
        <infon key="type">Transcription</infon>
        <location offset="3081" length="3090"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T131">
        <infon key="type">Transcription</infon>
        <location offset="3219" length="3228"></location>
        <text>expressed</text>
      </annotation>
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        <infon key="type">Regulation</infon>
        <location offset="3281" length="3291"></location>
        <text>difference</text>
      </annotation>
      <annotation id="T133">
        <infon key="type">Transcription</infon>
        <location offset="3301" length="3316"></location>
        <text>mRNA expression</text>
      </annotation>
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        <location offset="3540" length="3552"></location>
        <text>coexpression</text>
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        <location offset="3622" length="3625"></location>
        <text>low</text>
      </annotation>
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        <location offset="3632" length="3642"></location>
        <text>expression</text>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E3">
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      <infon key="type">Event</infon>
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    <relation id="E2">
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E5">
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      <infon key="type">Event</infon>
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    <relation id="E7">
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      <infon key="type">Event</infon>
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    <relation id="E9">
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      <infon key="type">Event</infon>
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    <relation id="E6">
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      <infon key="type">Event</infon>
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    <relation id="E8">
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
      <node refid="T95" role="Trigger"></node>
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    <relation id="E11">
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      <infon key="type">Event</infon>
      <node refid="T96" role="Trigger"></node>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E14">
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T98" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T98" role="Trigger"></node>
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    </relation>
    <relation id="E17">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T99" role="Trigger"></node>
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    <relation id="E18">
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      <infon key="type">Event</infon>
      <node refid="T99" role="Trigger"></node>
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    </relation>
    <relation id="E19">
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      <infon key="type">Event</infon>
      <node refid="T100" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
      <node refid="E17" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E22">
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      <infon key="type">Event</infon>
      <node refid="T101" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
      <node refid="T27" role="Cause"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T102" role="Trigger"></node>
      <node refid="T28" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T103" role="Trigger"></node>
      <node refid="T30" role="Theme"></node>
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    <relation id="E24">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T104" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="E25" role="Cause"></node>
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    <relation id="E26">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T104" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
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    <relation id="E25">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T105" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
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    <relation id="E27">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T105" role="Trigger"></node>
      <node refid="T32" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
      <node refid="E29" role="Theme"></node>
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    </relation>
    <relation id="E31">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
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    </relation>
    <relation id="E33">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
      <node refid="E34" role="Theme"></node>
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    </relation>
    <relation id="E35">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
      <node refid="E34" role="Theme"></node>
      <node refid="E32" role="Cause"></node>
    </relation>
    <relation id="E36">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
      <node refid="E37" role="Theme"></node>
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    </relation>
    <relation id="E38">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T106" role="Trigger"></node>
      <node refid="E37" role="Theme"></node>
      <node refid="E32" role="Cause"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T107" role="Trigger"></node>
      <node refid="T37" role="Theme"></node>
    </relation>
    <relation id="E34">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T108" role="Trigger"></node>
      <node refid="T38" role="Theme"></node>
    </relation>
    <relation id="E37">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T109" role="Trigger"></node>
      <node refid="T39" role="Theme"></node>
    </relation>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T110" role="Trigger"></node>
      <node refid="T40" role="Theme"></node>
    </relation>
    <relation id="E32">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T110" role="Trigger"></node>
      <node refid="T41" role="Theme"></node>
    </relation>
    <relation id="E39">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T111" role="Trigger"></node>
      <node refid="T42" role="Theme"></node>
    </relation>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T112" role="Trigger"></node>
      <node refid="E39" role="Theme"></node>
      <node refid="E41" role="Cause"></node>
    </relation>
    <relation id="E42">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T112" role="Trigger"></node>
      <node refid="E39" role="Theme"></node>
      <node refid="E43" role="Cause"></node>
    </relation>
    <relation id="E41">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T113" role="Trigger"></node>
      <node refid="T43" role="Theme"></node>
    </relation>
    <relation id="E43">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T113" role="Trigger"></node>
      <node refid="T44" role="Theme"></node>
    </relation>
    <relation id="E44">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T114" role="Trigger"></node>
      <node refid="E45" role="Theme"></node>
      <node refid="E46" role="Cause"></node>
    </relation>
    <relation id="E46">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T115" role="Trigger"></node>
      <node refid="T45" role="Theme"></node>
    </relation>
    <relation id="E45">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T116" role="Trigger"></node>
      <node refid="T46" role="Theme"></node>
    </relation>
    <relation id="E47">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T117" role="Trigger"></node>
      <node refid="T48" role="Theme"></node>
      <node refid="E48" role="Cause"></node>
    </relation>
    <relation id="E48">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T118" role="Trigger"></node>
      <node refid="T49" role="Theme"></node>
    </relation>
    <relation id="E49">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T119" role="Trigger"></node>
      <node refid="T50" role="Theme"></node>
    </relation>
    <relation id="E50">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T120" role="Trigger"></node>
      <node refid="T48" role="Theme"></node>
      <node refid="E49" role="Cause"></node>
    </relation>
    <relation id="E51">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T121" role="Trigger"></node>
      <node refid="T51" role="Theme"></node>
      <node refid="E52" role="Cause"></node>
    </relation>
    <relation id="E53">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T121" role="Trigger"></node>
      <node refid="T51" role="Theme"></node>
      <node refid="E54" role="Cause"></node>
    </relation>
    <relation id="E52">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T122" role="Trigger"></node>
      <node refid="T52" role="Theme"></node>
    </relation>
    <relation id="E54">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T122" role="Trigger"></node>
      <node refid="T53" role="Theme"></node>
    </relation>
    <relation id="E55">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T123" role="Trigger"></node>
      <node refid="T55" role="Theme"></node>
    </relation>
    <relation id="E56">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T123" role="Trigger"></node>
      <node refid="T56" role="Theme"></node>
    </relation>
    <relation id="E57">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T124" role="Trigger"></node>
      <node refid="E58" role="Theme"></node>
      <node refid="T57" role="Cause"></node>
    </relation>
    <relation id="E59">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T124" role="Trigger"></node>
      <node refid="E58" role="Theme"></node>
      <node refid="T58" role="Cause"></node>
    </relation>
    <relation id="E58">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T125" role="Trigger"></node>
      <node refid="T59" role="Theme"></node>
    </relation>
    <relation id="E60">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T126" role="Trigger"></node>
      <node refid="T60" role="Theme"></node>
    </relation>
    <relation id="E61">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T126" role="Trigger"></node>
      <node refid="T61" role="Theme"></node>
    </relation>
    <relation id="E62">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T126" role="Trigger"></node>
      <node refid="T62" role="Theme"></node>
    </relation>
    <relation id="E63">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T126" role="Trigger"></node>
      <node refid="T63" role="Theme"></node>
    </relation>
    <relation id="E64">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T126" role="Trigger"></node>
      <node refid="T64" role="Theme"></node>
    </relation>
    <relation id="E65">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T127" role="Trigger"></node>
      <node refid="T65" role="Theme"></node>
    </relation>
    <relation id="E66">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T127" role="Trigger"></node>
      <node refid="T66" role="Theme"></node>
    </relation>
    <relation id="E67">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E60" role="Theme"></node>
      <node refid="E65" role="Cause"></node>
    </relation>
    <relation id="E68">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E60" role="Theme"></node>
      <node refid="E66" role="Cause"></node>
    </relation>
    <relation id="E69">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E60" role="Theme"></node>
      <node refid="T71" role="Cause"></node>
    </relation>
    <relation id="E70">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E61" role="Theme"></node>
      <node refid="E65" role="Cause"></node>
    </relation>
    <relation id="E71">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E61" role="Theme"></node>
      <node refid="E66" role="Cause"></node>
    </relation>
    <relation id="E72">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E61" role="Theme"></node>
      <node refid="T71" role="Cause"></node>
    </relation>
    <relation id="E73">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E62" role="Theme"></node>
      <node refid="E65" role="Cause"></node>
    </relation>
    <relation id="E74">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E62" role="Theme"></node>
      <node refid="E66" role="Cause"></node>
    </relation>
    <relation id="E75">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E62" role="Theme"></node>
      <node refid="T71" role="Cause"></node>
    </relation>
    <relation id="E76">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T128" role="Trigger"></node>
      <node refid="E63" role="Theme"></node>
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  </document>
  <document>
    <id>PMC-2806624-03-RESULTS-02</id>
    <passage>
      <offset>0</offset>
      <text>RUNX1 and RUNX3 bind to the FOXP3 promoter</text>
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      <offset>43</offset>
      <text>Transcription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS). All three binding sites are conserved between human, mouse, and rat (Fig. S4). To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3. After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A). To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay. Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors. The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody. We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B). Although there was a similar and high degree of binding to the -333 and -287 sites, a lower degree of binding was detected when the oligonucleotide containing the -53 site in the Foxp3 promoter was used. This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B). The binding to the two single binding sites at -333 and -287 was comparable to the mixture of all three oligonucleotides. Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFbeta to FOXP3 promoter during the differentiation of naive T cells toward T reg cells. Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-beta as a Foxp3-inducing stimulation. Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFbeta were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C). Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.</text>
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  <document>
    <id>PMC-2806624-04-RESULTS-03</id>
    <passage>
      <offset>0</offset>
      <text>Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3</text>
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      <text>To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors. An increase in luciferase activity was observed only when the FOXP3 promoter (-511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A). The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1. Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (-511 to +176) luciferase construct were mutated (Fig. 4 A). In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-beta for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.</text>
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    <passage>
      <offset>921</offset>
      <text>To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination. No reduction in luciferase activity was observed when the -53 site was mutated and only a slight reduction when either the -287 or -333 site was mutated (Fig. 4 B). However, mutating the -53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter. Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h. This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-beta. Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).</text>
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      <infon key="type">Event</infon>
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      <node refid="E5" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T40" role="Site"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
      <node refid="T44" role="Site2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
      <node refid="T44" role="Site2"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="E16" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="E18" role="Cause"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="E20" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="E25" role="Cause"></node>
      <node refid="T51" role="Site"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="E27" role="Cause"></node>
      <node refid="T51" role="Site"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T52" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T52" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T53" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T54" role="Trigger"></node>
      <node refid="E28" role="Theme"></node>
    </relation>
    <relation id="E30">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T55" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E31">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T55" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E32">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T56" role="Trigger"></node>
      <node refid="E33" role="Theme"></node>
      <node refid="T23" role="Cause"></node>
    </relation>
    <relation id="E33">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T58" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
      <node refid="T57" role="Site"></node>
    </relation>
    <relation id="E34">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T59" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="E35">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T60" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="E36">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T61" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E37">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T62" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E38">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T64" role="Trigger"></node>
      <node refid="T28" role="Theme"></node>
      <node refid="T29" role="Theme2"></node>
      <node refid="T63" role="Site2"></node>
    </relation>
    <relation id="E39">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T65" role="Trigger"></node>
      <node refid="E40" role="Theme"></node>
      <node refid="E38" role="Cause"></node>
    </relation>
    <relation id="E40">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T66" role="Trigger"></node>
      <node refid="T29" role="Theme"></node>
      <node refid="T67" role="Site"></node>
    </relation>
    <relation id="E41">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T68" role="Trigger"></node>
      <node refid="T30" role="Theme"></node>
    </relation>
    <relation id="E42">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T69" role="Trigger"></node>
      <node refid="T32" role="Theme"></node>
      <node refid="E41" role="Cause"></node>
    </relation>
    <relation id="E43">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T70" role="Trigger"></node>
      <node refid="E42" role="Theme"></node>
      <node refid="T35" role="Cause"></node>
    </relation>
    <relation id="E44">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T71" role="Trigger"></node>
      <node refid="T37" role="Theme"></node>
    </relation>
    <relation id="E45">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T72" role="Trigger"></node>
      <node refid="T38" role="Theme"></node>
      <node refid="E44" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E34" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E39" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E43" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E45" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2806624-05-RESULTS-04</id>
    <passage>
      <offset>0</offset>
      <text>Role of CBFbeta in the induction of Foxp3</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="8" length="15"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="36" length="41"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="4"></location>
        <text>Role</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Gene_expression</infon>
        <location offset="23" length="32"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <passage>
      <offset>42</offset>
      <text>CBFbeta, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences. To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene. Retinoic acid and TGF-beta synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007). To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFbeta, naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-gamma expression. TGF-beta induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells. Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-beta and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-beta doses. The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-beta induction of Foxp3 in T reg cells (Fig. 5 A). There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-gamma were neutralized (Fig. S6).</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="42" length="49"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="76" length="80"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="186" length="190"></location>
        <text>Runx</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="243" length="248"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="285" length="289"></location>
        <text>Cbfb</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="350" length="357"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="387" length="395"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="426" length="431"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="493" length="497"></location>
        <text>Runx</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="520" length="525"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="560" length="567"></location>
        <text>CBFbeta</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="575" length="578"></location>
        <text>CD4</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="580" length="583"></location>
        <text>CD8</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="598" length="602"></location>
        <text>Cbfb</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="606" length="613"></location>
        <text>CD4-cre</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="626" length="630"></location>
        <text>Cbfb</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>CD4-cre</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="673" length="676"></location>
        <text>CD3</text>
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        <text>28</text>
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        <text>TGF-beta</text>
      </annotation>
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        <text>Foxp3</text>
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        <text>TGF-beta</text>
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        <location offset="897" length="902"></location>
        <text>Foxp3</text>
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        <location offset="906" length="910"></location>
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        <location offset="914" length="921"></location>
        <text>CD4-cre</text>
      </annotation>
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        <location offset="994" length="998"></location>
        <text>Cbfb</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1002" length="1009"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="1040" length="1045"></location>
        <text>Foxp3</text>
      </annotation>
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        <location offset="1077" length="1085"></location>
        <text>TGF-beta</text>
      </annotation>
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        <location offset="1111" length="1114"></location>
        <text>CD4</text>
      </annotation>
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        <location offset="1129" length="1133"></location>
        <text>Cbfb</text>
      </annotation>
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        <location offset="1137" length="1144"></location>
        <text>CD4-cre</text>
      </annotation>
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        <location offset="1157" length="1162"></location>
        <text>Foxp3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1185" length="1193"></location>
        <text>TGF-beta</text>
      </annotation>
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        <location offset="1218" length="1223"></location>
        <text>Foxp3</text>
      </annotation>
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        <location offset="1257" length="1261"></location>
        <text>Cbfb</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1265" length="1272"></location>
        <text>CD4-cre</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1273" length="1276"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="1358" length="1362"></location>
        <text>Runx</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="1390" length="1398"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="1412" length="1417"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="1489" length="1494"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="1511" length="1515"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Protein</infon>
        <location offset="1520" length="1529"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Regulation</infon>
        <location offset="214" length="222"></location>
        <text>involved</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Positive_regulation</infon>
        <location offset="230" length="239"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Negative_regulation</infon>
        <location offset="303" length="314"></location>
        <text>inactivated</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Gene_expression</infon>
        <location offset="334" length="344"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Positive_regulation</infon>
        <location offset="396" length="405"></location>
        <text>synergize</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Gene_expression</infon>
        <location offset="413" length="422"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T73">
        <infon key="type">Positive_regulation</infon>
        <location offset="498" length="506"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T74">
        <infon key="type">Gene_expression</infon>
        <location offset="507" length="516"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T75">
        <infon key="type">Positive_regulation</infon>
        <location offset="529" length="538"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T76">
        <infon key="type">Gene_expression</infon>
        <location offset="546" length="556"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T77">
        <infon key="type">Gene_expression</infon>
        <location offset="868" length="878"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Positive_regulation</infon>
        <location offset="889" length="896"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T79">
        <infon key="type">Negative_regulation</infon>
        <location offset="983" length="990"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Positive_regulation</infon>
        <location offset="1031" length="1039"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Gene_expression</infon>
        <location offset="1046" length="1056"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Gene_expression</infon>
        <location offset="1205" length="1214"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Negative_regulation</infon>
        <location offset="1248" length="1253"></location>
        <text>lower</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Binding</infon>
        <location offset="1363" length="1370"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Regulation</infon>
        <location offset="1378" length="1385"></location>
        <text>affects</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Positive_regulation</infon>
        <location offset="1399" length="1408"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T87">
        <infon key="type">Regulation</infon>
        <location offset="1458" length="1468"></location>
        <text>difference</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="1476" length="1485"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T89">
        <infon key="type">Negative_regulation</infon>
        <location offset="1535" length="1546"></location>
        <text>neutralized</text>
      </annotation>
    </passage>
    <passage>
      <offset>1559</offset>
      <text>The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined. Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2-/- mice. 6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B). There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C). These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.</text>
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        <infon key="type">Protein</infon>
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        <infon key="type">Protein</infon>
        <location offset="1640" length="1643"></location>
        <text>CD4</text>
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      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="1693" length="1697"></location>
        <text>Cbfb</text>
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      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="1704" length="1708"></location>
        <text>Cbfb</text>
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      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="1712" length="1719"></location>
        <text>CD4-cre</text>
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        <location offset="1747" length="1752"></location>
        <text>Foxp3</text>
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        <location offset="1758" length="1761"></location>
        <text>GFP</text>
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        <location offset="1802" length="1806"></location>
        <text>Rag2</text>
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        <infon key="type">Protein</infon>
        <location offset="1828" length="1831"></location>
        <text>CD4</text>
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        <text>Foxp3-GFP</text>
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        <infon key="type">Protein</infon>
        <location offset="2013" length="2016"></location>
        <text>CD4</text>
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        <location offset="2045" length="2050"></location>
        <text>Foxp3</text>
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      <annotation id="T60">
        <infon key="type">Protein</infon>
        <location offset="2135" length="2139"></location>
        <text>Cbfb</text>
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      <annotation id="T61">
        <infon key="type">Protein</infon>
        <location offset="2143" length="2150"></location>
        <text>CD4-cre</text>
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      <annotation id="T62">
        <infon key="type">Protein</infon>
        <location offset="2242" length="2246"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Protein</infon>
        <location offset="2286" length="2289"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
        <location offset="2291" length="2296"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T90">
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        <location offset="1945" length="1955"></location>
        <text>expression</text>
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      <annotation id="T91">
        <infon key="type">Gene_expression</infon>
        <location offset="2051" length="2061"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T92">
        <infon key="type">Positive_regulation</infon>
        <location offset="2226" length="2238"></location>
        <text>significance</text>
      </annotation>
      <annotation id="T93">
        <infon key="type">Gene_expression</infon>
        <location offset="2272" length="2282"></location>
        <text>generation</text>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
      <node refid="T77" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T77" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
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      <infon key="type">Event</infon>
      <node refid="T78" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T79" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T80" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="T32" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T31" role="Theme"></node>
    </relation>
    <relation id="E19">
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      <infon key="type">Event</infon>
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      <node refid="T38" role="Theme"></node>
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    <relation id="E20">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T83" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
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    <relation id="E21">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T84" role="Trigger"></node>
      <node refid="T42" role="Theme"></node>
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    <relation id="E22">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E23">
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      <infon key="type">Event</infon>
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      <node refid="T44" role="Theme"></node>
      <node refid="T43" role="Cause"></node>
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    <relation id="E24">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T87" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="E26" role="Cause"></node>
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    <relation id="E27">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T87" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="E28" role="Cause"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T88" role="Trigger"></node>
      <node refid="T45" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T89" role="Trigger"></node>
      <node refid="T46" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T89" role="Trigger"></node>
      <node refid="T47" role="Theme"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T90" role="Trigger"></node>
      <node refid="T57" role="Theme"></node>
    </relation>
    <relation id="E30">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T91" role="Trigger"></node>
      <node refid="T59" role="Theme"></node>
    </relation>
    <relation id="E31">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T92" role="Trigger"></node>
      <node refid="E32" role="Theme"></node>
      <node refid="T62" role="Cause"></node>
    </relation>
    <relation id="E33">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T92" role="Trigger"></node>
      <node refid="E34" role="Theme"></node>
      <node refid="T62" role="Cause"></node>
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    <relation id="E32">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T93" role="Trigger"></node>
      <node refid="T64" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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    </relation>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
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  </document>
  <document>
    <id>PMC-2806624-06-RESULTS-05</id>
    <passage>
      <offset>0</offset>
      <text>CBFbeta, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells</text>
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        <infon key="type">Protein</infon>
        <location offset="0" length="7"></location>
        <text>CBFbeta</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="9" length="14"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="20" length="25"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="72" length="76"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="78" length="83"></location>
        <text>Foxp3</text>
      </annotation>
    </passage>
    <passage>
      <offset>97</offset>
      <text>Even though Cbfb-deficient CD4+ T cells had impaired induction of Foxp3 after stimulation with anti-CD3/28 mAbs and TGF-beta, sufficient numbers of Foxp3+ cells could be generated to permit analysis of their suppressive activity. Purified naive CD4+ T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice (Cd45.2) harboring a Foxp3-ires-GFP allele (Bettelli et al., 2006) were stimulated in vitro with anti-CD3/28 mAbs, IL-2, and TGF-beta. After 3 d, Foxp3+-GFP+ cells were sorted by flow cytometry and mixed with CFSE-labeled naive CD45.1+ CD4+ cells at ratios of 1:4, 1:2, and 1:1. The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days. CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A). CbfbF/+ CD4-cre CD4+ T cells activated in the presence of TGF-beta showed a clear suppression of T cell proliferation that became even more apparent when an increased ratio of FOXP3+/CD25- cells was used (Fig. 6 B). The suppression was significantly reduced when cells from CbfbF/F CD4-cre mice were used, demonstrating that TGF-beta-induced Runx complexes are important for the suppressive activity of Foxp3+ T reg cells (Fig. 6 B). As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-labeled naive CD45.1+ CD4+ cells. No suppression could be observed in all control groups without TGF-beta at all tested ratios (Fig. 6 C). The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation. Foxp3+ and Foxp3- cells generated from both CbfbF/F CD4-cre and control cells all displayed similar proliferation rates and cell death as measured by CFSE dilution and annexin V staining, respectively (Fig. S7, A and B).</text>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="109" length="113"></location>
        <text>Cbfb</text>
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        <infon key="type">Protein</infon>
        <location offset="124" length="127"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="163" length="168"></location>
        <text>Foxp3</text>
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      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="197" length="200"></location>
        <text>CD3</text>
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        <location offset="201" length="203"></location>
        <text>28</text>
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        <location offset="213" length="221"></location>
        <text>TGF-beta</text>
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        <location offset="245" length="250"></location>
        <text>Foxp3</text>
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        <location offset="342" length="345"></location>
        <text>CD4</text>
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        <location offset="360" length="364"></location>
        <text>Cbfb</text>
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        <location offset="368" length="375"></location>
        <text>CD4-cre</text>
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        <location offset="388" length="392"></location>
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        <infon key="type">Protein</infon>
        <location offset="396" length="403"></location>
        <text>CD4-cre</text>
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        <location offset="410" length="416"></location>
        <text>Cd45.2</text>
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        <location offset="430" length="435"></location>
        <text>Foxp3</text>
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        <location offset="441" length="444"></location>
        <text>GFP</text>
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        <location offset="511" length="514"></location>
        <text>CD3</text>
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        <location offset="515" length="517"></location>
        <text>28</text>
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        <text>Cbfb</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="866" length="873"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="874" length="877"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="916" length="924"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="1034" length="1039"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="1041" length="1045"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="1132" length="1136"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Protein</infon>
        <location offset="1140" length="1147"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Protein</infon>
        <location offset="1183" length="1191"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Protein</infon>
        <location offset="1261" length="1266"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Protein</infon>
        <location offset="1306" length="1310"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="1314" length="1321"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="1322" length="1325"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="1359" length="1367"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="1403" length="1409"></location>
        <text>CD45.1</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="1411" length="1414"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Protein</infon>
        <location offset="1486" length="1494"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Protein</infon>
        <location offset="1566" length="1570"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Protein</infon>
        <location offset="1574" length="1581"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="1661" length="1666"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="1672" length="1677"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="1705" length="1709"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Protein</infon>
        <location offset="1713" length="1720"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Protein</infon>
        <location offset="1829" length="1838"></location>
        <text>annexin V</text>
      </annotation>
      <annotation id="T73">
        <infon key="type">Negative_regulation</infon>
        <location offset="114" length="123"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T74">
        <infon key="type">Negative_regulation</infon>
        <location offset="141" length="149"></location>
        <text>impaired</text>
      </annotation>
      <annotation id="T75">
        <infon key="type">Positive_regulation</infon>
        <location offset="150" length="159"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <passage>
      <offset>1883</offset>
      <text>In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells. We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA. Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb. Cells in which RUNX1 and RUNX3 were knocked down showed markedly lower suppressive activity compared with control iT reg cells at a T reg/CD4+ T responder cell ratio of 1:20, but not when the T reg/CD4+ T responder cell ratio was increased to 1:5 (Fig. 6 D). These results demonstrate the important role of RUNX1 and RUNX3 not only for the induction of FOXP3, but also for the suppressive capacity of iT reg cells both in humans and in mice. The data suggest both quantity and quality of T reg cells are hampered. Reduced intrinsic suppressive capacity of iT reg cells was demonstrated in mice, because Foxp3-GFP+ cells were FACS sorted and same numbers of iT reg cells are included in control experiments. In the suppression experiment with human cells, reduced suppressive activity was caused by reduced FOXP3 expression in T cells by siRNA inhibition of RUNX1 and RUNX3.</text>
      <annotation id="T55">
        <infon key="type">Protein</infon>
        <location offset="1925" length="1930"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Protein</infon>
        <location offset="1935" length="1940"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Protein</infon>
        <location offset="2036" length="2039"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Protein</infon>
        <location offset="2092" length="2097"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T59">
        <infon key="type">Protein</infon>
        <location offset="2102" length="2107"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T60">
        <infon key="type">Protein</infon>
        <location offset="2238" length="2241"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Protein</infon>
        <location offset="2276" length="2279"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T62">
        <infon key="type">Protein</infon>
        <location offset="2300" length="2305"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Protein</infon>
        <location offset="2310" length="2315"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
        <location offset="2423" length="2426"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Protein</infon>
        <location offset="2483" length="2486"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Protein</infon>
        <location offset="2592" length="2597"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Protein</infon>
        <location offset="2602" length="2607"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Protein</infon>
        <location offset="2638" length="2643"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Protein</infon>
        <location offset="2888" length="2897"></location>
        <text>Foxp3-GFP</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Protein</infon>
        <location offset="3091" length="3096"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Protein</infon>
        <location offset="3142" length="3147"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Protein</infon>
        <location offset="3152" length="3157"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T76">
        <infon key="type">Negative_regulation</infon>
        <location offset="2108" length="2113"></location>
        <text>siRNA</text>
      </annotation>
      <annotation id="T77">
        <infon key="type">Negative_regulation</infon>
        <location offset="2321" length="2333"></location>
        <text>knocked down</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Positive_regulation</infon>
        <location offset="2574" length="2583"></location>
        <text>important</text>
      </annotation>
      <annotation id="T79">
        <infon key="type">Gene_expression</infon>
        <location offset="2625" length="2634"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Negative_regulation</infon>
        <location offset="3083" length="3090"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Gene_expression</infon>
        <location offset="3097" length="3107"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Negative_regulation</infon>
        <location offset="3128" length="3138"></location>
        <text>inhibition</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T73" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T74" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T75" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T76" role="Trigger"></node>
      <node refid="T58" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T76" role="Trigger"></node>
      <node refid="T59" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T77" role="Trigger"></node>
      <node refid="T62" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T77" role="Trigger"></node>
      <node refid="T63" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T78" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T66" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T78" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T67" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T79" role="Trigger"></node>
      <node refid="T68" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T80" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="E13" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T80" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="E15" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T81" role="Trigger"></node>
      <node refid="T70" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T82" role="Trigger"></node>
      <node refid="T71" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T82" role="Trigger"></node>
      <node refid="T72" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2806624-07-DISCUSSION</id>
    <passage>
      <offset>0</offset>
      <text>DISCUSSION</text>
    </passage>
    <passage>
      <offset>11</offset>
      <text>This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-beta. TGF-beta mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells. There are three putative RUNX binding sites in the proximal FOXP3 promoter. One binding site was predicted as a binding site for RUNX2. Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites. This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family. The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development. RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003). Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent. The most striking effect was observed when all three binding sites were mutated. We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="40" length="68"></location>
        <text>RUNX transcription factors 1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="73" length="74"></location>
        <text>3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="119" length="124"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="152" length="160"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="170" length="174"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="189" length="194"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="226" length="231"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="236" length="241"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="251" length="254"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="325" length="330"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="394" length="399"></location>
        <text>RUNX2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="460" length="465"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="470" length="475"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="629" length="633"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="766" length="770"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="783" length="787"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="836" length="841"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="907" length="911"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="967" length="971"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1277" length="1282"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T173">
        <infon key="type">Positive_regulation</infon>
        <location offset="161" length="169"></location>
        <text>mediates</text>
      </annotation>
      <annotation id="T174">
        <infon key="type">Gene_expression</infon>
        <location offset="175" length="184"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T175">
        <infon key="type">Positive_regulation</infon>
        <location offset="210" length="222"></location>
        <text>up-regulated</text>
      </annotation>
      <annotation id="T176">
        <infon key="type">Binding</infon>
        <location offset="449" length="456"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T177">
        <infon key="type">Binding</infon>
        <location offset="643" length="647"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T178">
        <infon key="type">Entity</infon>
        <location offset="711" length="722"></location>
        <text>runt domain</text>
      </annotation>
      <annotation id="T179">
        <infon key="type">Positive_regulation</infon>
        <location offset="810" length="817"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T180">
        <infon key="type">Gene_expression</infon>
        <location offset="822" length="832"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T181">
        <infon key="type">Regulation</infon>
        <location offset="861" length="870"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T182">
        <infon key="type">Entity</infon>
        <location offset="1442" length="1455"></location>
        <text>binding sites</text>
      </annotation>
      <annotation id="T183">
        <infon key="type">Binding</infon>
        <location offset="1496" length="1503"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T184">
        <infon key="type">Positive_regulation</infon>
        <location offset="1538" length="1547"></location>
        <text>necessary</text>
      </annotation>
      <annotation id="T185">
        <infon key="type">Entity</infon>
        <location offset="1557" length="1565"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T186">
        <infon key="type">Positive_regulation</infon>
        <location offset="1566" length="1576"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <passage>
      <offset>1579</offset>
      <text>TGF-beta promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells. It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massague, 2003). It was shown that TGF-beta is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007). RUNX transcription factors are targets of the TGF-beta superfamily and they are involved in the TGF-beta pathway. They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003). TGF-beta can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004). It was shown that RUNX2 regulates the expression of TGF-beta type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved. The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-beta inhibition of leukemic cell growth. RUNX3 plays an important role in TGF-beta-mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-beta and hyperproliferation of the gastric mucosa (Blyth et al., 2005). The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1. This could be dependent on the stage of the cells and organ from which they were isolated.</text>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1579" length="1587"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1839" length="1847"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1931" length="1936"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1987" length="1991"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="2195" length="2203"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="2217" length="2221"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="2334" length="2338"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="2386" length="2391"></location>
        <text>RUNX2</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="2420" length="2444"></location>
        <text>TGF-beta type I receptor</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="2555" length="2565"></location>
        <text>RUNX1-EVI1</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="2570" length="2579"></location>
        <text>RUNX1-ETO</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="2586" length="2594"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="2631" length="2636"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="2664" length="2672"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="2729" length="2734"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="2769" length="2777"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="2881" length="2886"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Protein</infon>
        <location offset="2985" length="2990"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T187">
        <infon key="type">Gene_expression</infon>
        <location offset="1705" length="1713"></location>
        <text>produced</text>
      </annotation>
      <annotation id="T188">
        <infon key="type">Positive_regulation</infon>
        <location offset="1851" length="1860"></location>
        <text>mandatory</text>
      </annotation>
      <annotation id="T189">
        <infon key="type">Gene_expression</infon>
        <location offset="1917" length="1927"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T190">
        <infon key="type">Regulation</infon>
        <location offset="2018" length="2025"></location>
        <text>targets</text>
      </annotation>
      <annotation id="T191">
        <infon key="type">Regulation</infon>
        <location offset="2067" length="2075"></location>
        <text>involved</text>
      </annotation>
      <annotation id="T192">
        <infon key="type">Binding</infon>
        <location offset="2106" length="2114"></location>
        <text>interact</text>
      </annotation>
      <annotation id="T193">
        <infon key="type">Positive_regulation</infon>
        <location offset="2208" length="2216"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T194">
        <infon key="type">Transcription</infon>
        <location offset="2235" length="2250"></location>
        <text>transcriptional</text>
      </annotation>
      <annotation id="T195">
        <infon key="type">Positive_regulation</infon>
        <location offset="2303" length="2313"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T196">
        <infon key="type">Regulation</infon>
        <location offset="2392" length="2401"></location>
        <text>regulates</text>
      </annotation>
      <annotation id="T197">
        <infon key="type">Gene_expression</infon>
        <location offset="2406" length="2416"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T198">
        <infon key="type">Negative_regulation</infon>
        <location offset="2721" length="2725"></location>
        <text>loss</text>
      </annotation>
      <annotation id="T199">
        <infon key="type">Gene_expression</infon>
        <location offset="2887" length="2897"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>3084</offset>
      <text>We observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins. For this reason, we decided to use CbfbetaF/F CD4-cre mice. Foxp3 induction by TGF-beta is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice. Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue. It inhibits the IL-6-driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007). We observed an increased number of Foxp3+ cells by retinoic acid and TGF-beta compared with TGF-beta treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice. In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2-/- mice. These data in mice confirm the human data that RUNX proteins play an important role for TGF-beta-dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells. As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-beta and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells. In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.</text>
      <annotation id="T39">
        <infon key="type">Protein</infon>
        <location offset="3137" length="3142"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Protein</infon>
        <location offset="3146" length="3151"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Protein</infon>
        <location offset="3185" length="3190"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="3304" length="3311"></location>
        <text>Cbfbeta</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="3315" length="3322"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="3329" length="3334"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="3348" length="3356"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="3371" length="3374"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Protein</infon>
        <location offset="3387" length="3391"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Protein</infon>
        <location offset="3395" length="3402"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Protein</infon>
        <location offset="3422" length="3426"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="3430" length="3437"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="3556" length="3560"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="3648" length="3653"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Protein</infon>
        <location offset="3725" length="3730"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Protein</infon>
        <location offset="3759" length="3767"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T55">
        <infon key="type">Protein</infon>
        <location offset="3782" length="3790"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Protein</infon>
        <location offset="3810" length="3814"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Protein</infon>
        <location offset="3818" length="3825"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Protein</infon>
        <location offset="3835" length="3839"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T59">
        <infon key="type">Protein</infon>
        <location offset="3843" length="3850"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T60">
        <infon key="type">Protein</infon>
        <location offset="3931" length="3935"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Protein</infon>
        <location offset="3946" length="3949"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T62">
        <infon key="type">Protein</infon>
        <location offset="3962" length="3966"></location>
        <text>Rag2</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Protein</infon>
        <location offset="4023" length="4027"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
        <location offset="4064" length="4072"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Protein</infon>
        <location offset="4083" length="4088"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Protein</infon>
        <location offset="4221" length="4226"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Protein</infon>
        <location offset="4245" length="4250"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Protein</infon>
        <location offset="4297" length="4305"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Protein</infon>
        <location offset="4315" length="4318"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Protein</infon>
        <location offset="4328" length="4332"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Protein</infon>
        <location offset="4362" length="4365"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Protein</infon>
        <location offset="4415" length="4420"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T200">
        <infon key="type">Negative_regulation</infon>
        <location offset="3114" length="3126"></location>
        <text>interference</text>
      </annotation>
      <annotation id="T201">
        <infon key="type">Negative_regulation</infon>
        <location offset="3173" length="3181"></location>
        <text>decrease</text>
      </annotation>
      <annotation id="T202">
        <infon key="type">Positive_regulation</infon>
        <location offset="3203" length="3212"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T203">
        <infon key="type">Positive_regulation</infon>
        <location offset="3335" length="3344"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T204">
        <infon key="type">Negative_regulation</infon>
        <location offset="3360" length="3367"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T205">
        <infon key="type">Positive_regulation</infon>
        <location offset="3705" length="3714"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T206">
        <infon key="type">Negative_regulation</infon>
        <location offset="3936" length="3945"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T207">
        <infon key="type">Positive_regulation</infon>
        <location offset="4045" length="4059"></location>
        <text>important role</text>
      </annotation>
      <annotation id="T208">
        <infon key="type">Positive_regulation</infon>
        <location offset="4073" length="4082"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T209">
        <infon key="type">Gene_expression</infon>
        <location offset="4089" length="4098"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T210">
        <infon key="type">Gene_expression</infon>
        <location offset="4203" length="4217"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T211">
        <infon key="type">Positive_regulation</infon>
        <location offset="4227" length="4234"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T212">
        <infon key="type">Positive_regulation</infon>
        <location offset="4235" length="4244"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T213">
        <infon key="type">Gene_expression</infon>
        <location offset="4259" length="4269"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T214">
        <infon key="type">Positive_regulation</infon>
        <location offset="4282" length="4293"></location>
        <text>requirement</text>
      </annotation>
      <annotation id="T215">
        <infon key="type">Negative_regulation</infon>
        <location offset="4407" length="4414"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T216">
        <infon key="type">Gene_expression</infon>
        <location offset="4421" length="4431"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>4494</offset>
      <text>In a recent study, the role of Runx-CBFbeta was investigated in nT reg cell development in the thymus (Rudra et al., 2009). It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx-CBFbeta complexes. Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study. The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment. Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo. This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007). Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non-T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.</text>
      <annotation id="T73">
        <infon key="type">Protein</infon>
        <location offset="4525" length="4529"></location>
        <text>Runx</text>
      </annotation>
      <annotation id="T74">
        <infon key="type">Protein</infon>
        <location offset="4530" length="4537"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T75">
        <infon key="type">Protein</infon>
        <location offset="4639" length="4644"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T76">
        <infon key="type">Protein</infon>
        <location offset="4707" length="4714"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T77">
        <infon key="type">Protein</infon>
        <location offset="4726" length="4730"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Protein</infon>
        <location offset="4773" length="4778"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T79">
        <infon key="type">Protein</infon>
        <location offset="4841" length="4845"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Protein</infon>
        <location offset="4903" length="4907"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Protein</infon>
        <location offset="4918" length="4925"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Protein</infon>
        <location offset="5026" length="5031"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Protein</infon>
        <location offset="5164" length="5169"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Protein</infon>
        <location offset="5297" length="5302"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Protein</infon>
        <location offset="5390" length="5394"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Protein</infon>
        <location offset="5405" length="5412"></location>
        <text>CD4-cre</text>
      </annotation>
      <annotation id="T87">
        <infon key="type">Protein</infon>
        <location offset="5442" length="5447"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T88">
        <infon key="type">Protein</infon>
        <location offset="5489" length="5493"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T89">
        <infon key="type">Protein</infon>
        <location offset="5550" length="5554"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T90">
        <infon key="type">Protein</infon>
        <location offset="5584" length="5589"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T217">
        <infon key="type">Gene_expression</infon>
        <location offset="4645" length="4655"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T218">
        <infon key="type">Negative_regulation</infon>
        <location offset="4731" length="4740"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T219">
        <infon key="type">Negative_regulation</infon>
        <location offset="4768" length="4772"></location>
        <text>lose</text>
      </annotation>
      <annotation id="T220">
        <infon key="type">Negative_regulation</infon>
        <location offset="4846" length="4855"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T221">
        <infon key="type">Negative_regulation</infon>
        <location offset="4908" length="4917"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T222">
        <infon key="type">Positive_regulation</infon>
        <location offset="5013" length="5022"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T223">
        <infon key="type">Gene_expression</infon>
        <location offset="5032" length="5042"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T224">
        <infon key="type">Positive_regulation</infon>
        <location offset="5154" length="5163"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T225">
        <infon key="type">Positive_regulation</infon>
        <location offset="5303" length="5312"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T226">
        <infon key="type">Negative_regulation</infon>
        <location offset="5367" length="5377"></location>
        <text>diminished</text>
      </annotation>
      <annotation id="T227">
        <infon key="type">Negative_regulation</infon>
        <location offset="5395" length="5404"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T228">
        <infon key="type">Gene_expression</infon>
        <location offset="5428" length="5438"></location>
        <text>generation</text>
      </annotation>
      <annotation id="T229">
        <infon key="type">Positive_regulation</infon>
        <location offset="5455" length="5461"></location>
        <text>caused</text>
      </annotation>
      <annotation id="T230">
        <infon key="type">Negative_regulation</infon>
        <location offset="5494" length="5503"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T231">
        <infon key="type">Negative_regulation</infon>
        <location offset="5555" length="5564"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T232">
        <infon key="type">Positive_regulation</infon>
        <location offset="5590" length="5599"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <passage>
      <offset>5627</offset>
      <text>The involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcon-Riquelme, 2003). A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002). Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003). RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004). These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004). Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation. Runx3/Cbfbeta are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007). Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002). Runx1 regulates the transitions of developing thymocytes from the CD4- CD8- double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007). Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages. In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated. Furthermore, it was shown that Runx1 activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells by binding to their respective promoter. RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-gamma production and up-regulation of T reg cell-associated molecules (Ono et al., 2007).</text>
      <annotation id="T91">
        <infon key="type">Protein</infon>
        <location offset="5646" length="5650"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T92">
        <infon key="type">Protein</infon>
        <location offset="5795" length="5818"></location>
        <text>programmed cell death 1</text>
      </annotation>
      <annotation id="T93">
        <infon key="type">Protein</infon>
        <location offset="5825" length="5831"></location>
        <text>PDCD-1</text>
      </annotation>
      <annotation id="T94">
        <infon key="type">Protein</infon>
        <location offset="5949" length="5954"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T95">
        <infon key="type">Protein</infon>
        <location offset="6170" length="6175"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T96">
        <infon key="type">Protein</infon>
        <location offset="6371" length="6376"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T97">
        <infon key="type">Protein</infon>
        <location offset="6532" length="6537"></location>
        <text>T-bet</text>
      </annotation>
      <annotation id="T98">
        <infon key="type">Protein</infon>
        <location offset="6552" length="6557"></location>
        <text>Runx3</text>
      </annotation>
      <annotation id="T99">
        <infon key="type">Protein</infon>
        <location offset="6594" length="6599"></location>
        <text>Runx3</text>
      </annotation>
      <annotation id="T100">
        <infon key="type">Protein</infon>
        <location offset="6644" length="6649"></location>
        <text>Runx3</text>
      </annotation>
      <annotation id="T101">
        <infon key="type">Protein</infon>
        <location offset="6650" length="6657"></location>
        <text>Cbfbeta</text>
      </annotation>
      <annotation id="T102">
        <infon key="type">Protein</infon>
        <location offset="6702" length="6706"></location>
        <text>Ifng</text>
      </annotation>
      <annotation id="T103">
        <infon key="type">Protein</infon>
        <location offset="6733" length="6736"></location>
        <text>Il4</text>
      </annotation>
      <annotation id="T104">
        <infon key="type">Protein</infon>
        <location offset="6799" length="6803"></location>
        <text>Runx</text>
      </annotation>
      <annotation id="T105">
        <infon key="type">Protein</infon>
        <location offset="6916" length="6921"></location>
        <text>Runx1</text>
      </annotation>
      <annotation id="T106">
        <infon key="type">Protein</infon>
        <location offset="6982" length="6985"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T107">
        <infon key="type">Protein</infon>
        <location offset="6987" length="6990"></location>
        <text>CD8</text>
      </annotation>
      <annotation id="T108">
        <infon key="type">Protein</infon>
        <location offset="7021" length="7024"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T109">
        <infon key="type">Protein</infon>
        <location offset="7026" length="7029"></location>
        <text>CD8</text>
      </annotation>
      <annotation id="T110">
        <infon key="type">Protein</infon>
        <location offset="7133" length="7138"></location>
        <text>Runx1</text>
      </annotation>
      <annotation id="T111">
        <infon key="type">Protein</infon>
        <location offset="7143" length="7148"></location>
        <text>Runx3</text>
      </annotation>
      <annotation id="T112">
        <infon key="type">Protein</infon>
        <location offset="7227" length="7230"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T113">
        <infon key="type">Protein</infon>
        <location offset="7243" length="7246"></location>
        <text>CD8</text>
      </annotation>
      <annotation id="T114">
        <infon key="type">Protein</infon>
        <location offset="7309" length="7314"></location>
        <text>Runx1</text>
      </annotation>
      <annotation id="T115">
        <infon key="type">Protein</infon>
        <location offset="7319" length="7324"></location>
        <text>Runx3</text>
      </annotation>
      <annotation id="T116">
        <infon key="type">Protein</infon>
        <location offset="7407" length="7410"></location>
        <text>CD8</text>
      </annotation>
      <annotation id="T117">
        <infon key="type">Protein</infon>
        <location offset="7459" length="7463"></location>
        <text>Runx</text>
      </annotation>
      <annotation id="T118">
        <infon key="type">Protein</infon>
        <location offset="7706" length="7711"></location>
        <text>Runx1</text>
      </annotation>
      <annotation id="T119">
        <infon key="type">Protein</infon>
        <location offset="7722" length="7726"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T120">
        <infon key="type">Protein</infon>
        <location offset="7731" length="7740"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T121">
        <infon key="type">Protein</infon>
        <location offset="7773" length="7776"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T122">
        <infon key="type">Protein</infon>
        <location offset="7827" length="7832"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T123">
        <infon key="type">Protein</infon>
        <location offset="7859" length="7864"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T124">
        <infon key="type">Protein</infon>
        <location offset="7964" length="7968"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T125">
        <infon key="type">Protein</infon>
        <location offset="7973" length="7982"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T233">
        <infon key="type">Regulation</infon>
        <location offset="5631" length="5642"></location>
        <text>involvement</text>
      </annotation>
      <annotation id="T234">
        <infon key="type">Regulation</infon>
        <location offset="5943" length="5948"></location>
        <text>alter</text>
      </annotation>
      <annotation id="T235">
        <infon key="type">Binding</infon>
        <location offset="5955" length="5962"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T236">
        <infon key="type">Negative_regulation</infon>
        <location offset="6176" length="6185"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T237">
        <infon key="type">Negative_regulation</infon>
        <location offset="6358" length="6367"></location>
        <text>depletion</text>
      </annotation>
      <annotation id="T238">
        <infon key="type">Gene_expression</infon>
        <location offset="6377" length="6387"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T239">
        <infon key="type">Positive_regulation</infon>
        <location offset="6544" length="6551"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T240">
        <infon key="type">Binding</infon>
        <location offset="6580" length="6588"></location>
        <text>partners</text>
      </annotation>
      <annotation id="T241">
        <infon key="type">Positive_regulation</infon>
        <location offset="6667" length="6675"></location>
        <text>required</text>
      </annotation>
      <annotation id="T242">
        <infon key="type">Positive_regulation</infon>
        <location offset="6684" length="6694"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T243">
        <infon key="type">Negative_regulation</infon>
        <location offset="6716" length="6725"></location>
        <text>silencing</text>
      </annotation>
      <annotation id="T244">
        <infon key="type">Negative_regulation</infon>
        <location offset="7149" length="7161"></location>
        <text>deficiencies</text>
      </annotation>
      <annotation id="T245">
        <infon key="type">Negative_regulation</infon>
        <location offset="7288" length="7300"></location>
        <text>inactivation</text>
      </annotation>
      <annotation id="T246">
        <infon key="type">Positive_regulation</infon>
        <location offset="7712" length="7721"></location>
        <text>activates</text>
      </annotation>
      <annotation id="T247">
        <infon key="type">Gene_expression</infon>
        <location offset="7746" length="7756"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T248">
        <infon key="type">Binding</infon>
        <location offset="7789" length="7796"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T249">
        <infon key="type">Binding</infon>
        <location offset="7833" length="7842"></location>
        <text>interacts</text>
      </annotation>
      <annotation id="T250">
        <infon key="type">Positive_regulation</infon>
        <location offset="7929" length="7940"></location>
        <text>responsible</text>
      </annotation>
      <annotation id="T251">
        <infon key="type">Negative_regulation</infon>
        <location offset="7949" length="7960"></location>
        <text>suppression</text>
      </annotation>
      <annotation id="T252">
        <infon key="type">Gene_expression</infon>
        <location offset="7983" length="7993"></location>
        <text>production</text>
      </annotation>
    </passage>
    <passage>
      <offset>8068</offset>
      <text>It has been shown that Foxp3 also influences Th17 differentiation. Specifically, Foxp3 physically interacts with RORgammat, and this interaction inhibits RORgammat function (Zhou et al., 2008). This relationship of RORgammat and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006). Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORgammat to activate transcription (Zhang et al., 2008).</text>
      <annotation id="T126">
        <infon key="type">Protein</infon>
        <location offset="8091" length="8096"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T127">
        <infon key="type">Protein</infon>
        <location offset="8149" length="8154"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T128">
        <infon key="type">Protein</infon>
        <location offset="8181" length="8190"></location>
        <text>RORgammat</text>
      </annotation>
      <annotation id="T129">
        <infon key="type">Protein</infon>
        <location offset="8222" length="8231"></location>
        <text>RORgammat</text>
      </annotation>
      <annotation id="T130">
        <infon key="type">Protein</infon>
        <location offset="8283" length="8292"></location>
        <text>RORgammat</text>
      </annotation>
      <annotation id="T131">
        <infon key="type">Protein</infon>
        <location offset="8297" length="8302"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T132">
        <infon key="type">Protein</infon>
        <location offset="8506" length="8511"></location>
        <text>Runx1</text>
      </annotation>
      <annotation id="T133">
        <infon key="type">Protein</infon>
        <location offset="8547" length="8551"></location>
        <text>Il17</text>
      </annotation>
      <annotation id="T134">
        <infon key="type">Protein</infon>
        <location offset="8595" length="8604"></location>
        <text>RORgammat</text>
      </annotation>
      <annotation id="T253">
        <infon key="type">Binding</infon>
        <location offset="8166" length="8175"></location>
        <text>interacts</text>
      </annotation>
      <annotation id="T254">
        <infon key="type">Negative_regulation</infon>
        <location offset="8213" length="8221"></location>
        <text>inhibits</text>
      </annotation>
      <annotation id="T255">
        <infon key="type">Regulation</infon>
        <location offset="8536" length="8546"></location>
        <text>regulating</text>
      </annotation>
      <annotation id="T256">
        <infon key="type">Transcription</infon>
        <location offset="8552" length="8565"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>8654</offset>
      <text>The Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005). Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007). All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role. We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3. RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-beta pathway is of particular interest.</text>
      <annotation id="T135">
        <infon key="type">Protein</infon>
        <location offset="8658" length="8663"></location>
        <text>Runx3</text>
      </annotation>
      <annotation id="T136">
        <infon key="type">Protein</infon>
        <location offset="8789" length="8793"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T137">
        <infon key="type">Protein</infon>
        <location offset="8797" length="8800"></location>
        <text>Cd4</text>
      </annotation>
      <annotation id="T138">
        <infon key="type">Protein</infon>
        <location offset="9164" length="9169"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T139">
        <infon key="type">Protein</infon>
        <location offset="9200" length="9205"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T140">
        <infon key="type">Protein</infon>
        <location offset="9210" length="9215"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T141">
        <infon key="type">Protein</infon>
        <location offset="9217" length="9221"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T142">
        <infon key="type">Protein</infon>
        <location offset="9338" length="9346"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T257">
        <infon key="type">Negative_regulation</infon>
        <location offset="8664" length="8673"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T258">
        <infon key="type">Gene_expression</infon>
        <location offset="9170" length="9179"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T259">
        <infon key="type">Binding</infon>
        <location offset="9317" length="9328"></location>
        <text>interaction</text>
      </annotation>
    </passage>
    <passage>
      <offset>9383</offset>
      <text>Foxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-kappaB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008). NFAT forms a complex with AP-1 and NF-kappaB and regulates the expression of IL-2, IL-4, IFN-gamma, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007). The NFAT-AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006). It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-beta response element was identified in the Foxp3 gene or in the surrounding regions. The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24-48 h after stimulation. The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006). RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008). Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.</text>
      <annotation id="T143">
        <infon key="type">Protein</infon>
        <location offset="9383" length="9388"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T144">
        <infon key="type">Protein</infon>
        <location offset="9421" length="9425"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T145">
        <infon key="type">Protein</infon>
        <location offset="9568" length="9573"></location>
        <text>TIP60</text>
      </annotation>
      <annotation id="T146">
        <infon key="type">Protein</infon>
        <location offset="9719" length="9723"></location>
        <text>AP-1</text>
      </annotation>
      <annotation id="T147">
        <infon key="type">Protein</infon>
        <location offset="9770" length="9774"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T148">
        <infon key="type">Protein</infon>
        <location offset="9776" length="9780"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T149">
        <infon key="type">Protein</infon>
        <location offset="9782" length="9791"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T150">
        <infon key="type">Protein</infon>
        <location offset="9797" length="9802"></location>
        <text>CTLA4</text>
      </annotation>
      <annotation id="T151">
        <infon key="type">Protein</infon>
        <location offset="9949" length="9953"></location>
        <text>AP-1</text>
      </annotation>
      <annotation id="T152">
        <infon key="type">Protein</infon>
        <location offset="9980" length="9985"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T153">
        <infon key="type">Protein</infon>
        <location offset="10111" length="10116"></location>
        <text>Smad3</text>
      </annotation>
      <annotation id="T154">
        <infon key="type">Protein</infon>
        <location offset="10137" length="10142"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T155">
        <infon key="type">Protein</infon>
        <location offset="10251" length="10256"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T156">
        <infon key="type">Protein</infon>
        <location offset="10318" length="10323"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T157">
        <infon key="type">Protein</infon>
        <location offset="10328" length="10333"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T158">
        <infon key="type">Protein</infon>
        <location offset="10399" length="10404"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T159">
        <infon key="type">Protein</infon>
        <location offset="10481" length="10486"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T160">
        <infon key="type">Protein</infon>
        <location offset="10549" length="10554"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T161">
        <infon key="type">Protein</infon>
        <location offset="10632" length="10637"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T162">
        <infon key="type">Protein</infon>
        <location offset="10687" length="10691"></location>
        <text>p300</text>
      </annotation>
      <annotation id="T163">
        <infon key="type">Protein</infon>
        <location offset="10696" length="10700"></location>
        <text>CREB</text>
      </annotation>
      <annotation id="T164">
        <infon key="type">Protein</infon>
        <location offset="10784" length="10788"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T165">
        <infon key="type">Protein</infon>
        <location offset="10882" length="10887"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T166">
        <infon key="type">Protein</infon>
        <location offset="10891" length="10896"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T167">
        <infon key="type">Protein</infon>
        <location offset="10909" length="10914"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T260">
        <infon key="type">Binding</infon>
        <location offset="9397" length="9406"></location>
        <text>interacts</text>
      </annotation>
      <annotation id="T261">
        <infon key="type">Binding</infon>
        <location offset="9698" length="9713"></location>
        <text>forms a complex</text>
      </annotation>
      <annotation id="T262">
        <infon key="type">Regulation</infon>
        <location offset="9742" length="9751"></location>
        <text>regulates</text>
      </annotation>
      <annotation id="T263">
        <infon key="type">Gene_expression</infon>
        <location offset="9756" length="9766"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T264">
        <infon key="type">Binding</infon>
        <location offset="9967" length="9972"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T265">
        <infon key="type">Entity</infon>
        <location offset="9986" length="9994"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T266">
        <infon key="type">Positive_regulation</infon>
        <location offset="10020" length="10029"></location>
        <text>regulates</text>
      </annotation>
      <annotation id="T267">
        <infon key="type">Gene_expression</infon>
        <location offset="10039" length="10049"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T268">
        <infon key="type">Positive_regulation</infon>
        <location offset="10130" length="10136"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T269">
        <infon key="type">Gene_expression</infon>
        <location offset="10143" length="10153"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T270">
        <infon key="type">Positive_regulation</infon>
        <location offset="10305" length="10314"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T271">
        <infon key="type">Binding</infon>
        <location offset="10353" length="10360"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T272">
        <infon key="type">Entity</infon>
        <location offset="10405" length="10413"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T273">
        <infon key="type">Transcription</infon>
        <location offset="10468" length="10477"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T274">
        <infon key="type">Binding</infon>
        <location offset="10534" length="10545"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T275">
        <infon key="type">Regulation</infon>
        <location offset="10567" length="10576"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T276">
        <infon key="type">Binding</infon>
        <location offset="10598" length="10605"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T277">
        <infon key="type">Binding</infon>
        <location offset="10666" length="10677"></location>
        <text>interacting</text>
      </annotation>
      <annotation id="T278">
        <infon key="type">Binding</infon>
        <location offset="10701" length="10708"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T279">
        <infon key="type">Positive_regulation</infon>
        <location offset="10771" length="10779"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T280">
        <infon key="type">Entity</infon>
        <location offset="10789" length="10797"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T281">
        <infon key="type">Positive_regulation</infon>
        <location offset="10900" length="10908"></location>
        <text>activate</text>
      </annotation>
    </passage>
    <passage>
      <offset>10981</offset>
      <text>In conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function. The induction of the transcription factors RUNX1 and RUNX3 by TGF-beta and the subsequent up-regulation of Foxp3 play a role in iT reg cell generation and its suppressive capacity.</text>
      <annotation id="T168">
        <infon key="type">Protein</infon>
        <location offset="11031" length="11035"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T169">
        <infon key="type">Protein</infon>
        <location offset="11129" length="11134"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T170">
        <infon key="type">Protein</infon>
        <location offset="11139" length="11144"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T171">
        <infon key="type">Protein</infon>
        <location offset="11148" length="11156"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T172">
        <infon key="type">Protein</infon>
        <location offset="11193" length="11198"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T282">
        <infon key="type">Positive_regulation</infon>
        <location offset="11090" length="11099"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T283">
        <infon key="type">Positive_regulation</infon>
        <location offset="11176" length="11189"></location>
        <text>up-regulation</text>
      </annotation>
    </passage>
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      <infon key="type">Equiv</infon>
      <node refid="T92" role="Event"></node>
      <node refid="T93" role="Event"></node>
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    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T173" role="Trigger"></node>
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      <node refid="T4" role="Cause"></node>
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    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T174" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T175" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T7" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T175" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T176" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
      <node refid="T12" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T176" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
      <node refid="T13" role="Theme2"></node>
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    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T177" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
      <node refid="T178" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T179" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
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    <relation id="E9">
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      <infon key="type">Event</infon>
      <node refid="T180" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
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    <relation id="E10">
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      <infon key="type">Event</infon>
      <node refid="T181" role="Trigger"></node>
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      <infon key="type">Event</infon>
      <node refid="T183" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
      <node refid="T182" role="Site"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T184" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
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    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T186" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
      <node refid="T185" role="Site"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T187" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T188" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
      <node refid="T22" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T189" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T190" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T191" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T192" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T193" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T194" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T195" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T196" role="Trigger"></node>
      <node refid="E24" role="Theme"></node>
      <node refid="T28" role="Cause"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T197" role="Trigger"></node>
      <node refid="T29" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T198" role="Trigger"></node>
      <node refid="T35" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T199" role="Trigger"></node>
      <node refid="T37" role="Theme"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T199" role="Trigger"></node>
      <node refid="T38" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T200" role="Trigger"></node>
      <node refid="T39" role="Theme"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T200" role="Trigger"></node>
      <node refid="T40" role="Theme"></node>
    </relation>
    <relation id="E30">
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      <infon key="type">Event</infon>
      <node refid="T201" role="Trigger"></node>
      <node refid="E31" role="Theme"></node>
      <node refid="E28" role="Cause"></node>
    </relation>
    <relation id="E32">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T201" role="Trigger"></node>
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      <node refid="E29" role="Cause"></node>
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    <relation id="E31">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T202" role="Trigger"></node>
      <node refid="T41" role="Theme"></node>
    </relation>
    <relation id="E33">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T203" role="Trigger"></node>
      <node refid="T44" role="Theme"></node>
      <node refid="T45" role="Cause"></node>
    </relation>
    <relation id="E34">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T204" role="Trigger"></node>
      <node refid="E33" role="Theme"></node>
    </relation>
    <relation id="E35">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T205" role="Trigger"></node>
      <node refid="T53" role="Theme"></node>
      <node refid="T54" role="Cause"></node>
    </relation>
    <relation id="E36">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T205" role="Trigger"></node>
      <node refid="T53" role="Theme"></node>
      <node refid="T55" role="Cause"></node>
    </relation>
    <relation id="E37">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T206" role="Trigger"></node>
      <node refid="T60" role="Theme"></node>
    </relation>
    <relation id="E38">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T207" role="Trigger"></node>
      <node refid="E39" role="Theme"></node>
      <node refid="T63" role="Cause"></node>
    </relation>
    <relation id="E40">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T208" role="Trigger"></node>
      <node refid="E39" role="Theme"></node>
      <node refid="T64" role="Cause"></node>
    </relation>
    <relation id="E39">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T209" role="Trigger"></node>
      <node refid="T65" role="Theme"></node>
    </relation>
    <relation id="E41">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T210" role="Trigger"></node>
      <node refid="T66" role="Theme"></node>
    </relation>
    <relation id="E42">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T211" role="Trigger"></node>
      <node refid="E43" role="Theme"></node>
      <node refid="T66" role="Cause"></node>
    </relation>
    <relation id="E43">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T212" role="Trigger"></node>
      <node refid="E44" role="Theme"></node>
    </relation>
    <relation id="E44">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T213" role="Trigger"></node>
      <node refid="T67" role="Theme"></node>
    </relation>
    <relation id="E45">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T214" role="Trigger"></node>
      <node refid="E42" role="Theme"></node>
      <node refid="T68" role="Cause"></node>
    </relation>
    <relation id="E46">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T215" role="Trigger"></node>
      <node refid="E47" role="Theme"></node>
    </relation>
    <relation id="E47">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T216" role="Trigger"></node>
      <node refid="T72" role="Theme"></node>
    </relation>
    <relation id="E48">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T217" role="Trigger"></node>
      <node refid="T75" role="Theme"></node>
    </relation>
    <relation id="E49">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T218" role="Trigger"></node>
      <node refid="T77" role="Theme"></node>
    </relation>
    <relation id="E50">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T219" role="Trigger"></node>
      <node refid="T78" role="Theme"></node>
      <node refid="E49" role="Cause"></node>
    </relation>
    <relation id="E51">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T220" role="Trigger"></node>
      <node refid="T79" role="Theme"></node>
    </relation>
    <relation id="E52">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T221" role="Trigger"></node>
      <node refid="T80" role="Theme"></node>
    </relation>
    <relation id="E53">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T222" role="Trigger"></node>
      <node refid="E54" role="Theme"></node>
    </relation>
    <relation id="E54">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T223" role="Trigger"></node>
      <node refid="T82" role="Theme"></node>
    </relation>
    <relation id="E55">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T224" role="Trigger"></node>
      <node refid="T83" role="Theme"></node>
    </relation>
    <relation id="E56">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T225" role="Trigger"></node>
      <node refid="T84" role="Theme"></node>
    </relation>
    <relation id="E57">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T226" role="Trigger"></node>
      <node refid="E58" role="Theme"></node>
    </relation>
    <relation id="E59">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T227" role="Trigger"></node>
      <node refid="T85" role="Theme"></node>
    </relation>
    <relation id="E58">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T228" role="Trigger"></node>
      <node refid="T87" role="Theme"></node>
    </relation>
    <relation id="E60">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T229" role="Trigger"></node>
      <node refid="E57" role="Theme"></node>
    </relation>
    <relation id="E61">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T230" role="Trigger"></node>
      <node refid="T88" role="Theme"></node>
    </relation>
    <relation id="E62">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T231" role="Trigger"></node>
      <node refid="T89" role="Theme"></node>
    </relation>
    <relation id="E63">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T232" role="Trigger"></node>
      <node refid="T90" role="Theme"></node>
    </relation>
    <relation id="E64">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T233" role="Trigger"></node>
      <node refid="T91" role="Theme"></node>
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    <relation id="E65">
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      <infon key="type">Event</infon>
      <node refid="T234" role="Trigger"></node>
      <node refid="E66" role="Theme"></node>
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    <relation id="E66">
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      <infon key="type">Event</infon>
      <node refid="T235" role="Trigger"></node>
      <node refid="T94" role="Theme"></node>
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    <relation id="E67">
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      <infon key="type">Event</infon>
      <node refid="T236" role="Trigger"></node>
      <node refid="T95" role="Theme"></node>
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    <relation id="E68">
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      <infon key="type">Event</infon>
      <node refid="T237" role="Trigger"></node>
      <node refid="E69" role="Theme"></node>
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    <relation id="E69">
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      <infon key="type">Event</infon>
      <node refid="T238" role="Trigger"></node>
      <node refid="T96" role="Theme"></node>
    </relation>
    <relation id="E70">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T239" role="Trigger"></node>
      <node refid="T98" role="Theme"></node>
      <node refid="T97" role="Cause"></node>
    </relation>
    <relation id="E71">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T240" role="Trigger"></node>
      <node refid="T97" role="Theme"></node>
      <node refid="T99" role="Theme2"></node>
    </relation>
    <relation id="E72">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T241" role="Trigger"></node>
      <node refid="E73" role="Theme"></node>
      <node refid="T100" role="Cause"></node>
    </relation>
    <relation id="E74">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T241" role="Trigger"></node>
      <node refid="E73" role="Theme"></node>
      <node refid="T101" role="Cause"></node>
    </relation>
    <relation id="E75">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T241" role="Trigger"></node>
      <node refid="E76" role="Theme"></node>
      <node refid="T100" role="Cause"></node>
    </relation>
    <relation id="E77">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T241" role="Trigger"></node>
      <node refid="E76" role="Theme"></node>
      <node refid="T101" role="Cause"></node>
    </relation>
    <relation id="E73">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T242" role="Trigger"></node>
      <node refid="T102" role="Theme"></node>
    </relation>
    <relation id="E76">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T243" role="Trigger"></node>
      <node refid="T103" role="Theme"></node>
    </relation>
    <relation id="E78">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T244" role="Trigger"></node>
      <node refid="T110" role="Theme"></node>
    </relation>
    <relation id="E79">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T244" role="Trigger"></node>
      <node refid="T111" role="Theme"></node>
    </relation>
    <relation id="E80">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T245" role="Trigger"></node>
      <node refid="T114" role="Theme"></node>
    </relation>
    <relation id="E81">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T245" role="Trigger"></node>
      <node refid="T115" role="Theme"></node>
    </relation>
    <relation id="E82">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T246" role="Trigger"></node>
      <node refid="E83" role="Theme"></node>
      <node refid="E84" role="Cause"></node>
    </relation>
    <relation id="E85">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T246" role="Trigger"></node>
      <node refid="E86" role="Theme"></node>
      <node refid="E84" role="Cause"></node>
    </relation>
    <relation id="E83">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T247" role="Trigger"></node>
      <node refid="T119" role="Theme"></node>
    </relation>
    <relation id="E86">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T247" role="Trigger"></node>
      <node refid="T120" role="Theme"></node>
    </relation>
    <relation id="E84">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T248" role="Trigger"></node>
      <node refid="T118" role="Theme"></node>
    </relation>
    <relation id="E87">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T249" role="Trigger"></node>
      <node refid="T122" role="Theme"></node>
      <node refid="T123" role="Theme2"></node>
    </relation>
    <relation id="E88">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T250" role="Trigger"></node>
      <node refid="E89" role="Theme"></node>
      <node refid="E87" role="Cause"></node>
    </relation>
    <relation id="E90">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T250" role="Trigger"></node>
      <node refid="E91" role="Theme"></node>
      <node refid="E87" role="Cause"></node>
    </relation>
    <relation id="E89">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T251" role="Trigger"></node>
      <node refid="E92" role="Theme"></node>
    </relation>
    <relation id="E91">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T251" role="Trigger"></node>
      <node refid="E93" role="Theme"></node>
    </relation>
    <relation id="E92">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T252" role="Trigger"></node>
      <node refid="T124" role="Theme"></node>
    </relation>
    <relation id="E93">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T252" role="Trigger"></node>
      <node refid="T125" role="Theme"></node>
    </relation>
    <relation id="E94">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T253" role="Trigger"></node>
      <node refid="T127" role="Theme"></node>
      <node refid="T128" role="Theme2"></node>
    </relation>
    <relation id="E95">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T254" role="Trigger"></node>
      <node refid="T129" role="Theme"></node>
      <node refid="E94" role="Cause"></node>
    </relation>
    <relation id="E96">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T255" role="Trigger"></node>
      <node refid="E97" role="Theme"></node>
      <node refid="T132" role="Cause"></node>
    </relation>
    <relation id="E97">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T256" role="Trigger"></node>
      <node refid="T133" role="Theme"></node>
    </relation>
    <relation id="E98">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T257" role="Trigger"></node>
      <node refid="T135" role="Theme"></node>
    </relation>
    <relation id="E99">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T258" role="Trigger"></node>
      <node refid="T138" role="Theme"></node>
    </relation>
    <relation id="E100">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T259" role="Trigger"></node>
      <node refid="T141" role="Theme"></node>
      <node refid="T142" role="Theme2"></node>
    </relation>
    <relation id="E101">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T260" role="Trigger"></node>
      <node refid="T143" role="Theme"></node>
      <node refid="T144" role="Theme2"></node>
    </relation>
    <relation id="E102">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T260" role="Trigger"></node>
      <node refid="T143" role="Theme"></node>
      <node refid="T145" role="Theme2"></node>
    </relation>
    <relation id="E103">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T261" role="Trigger"></node>
      <node refid="T146" role="Theme"></node>
    </relation>
    <relation id="E104">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T262" role="Trigger"></node>
      <node refid="E105" role="Theme"></node>
      <node refid="E103" role="Cause"></node>
    </relation>
    <relation id="E106">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T262" role="Trigger"></node>
      <node refid="E107" role="Theme"></node>
      <node refid="E103" role="Cause"></node>
    </relation>
    <relation id="E108">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T262" role="Trigger"></node>
      <node refid="E109" role="Theme"></node>
      <node refid="E103" role="Cause"></node>
    </relation>
    <relation id="E110">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T262" role="Trigger"></node>
      <node refid="E111" role="Theme"></node>
      <node refid="E103" role="Cause"></node>
    </relation>
    <relation id="E105">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T263" role="Trigger"></node>
      <node refid="T147" role="Theme"></node>
    </relation>
    <relation id="E107">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T263" role="Trigger"></node>
      <node refid="T148" role="Theme"></node>
    </relation>
    <relation id="E109">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T263" role="Trigger"></node>
      <node refid="T149" role="Theme"></node>
    </relation>
    <relation id="E111">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T263" role="Trigger"></node>
      <node refid="T150" role="Theme"></node>
    </relation>
    <relation id="E112">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T264" role="Trigger"></node>
      <node refid="T151" role="Theme"></node>
      <node refid="T152" role="Theme2"></node>
      <node refid="T265" role="Site2"></node>
    </relation>
    <relation id="E113">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T266" role="Trigger"></node>
      <node refid="E114" role="Theme"></node>
      <node refid="E112" role="Cause"></node>
    </relation>
    <relation id="E114">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T267" role="Trigger"></node>
      <node refid="T152" role="Theme"></node>
    </relation>
    <relation id="E115">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T268" role="Trigger"></node>
      <node refid="E116" role="Theme"></node>
      <node refid="T153" role="Cause"></node>
    </relation>
    <relation id="E116">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T269" role="Trigger"></node>
      <node refid="T154" role="Theme"></node>
    </relation>
    <relation id="E117">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T270" role="Trigger"></node>
      <node refid="T156" role="Theme"></node>
    </relation>
    <relation id="E118">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T270" role="Trigger"></node>
      <node refid="T157" role="Theme"></node>
    </relation>
    <relation id="E119">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T271" role="Trigger"></node>
      <node refid="T156" role="Theme"></node>
      <node refid="T158" role="Theme2"></node>
      <node refid="T272" role="Site2"></node>
    </relation>
    <relation id="E120">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T271" role="Trigger"></node>
      <node refid="T157" role="Theme"></node>
      <node refid="T158" role="Theme2"></node>
      <node refid="T272" role="Site2"></node>
    </relation>
    <relation id="E121">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T273" role="Trigger"></node>
      <node refid="T159" role="Theme"></node>
    </relation>
    <relation id="E122">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T274" role="Trigger"></node>
      <node refid="T160" role="Theme"></node>
    </relation>
    <relation id="E123">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T275" role="Trigger"></node>
      <node refid="E122" role="Theme"></node>
      <node refid="E124" role="Cause"></node>
    </relation>
    <relation id="E124">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T276" role="Trigger"></node>
      <node refid="T160" role="Theme"></node>
    </relation>
    <relation id="E125">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T277" role="Trigger"></node>
      <node refid="T161" role="Theme"></node>
      <node refid="T162" role="Theme2"></node>
    </relation>
    <relation id="E126">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T278" role="Trigger"></node>
      <node refid="T163" role="Theme"></node>
    </relation>
    <relation id="E127">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T279" role="Trigger"></node>
      <node refid="T164" role="Theme"></node>
      <node refid="T161" role="Cause"></node>
      <node refid="T280" role="Site"></node>
    </relation>
    <relation id="E128">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T279" role="Trigger"></node>
      <node refid="T164" role="Theme"></node>
      <node refid="T162" role="Cause"></node>
      <node refid="T280" role="Site"></node>
    </relation>
    <relation id="E129">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T281" role="Trigger"></node>
      <node refid="T167" role="Theme"></node>
    </relation>
    <relation id="E130">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T282" role="Trigger"></node>
      <node refid="T169" role="Theme"></node>
      <node refid="T171" role="Cause"></node>
    </relation>
    <relation id="E131">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T282" role="Trigger"></node>
      <node refid="T170" role="Theme"></node>
      <node refid="T171" role="Cause"></node>
    </relation>
    <relation id="E132">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T283" role="Trigger"></node>
      <node refid="T172" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E45" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E60" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E64" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E88" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E90" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E96" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E127" role="Event"></node>
    </relation>
    <relation id="M8">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E128" role="Event"></node>
    </relation>
    <relation id="M9">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E129" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2806624-08-MATERIALS_AND_METHODS-01</id>
    <passage>
      <offset>0</offset>
      <text>Mice.</text>
    </passage>
    <passage>
      <offset>6</offset>
      <text>CbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007). Cd45.1 and Rag2-/- mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively. For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice. 5 x 106 cells were used per transfer. 6 wk later, TCRbeta+CD4+ gated cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine were analyzed for Foxp3-GFP expression. All analyses and experiments were performed on animals at 6-8 wk of age. Animals were housed under specific pathogen-free conditions at the animal facility of the Skirball Institute, and experiments were performed in accordance with approved protocols for the New York University Institutional Animal Care and Usage Committee.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="6" length="10"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="14" length="17"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="18" length="21"></location>
        <text>cre</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="26" length="31"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="31" length="34"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="115" length="121"></location>
        <text>Cd45.1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="126" length="130"></location>
        <text>Rag2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="238" length="243"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="268" length="271"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="286" length="290"></location>
        <text>Cbfb</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="291" length="294"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="295" length="298"></location>
        <text>cre</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="328" length="333"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="339" length="342"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="384" length="387"></location>
        <text>Rag</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="454" length="461"></location>
        <text>TCRbeta</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="462" length="465"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="585" length="594"></location>
        <text>Foxp3-GFP</text>
      </annotation>
      <annotation id="T253">
        <infon key="type">Negative_regulation</infon>
        <location offset="388" length="397"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T254">
        <infon key="type">Gene_expression</infon>
        <location offset="595" length="605"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>935</offset>
      <text>Isolation of PBMCs, CD4+ T cells, and culture conditions.</text>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="955" length="958"></location>
        <text>CD4</text>
      </annotation>
    </passage>
    <passage>
      <offset>993</offset>
      <text>Human PBMCs were isolated by Ficoll (Biochrom) density gradient centrifugation and CD4+ T cells were then isolated using the Dynal CD4+ Isolation kit (Invitrogen) according to the manufacturer's instructions. The purity of CD4+ T cells was initially tested by flow cytometry and was &gt;/=95%. Cells were stimulated with the following combination of mAbs to T cell surface molecules (Meiler et al., 2008): anti-CD2 (clone 4B2 and 6G4; 0.5 microg/ml), anti-CD3 (clone OKT3; 0.5 microg/ml), and anti-CD28 mAb (clone B7G5; 0.5 microg/ml; all from Sanquin) and cultured in serum-free AIM -V medium (Life Technologies) with the addition of 1 nmol/liter IL-2 (Roche). TGF-beta (R&amp;D Systems) was used at 5 ng/ml, if not stated otherwise. A combination of PMA (25 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich) was used.</text>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1076" length="1079"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1124" length="1127"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1139" length="1142"></location>
        <text>kit</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1216" length="1219"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1401" length="1404"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1446" length="1449"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1488" length="1492"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1638" length="1642"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1652" length="1660"></location>
        <text>TGF-beta</text>
      </annotation>
    </passage>
    <passage>
      <offset>1803</offset>
      <text>Human CD4+ CD127- CD25high and CD4+ CD127+ CD25neg cells were purified by flow cytometry using anti-CD127, anti-CD25-PC5 (Beckman Coulter), and anti-CD4-FITC antibodies (Dako).</text>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="1809" length="1812"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="1814" length="1819"></location>
        <text>CD127</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="1821" length="1825"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="1834" length="1837"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="1839" length="1844"></location>
        <text>CD127</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="1846" length="1850"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="1903" length="1908"></location>
        <text>CD127</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="1915" length="1919"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="1952" length="1955"></location>
        <text>CD4</text>
      </annotation>
    </passage>
    <passage>
      <offset>1981</offset>
      <text>Mouse naive (CD62Lhi44lo25-) CD4+ T cells were purified by flow cytometry and activated in vitro with 5 microg/ml plate-bound anti-CD3 and 1 microg/ml soluble anti-CD28 antibodies (eBioscience) in RPMI supplemented with 10% FCS, 5 mM beta-mercaptoethanol, and antibiotics. Neutralizing anti-IFN-gamma and anti-IL-4 mAbs (BD) were used at 1 microg/ml concentrations when indicated.</text>
      <annotation id="T38">
        <infon key="type">Protein</infon>
        <location offset="1994" length="1999"></location>
        <text>CD62L</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Protein</infon>
        <location offset="2001" length="2003"></location>
        <text>44</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Protein</infon>
        <location offset="2005" length="2007"></location>
        <text>25</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Protein</infon>
        <location offset="2010" length="2013"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Protein</infon>
        <location offset="2112" length="2115"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Protein</infon>
        <location offset="2145" length="2149"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Protein</infon>
        <location offset="2272" length="2281"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Protein</infon>
        <location offset="2291" length="2295"></location>
        <text>IL-4</text>
      </annotation>
    </passage>
    <passage>
      <offset>2363</offset>
      <text>In vitro T cell differentiation.</text>
    </passage>
    <passage>
      <offset>2396</offset>
      <text>CD4+ CD45RA+ magnetically sorted (CD45RO depletion with AutoMACS; Miltenyi Biotec) cells were stimulated with immobilized plate-bound anti-CD3 (1 microg/ml; OKT3; IgG1) and anti-CD28 (2 microg/ml). For Th1 differentiation conditions, cells were stimulated with the following: 40 ng/ml IL-2, 5 microg/ml anti-IL-4, and 25 ng/ml IL-12 (R&amp;D Systems). For Th2 conditions, cells were stimulated with the following: 40 ng/ml IL-2, 25 ng/ml IL-4, and 5 microg/ml anti-IL-12 (R&amp;D Systems). For T reg cell conditions, cells were stimulated with the following: 40 ng/ml IL-2, 5 ng/ml TGF-beta, 5 microg/ml anti-IL-12, 5 microg/ml anti-IL-4. For Th17 conditions, cells were stimulated with the following: 40 ng/ml IL-2, 20 ng/ml IL-6, 5 ng/ml TGF-beta, 20 ng/ml IL-23 (Alexis Biochemicals Corp.), 10 ng/ml IL-1beta, 5 microg/ml anti-IL-4, and 5 microg/ml anti-IL-12 were used. Proliferating cells were expanded in medium containing IL-2. The cytokine profile of these cells demonstrated that IFN-gamma is the predominant cytokine in Th1 cells, IL-4 and IL-13 in Th2 cells, and IL-17 in Th17 cells (Akdis et al., 2000; Burgler et al., 2009).</text>
      <annotation id="T46">
        <infon key="type">Protein</infon>
        <location offset="2396" length="2399"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Protein</infon>
        <location offset="2401" length="2407"></location>
        <text>CD45RA</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Protein</infon>
        <location offset="2430" length="2436"></location>
        <text>CD45RO</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Protein</infon>
        <location offset="2535" length="2538"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Protein</infon>
        <location offset="2574" length="2578"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Protein</infon>
        <location offset="2681" length="2685"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Protein</infon>
        <location offset="2704" length="2708"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Protein</infon>
        <location offset="2723" length="2728"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Protein</infon>
        <location offset="2815" length="2819"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T55">
        <infon key="type">Protein</infon>
        <location offset="2830" length="2834"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Protein</infon>
        <location offset="2857" length="2862"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Protein</infon>
        <location offset="2956" length="2960"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T58">
        <infon key="type">Protein</infon>
        <location offset="2970" length="2978"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T59">
        <infon key="type">Protein</infon>
        <location offset="2997" length="3002"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T60">
        <infon key="type">Protein</infon>
        <location offset="3021" length="3025"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T61">
        <infon key="type">Protein</infon>
        <location offset="3099" length="3103"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T62">
        <infon key="type">Protein</infon>
        <location offset="3114" length="3118"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T63">
        <infon key="type">Protein</infon>
        <location offset="3128" length="3136"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T64">
        <infon key="type">Protein</infon>
        <location offset="3147" length="3152"></location>
        <text>IL-23</text>
      </annotation>
      <annotation id="T65">
        <infon key="type">Protein</infon>
        <location offset="3191" length="3199"></location>
        <text>IL-1beta</text>
      </annotation>
      <annotation id="T66">
        <infon key="type">Protein</infon>
        <location offset="3218" length="3222"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T67">
        <infon key="type">Protein</infon>
        <location offset="3245" length="3250"></location>
        <text>IL-12</text>
      </annotation>
      <annotation id="T68">
        <infon key="type">Protein</infon>
        <location offset="3317" length="3321"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T69">
        <infon key="type">Protein</infon>
        <location offset="3377" length="3386"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T70">
        <infon key="type">Protein</infon>
        <location offset="3429" length="3433"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T71">
        <infon key="type">Protein</infon>
        <location offset="3438" length="3443"></location>
        <text>IL-13</text>
      </annotation>
      <annotation id="T72">
        <infon key="type">Protein</infon>
        <location offset="3462" length="3467"></location>
        <text>IL-17</text>
      </annotation>
      <annotation id="T255">
        <infon key="type">Negative_regulation</infon>
        <location offset="2437" length="2446"></location>
        <text>depletion</text>
      </annotation>
    </passage>
    <passage>
      <offset>3527</offset>
      <text>Immunohistochemistry.</text>
    </passage>
    <passage>
      <offset>3549</offset>
      <text>Human tonsils were obtained from tonsillectomy samples of hypertrophic and obstructive tonsils without a current infection. Ethical permission was obtained from Cantonal Ethics Commission, and informed consent was obtained from patients. Paraformaldehyde-fixed tonsil cryosections were stained with unconjugated rabbit IgG polyclonal antibody to human RUNX1 (Santa Cruz Biotechnology, Inc.) or unconjugated mouse IgG1 mAb to human RUNX3 (Abcam). After a washing step, the sections were stained with the corresponding secondary antibodies. RUNX1-binding antibodies were detected by using Alexa Fluor 633-conjugated goat anti-rabbit IgG and RUNX3-binding antibodies were detected by using Alexa Fluor 532-conjugated goat anti-mouse IgG1. Afterward, the sections were washed and in the case of RUNX3 staining a blocking step with an unconjugated mouse IgG1 mAb was used. Finally, the sections were stained with Alexa Fluor 488-conjugated mouse IgG1 mAb to human FOXP3 (eBioscience) or the corresponding isotype control. Tissue sections were stained with DAPI for the demonstration of nuclei and mounted with Prolong antifade (Invitrogen). Images were acquired and analyzed using the confocal microscope DMI 4000B and the TCS SPE system (both from Leica).</text>
      <annotation id="T73">
        <infon key="type">Protein</infon>
        <location offset="3901" length="3906"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T74">
        <infon key="type">Protein</infon>
        <location offset="3980" length="3985"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T75">
        <infon key="type">Protein</infon>
        <location offset="4088" length="4093"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T76">
        <infon key="type">Protein</infon>
        <location offset="4188" length="4193"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T77">
        <infon key="type">Protein</infon>
        <location offset="4340" length="4345"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T78">
        <infon key="type">Protein</infon>
        <location offset="4508" length="4513"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T256">
        <infon key="type">Binding</infon>
        <location offset="4094" length="4101"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T257">
        <infon key="type">Binding</infon>
        <location offset="4194" length="4201"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <passage>
      <offset>4802</offset>
      <text>In vitro suppression assays.</text>
    </passage>
    <passage>
      <offset>4831</offset>
      <text>Mouse Foxp3+ CD4+ CD8- T cells were FACS purified based on GFP expressed from a Foxp3-IRES-GFP knock-in allele (Foxp3GFP). Naive (CD62Lhi44lo25-) CD4+ T cells (effectors) were FACS-purified from Cd45.1 mice, then loaded with 5 microM CFSE (Invitrogen). Total splenocytes from C57BL/6 mice inactivated with 50 microg/ml mitomycin C (Sigma-Aldrich) for 45 min were used as APCs. A total of 4 x 105 CD4+ cells (CFSE-loaded CD25- plus Foxp3-GFP+) was mixed with 105 APCs + 1 microg/ml anti-CD3 mAb per well of a 96 well round bottom plate. Proliferation of the effector cells was analyzed by CFSE dilution. Apoptosis of the cells was investigated by annexin V staining and flow cytometry. Positive and negative control gates were made according to T cells cultured only in the presence of IL-2 without anti-CD3/28 stimulation.</text>
      <annotation id="T79">
        <infon key="type">Protein</infon>
        <location offset="4837" length="4842"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T80">
        <infon key="type">Protein</infon>
        <location offset="4844" length="4847"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T81">
        <infon key="type">Protein</infon>
        <location offset="4849" length="4852"></location>
        <text>CD8</text>
      </annotation>
      <annotation id="T82">
        <infon key="type">Protein</infon>
        <location offset="4890" length="4893"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T83">
        <infon key="type">Protein</infon>
        <location offset="4911" length="4916"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T84">
        <infon key="type">Protein</infon>
        <location offset="4922" length="4925"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T85">
        <infon key="type">Protein</infon>
        <location offset="4943" length="4948"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T86">
        <infon key="type">Protein</infon>
        <location offset="4948" length="4951"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T87">
        <infon key="type">Protein</infon>
        <location offset="4961" length="4966"></location>
        <text>CD62L</text>
      </annotation>
      <annotation id="T88">
        <infon key="type">Protein</infon>
        <location offset="4968" length="4970"></location>
        <text>44</text>
      </annotation>
      <annotation id="T89">
        <infon key="type">Protein</infon>
        <location offset="4972" length="4974"></location>
        <text>25</text>
      </annotation>
      <annotation id="T90">
        <infon key="type">Protein</infon>
        <location offset="4977" length="4980"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T91">
        <infon key="type">Protein</infon>
        <location offset="5026" length="5032"></location>
        <text>Cd45.1</text>
      </annotation>
      <annotation id="T92">
        <infon key="type">Protein</infon>
        <location offset="5227" length="5230"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T93">
        <infon key="type">Protein</infon>
        <location offset="5251" length="5255"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T94">
        <infon key="type">Protein</infon>
        <location offset="5262" length="5271"></location>
        <text>Foxp3-GFP</text>
      </annotation>
      <annotation id="T95">
        <infon key="type">Protein</infon>
        <location offset="5317" length="5320"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T96">
        <infon key="type">Protein</infon>
        <location offset="5477" length="5486"></location>
        <text>annexin V</text>
      </annotation>
      <annotation id="T97">
        <infon key="type">Protein</infon>
        <location offset="5616" length="5620"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T98">
        <infon key="type">Protein</infon>
        <location offset="5634" length="5637"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T99">
        <infon key="type">Protein</infon>
        <location offset="5638" length="5640"></location>
        <text>28</text>
      </annotation>
      <annotation id="T258">
        <infon key="type">Gene_expression</infon>
        <location offset="4894" length="4903"></location>
        <text>expressed</text>
      </annotation>
    </passage>
    <passage>
      <offset>5655</offset>
      <text>Human naive CD4+ T cells were isolated by negative selection by MACS from PBMCs and either transfected with a scrambled siRNA or with a combination of RUNX1 and RUNX3 siRNA. Cells were then cultured under iT reg cell differentiating conditions and mixed with 2 x 105 autologous irradiated PBMCs that were used as APCs and autologous CFSE-labeled CD4+ T cells. T reg cell to responder cell ratio was 1:20, 1:10, and 1:5. To check the proliferation of the CD4+ T cells without suppression, no T reg cells were added in a control group. Cells were stimulated with 2.5 microg/ml anti-CD3 mAb, cultured in a 96-well plate and the proliferation of the effector cells was determined by analyzing the CFSE dilution by flow cytometry after 5 d of culture. Gating on the CD4+CFSE+ T cells enabled the exclusion of APCs and T reg cells.</text>
      <annotation id="T100">
        <infon key="type">Protein</infon>
        <location offset="5667" length="5670"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T101">
        <infon key="type">Protein</infon>
        <location offset="5806" length="5811"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T102">
        <infon key="type">Protein</infon>
        <location offset="5816" length="5821"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T103">
        <infon key="type">Protein</infon>
        <location offset="6001" length="6004"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T104">
        <infon key="type">Protein</infon>
        <location offset="6109" length="6112"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T105">
        <infon key="type">Protein</infon>
        <location offset="6235" length="6238"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T106">
        <infon key="type">Protein</infon>
        <location offset="6416" length="6419"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T259">
        <infon key="type">Negative_regulation</infon>
        <location offset="5822" length="5827"></location>
        <text>siRNA</text>
      </annotation>
    </passage>
    <passage>
      <offset>6482</offset>
      <text>Cloning of the FOXP3 promoter, construction of mutant FOXP3 promoter, and RUNX expression plasmids.</text>
      <annotation id="T107">
        <infon key="type">Protein</infon>
        <location offset="6497" length="6502"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T108">
        <infon key="type">Protein</infon>
        <location offset="6536" length="6541"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T109">
        <infon key="type">Protein</infon>
        <location offset="6556" length="6560"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T260">
        <infon key="type">Gene_expression</infon>
        <location offset="6561" length="6571"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>6582</offset>
      <text>The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate pGL3 FOXP3 -511/+176 (Mantel et al., 2006). Site-directed mutagenesis for the three putative RUNX binding sites in the FOXP3 promoter region was introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions and confirmed by sequencing the DNA. The following primers and their complementary strands were used: foxp runx-333, forward 5'-CACTTTTGTTTTAAAAACTGTCCTTTCTCATGAGCCCTATTATC-3'; foxp runx-333 reverse 5'-GATAATAGGGCTCATGAGAAAGGACAGTTTTTAAAACAAAAGTG-3'; foxp runx-287 forward 5'-CCTCTCACCTCTGTCCTGAGGGGAAGAAATC-3'; foxp runx-287 reverse 5'-GATTTCTTCCCCTCAGGACAGAGGTGAGAGG-3'; foxp runx-53 forward 5'-GCTTCCACACCGTACAGCGTCCTTTTTCTTCTCGGTATAAAAG-3'; foxp runx-53 reverse 5'-CTTTTATACCGAGAAGAAAAAGGACGCTGTACGGTGTGGAAGC-3'.</text>
      <annotation id="T110">
        <infon key="type">Protein</infon>
        <location offset="6586" length="6591"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T111">
        <infon key="type">Protein</infon>
        <location offset="6674" length="6679"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T112">
        <infon key="type">Protein</infon>
        <location offset="6788" length="6793"></location>
        <text>FOXP3</text>
      </annotation>
    </passage>
    <passage>
      <offset>7428</offset>
      <text>The human RUNX1 fragment from the Addgene plasmid 12504 (Biggs et al., 2006) pFlagCMV2-AML1B was sub-cloned in the pEGFPN1 vector (Clontech Laboratories). The RUNX3 vector pCMV human RUNX3, which was a gift from K. Ito (Institute of Molecular and Cell Biology, Proteos, Singapore) was subcloned into pEGFPN1 vector (Clontech Laboratories; Yamamura et al., 2006).</text>
      <annotation id="T113">
        <infon key="type">Protein</infon>
        <location offset="7438" length="7443"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T114">
        <infon key="type">Protein</infon>
        <location offset="7587" length="7592"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T115">
        <infon key="type">Protein</infon>
        <location offset="7611" length="7616"></location>
        <text>RUNX3</text>
      </annotation>
    </passage>
    <passage>
      <offset>7792</offset>
      <text>Transfections and reporter gene assays.</text>
    </passage>
    <passage>
      <offset>7832</offset>
      <text>T cells were rested in serum-free AIM-V medium overnight. 3.5 microg of the FOXP3 promoter luciferase reporter vector or a combination together with the RUNX1, RUNX3 pEGFPN1 vector, and 0.5 microg phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 microl of Nucleofector solution (Lonza) and electroporated using the program U-15. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega) according to the manufacturer's instructions. PMA/ionomycin was used to stimulate the cells, because the transfection was only transient and the luciferase assay required a strong and fast stimulation of the cells. To evaluate the effect of overexpression of RUNX1 or RUNX3 on FOXP3 protein levels, CD4+ T cells were preactivated with 2 microg/ml phytohemagglutinin (Sigma-Aldrich) in serum-free AIM-V medium in the presence of 1 nmol/liter IL-2 (Roche) for 12 h, and then transfected with the vector pEGFPN1 containing the RUNX1 or RUNX3 fragment using the Nucleofector system (Amaxa Biosystems) and the program T-23. FOXP3 expression was evaluated by flow cytometry after 48 h of culture in AIM-V medium containing 1 nmol/liter IL-2.</text>
      <annotation id="T116">
        <infon key="type">Protein</infon>
        <location offset="7908" length="7913"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T117">
        <infon key="type">Protein</infon>
        <location offset="7923" length="7933"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T118">
        <infon key="type">Protein</infon>
        <location offset="7985" length="7990"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T119">
        <infon key="type">Protein</infon>
        <location offset="7992" length="7997"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T120">
        <infon key="type">Protein</infon>
        <location offset="8059" length="8062"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T121">
        <infon key="type">Protein</infon>
        <location offset="8261" length="8271"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T122">
        <infon key="type">Protein</infon>
        <location offset="8306" length="8316"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T123">
        <infon key="type">Protein</infon>
        <location offset="8485" length="8495"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T124">
        <infon key="type">Protein</infon>
        <location offset="8599" length="8604"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T125">
        <infon key="type">Protein</infon>
        <location offset="8608" length="8613"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T126">
        <infon key="type">Protein</infon>
        <location offset="8617" length="8622"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T127">
        <infon key="type">Protein</infon>
        <location offset="8639" length="8642"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T128">
        <infon key="type">Protein</infon>
        <location offset="8687" length="8705"></location>
        <text>phytohemagglutinin</text>
      </annotation>
      <annotation id="T129">
        <infon key="type">Protein</infon>
        <location offset="8781" length="8785"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T130">
        <infon key="type">Protein</infon>
        <location offset="8864" length="8869"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T131">
        <infon key="type">Protein</infon>
        <location offset="8873" length="8878"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T132">
        <infon key="type">Protein</infon>
        <location offset="8959" length="8964"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T133">
        <infon key="type">Protein</infon>
        <location offset="9070" length="9074"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T261">
        <infon key="type">Regulation</infon>
        <location offset="8571" length="8577"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T262">
        <infon key="type">Gene_expression</infon>
        <location offset="8581" length="8595"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T263">
        <infon key="type">Gene_expression</infon>
        <location offset="8813" length="8824"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T264">
        <infon key="type">Gene_expression</infon>
        <location offset="8965" length="8975"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>9077</offset>
      <text>RNA interference.</text>
    </passage>
    <passage>
      <offset>9095</offset>
      <text>CD4+ or naive CD4+ T cells were resuspended in 100 microl of Nucleofector solution (Lonza) and electroporated with 2 microM siRNA using the Nucleofector technology program U-14 (Lonza). Five different Silencer or Silencer Select Pre-designed siRNAs for RUNX1 (Applied Biosystems) and three Silencer Pre-designed siRNAs for RUNX3 (Applied Biosystems) were tested, and the best was selected for all further experiments. The Silencer Negative Control #1 siRNA (Applied Biosystems) was used for normalization. Cells were then left unstimulated or were stimulated after 12 h with anti-CD2, anti-CD3, and anti-CD28. Cells were cultured in serum-free AIM-V medium with the addition of 1 nmol/l IL-2 (Roche). Cells were harvested for mRNA detection of the target genes after 24 h and for protein detection after 48 h.</text>
      <annotation id="T134">
        <infon key="type">Protein</infon>
        <location offset="9095" length="9098"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T135">
        <infon key="type">Protein</infon>
        <location offset="9109" length="9112"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T136">
        <infon key="type">Protein</infon>
        <location offset="9348" length="9353"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T137">
        <infon key="type">Protein</infon>
        <location offset="9418" length="9423"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T138">
        <infon key="type">Protein</infon>
        <location offset="9675" length="9678"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T139">
        <infon key="type">Protein</infon>
        <location offset="9685" length="9688"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T140">
        <infon key="type">Protein</infon>
        <location offset="9699" length="9703"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T141">
        <infon key="type">Protein</infon>
        <location offset="9739" length="9744"></location>
        <text>AIM-V</text>
      </annotation>
      <annotation id="T142">
        <infon key="type">Protein</infon>
        <location offset="9782" length="9786"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T265">
        <infon key="type">Negative_regulation</infon>
        <location offset="9337" length="9343"></location>
        <text>siRNAs</text>
      </annotation>
      <annotation id="T266">
        <infon key="type">Negative_regulation</infon>
        <location offset="9407" length="9413"></location>
        <text>siRNAs</text>
      </annotation>
    </passage>
    <passage>
      <offset>9906</offset>
      <text>RNA isolation and cDNA synthesis.</text>
    </passage>
    <passage>
      <offset>9940</offset>
      <text>RNA was isolated using the RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol. Reverse transcription of human samples was performed with reverse-transcription reagents (Fermentas) with random hexamers according to the manufacturer's protocol.</text>
    </passage>
    <passage>
      <offset>10199</offset>
      <text>Real-time PCR.</text>
    </passage>
    <passage>
      <offset>10214</offset>
      <text>PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer, 5'-GAAACAGCACATTCCCAGAGTTC-3'; FOXP3 reverse primer, 5'-ATGGCCCAGCGGATGAG-3'; EF-1a forward primer, 5'-CTGAACCATCCAGGCCAAAT-3'; and EF-1a reverse primer, 5'-GCCGTGTGGCAATCCAAT-3', as previously described (Mantel et al., 2007). GATA3 forward primer, 5'-GCGGGCTCTATCACAAAATGA-3'; and GATA3 reverse primer 5'-GCTCTCCTGGCTGCAGACAGC-3' (Mantel et al., 2007). T-bet forward primer, 5'-GATGCGCCAGGAAGTTTCAT-3'; T-bet reverse primer, 5'-GCACAATCATCTGGGTCACATT-3'; RORC2 forward primer, 5'-CAGTCATGAGAACACAAATTGAAGTG-3'; and RORC2 reverse primer 5'-CAGGTGATAACCCCGTAGTGGAT-3'. The prepared cDNAs were amplified using SYBR green PCR master mix (Fermentas) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).</text>
      <annotation id="T143">
        <infon key="type">Protein</infon>
        <location offset="10372" length="10377"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T144">
        <infon key="type">Protein</infon>
        <location offset="10425" length="10430"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T145">
        <infon key="type">Protein</infon>
        <location offset="10472" length="10477"></location>
        <text>EF-1a</text>
      </annotation>
      <annotation id="T146">
        <infon key="type">Protein</infon>
        <location offset="10526" length="10531"></location>
        <text>EF-1a</text>
      </annotation>
      <annotation id="T147">
        <infon key="type">Protein</infon>
        <location offset="10621" length="10626"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T148">
        <infon key="type">Protein</infon>
        <location offset="10676" length="10681"></location>
        <text>GATA3</text>
      </annotation>
      <annotation id="T149">
        <infon key="type">Protein</infon>
        <location offset="10748" length="10753"></location>
        <text>T-bet</text>
      </annotation>
      <annotation id="T150">
        <infon key="type">Protein</infon>
        <location offset="10798" length="10803"></location>
        <text>T-bet</text>
      </annotation>
      <annotation id="T151">
        <infon key="type">Protein</infon>
        <location offset="10850" length="10855"></location>
        <text>RORC2</text>
      </annotation>
      <annotation id="T152">
        <infon key="type">Protein</infon>
        <location offset="10910" length="10915"></location>
        <text>RORC2</text>
      </annotation>
    </passage>
    <passage>
      <offset>11163</offset>
      <text>RUNX1 and RUNX3 mRNA was detected by using TaqMan Gene Expression Assays from Applied Biosystems and used according to the manufacturer's instruction using TaqMan master mix using a 7000 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding elongation factor (EF)-1alpha or by using the 18S rRNA Gene Expression Assay (Applied Biosystems) was performed to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative DeltaDeltaCT method (Applied Biosystems). The percentage of FOXP3 mRNA in siRNA-mediated RUNX knockdown cells was calculated in relation to cells, which were transfected with scrambled control siRNA. Arbitrary units show the 2-(Deltact) values multiplied by 10,000 incorporating the ct values of the gene of interest and the housekeeping gene.</text>
      <annotation id="T153">
        <infon key="type">Protein</infon>
        <location offset="11163" length="11168"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T154">
        <infon key="type">Protein</infon>
        <location offset="11173" length="11178"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T155">
        <infon key="type">Protein</infon>
        <location offset="11445" length="11474"></location>
        <text>elongation factor (EF)-1alpha</text>
      </annotation>
      <annotation id="T156">
        <infon key="type">Protein</infon>
        <location offset="11491" length="11499"></location>
        <text>18S rRNA</text>
      </annotation>
      <annotation id="T157">
        <infon key="type">Protein</infon>
        <location offset="11760" length="11765"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T158">
        <infon key="type">Protein</infon>
        <location offset="11789" length="11793"></location>
        <text>RUNX</text>
      </annotation>
      <annotation id="T267">
        <infon key="type">Transcription</infon>
        <location offset="11179" length="11183"></location>
        <text>mRNA</text>
      </annotation>
      <annotation id="T268">
        <infon key="type">Transcription</infon>
        <location offset="11766" length="11770"></location>
        <text>mRNA</text>
      </annotation>
      <annotation id="T269">
        <infon key="type">Positive_regulation</infon>
        <location offset="11780" length="11788"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T270">
        <infon key="type">Negative_regulation</infon>
        <location offset="11794" length="11803"></location>
        <text>knockdown</text>
      </annotation>
    </passage>
    <passage>
      <offset>12045</offset>
      <text>Flow cytometry.</text>
    </passage>
    <passage>
      <offset>12061</offset>
      <text>For analysis of human FOXP3 expression on the single-cell level, cells were first stained with the monoclonal CD4 mAb (Beckman Coulter), and after fixation and permeabilization, they were incubated with anti-human Foxp3-Alexa Fluor 488 antibody (BioLegend) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL; Beckman Coulter). A mouse IgG1 antibody (BioLegend) was used as an isotype control. Data were analyzed with the CXP software (Beckman Coulter). Cells were cultured with IL-2, and then left unstimulated or stimulated with anti-CD2/-CD3/-CD28 mAb.</text>
      <annotation id="T159">
        <infon key="type">Protein</infon>
        <location offset="12083" length="12088"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T160">
        <infon key="type">Protein</infon>
        <location offset="12171" length="12174"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T161">
        <infon key="type">Protein</infon>
        <location offset="12275" length="12280"></location>
        <text>Foxp3</text>
      </annotation>
      <annotation id="T162">
        <infon key="type">Protein</infon>
        <location offset="12568" length="12572"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T163">
        <infon key="type">Protein</infon>
        <location offset="12625" length="12628"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T164">
        <infon key="type">Protein</infon>
        <location offset="12630" length="12633"></location>
        <text>CD3</text>
      </annotation>
      <annotation id="T165">
        <infon key="type">Protein</infon>
        <location offset="12635" length="12639"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T271">
        <infon key="type">Gene_expression</infon>
        <location offset="12089" length="12099"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>12646</offset>
      <text>Flow cytometry analyses of the mouse cells were performed on an LSRII (BD) and cell sorting was performed on a FACSAria (BD). All antibodies for these experiments were purchased from eBioscience or BD.</text>
    </passage>
    <passage>
      <offset>12849</offset>
      <text>Western blotting.</text>
    </passage>
    <passage>
      <offset>12867</offset>
      <text>For human RUNX1 and RUNX3 analysis on the protein level, 106 cells were lysed and loaded next to a protein-mass ladder (Invitrogen) on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (GE Healthcare). Unspecific binding was blocked with 3% milk in TBS Tween, and the membranes were subsequently incubated with a 1:1,000 dilution of rabbit anti-RUNX1 (ab11903; Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50; Santa Cruz Biotechnology, Inc.) in blocking buffer containing 3% milk in TBS Tween overnight at 4degreesC. The blots were developed using an anti-rabbit IgG HRP-labeled mAb (Cell Signaling Technology) and visualized with a LAS-1000 gel documentation system (Fujifilm). To confirm sample loading and transfer membranes were incubated in stripping buffer and reblocked for 1 h and reprobed using anti-GAPDH (6C5; Ambion) and developed using an anti-mouse IgG HRP-labeled mAb (Cell Signaling Technology).</text>
      <annotation id="T166">
        <infon key="type">Protein</infon>
        <location offset="12877" length="12882"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T167">
        <infon key="type">Protein</infon>
        <location offset="12887" length="12892"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T168">
        <infon key="type">Protein</infon>
        <location offset="13258" length="13263"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T169">
        <infon key="type">Protein</infon>
        <location offset="13314" length="13319"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T170">
        <infon key="type">Protein</infon>
        <location offset="13726" length="13731"></location>
        <text>GAPDH</text>
      </annotation>
      <annotation id="T272">
        <infon key="type">Gene_expression</infon>
        <location offset="12909" length="12922"></location>
        <text>protein level</text>
      </annotation>
    </passage>
    <passage>
      <offset>13830</offset>
      <text>Pull-down assay.</text>
    </passage>
    <passage>
      <offset>13847</offset>
      <text>HEK293T cells were transfected with RUNX1 or RUNX3 using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. Cells were lysed by sonication in HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche Diagnostics). The cell lysate was precleared using streptavidin-agarose beads (GE Healthcare), incubated with biotinylated double-stranded oligonucleotides containing the wild-type or mutated RUNX binding sites, and polydeoxyinosinicdeoxycytidylic acid (Sigma-Aldrich). A combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used in the assay. DNA-bound proteins were collected with streptavidin-agarose beads, washed with HKMG buffer, and finally resuspended in NuPAGE loading buffer (Invitrogen Life Technologies), heated to 70degreesC for 10 min, and separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen Life Technologies). The proteins were electroblotted onto a PVDF membrane (GE Healthcare) and detected using RUNX1 or RUNX3 antibodies described in the previous section.</text>
      <annotation id="T171">
        <infon key="type">Protein</infon>
        <location offset="13883" length="13888"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T172">
        <infon key="type">Protein</infon>
        <location offset="13892" length="13897"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T173">
        <infon key="type">Protein</infon>
        <location offset="14939" length="14944"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T174">
        <infon key="type">Protein</infon>
        <location offset="14948" length="14953"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T273">
        <infon key="type">Gene_expression</infon>
        <location offset="13866" length="13877"></location>
        <text>transfected</text>
      </annotation>
    </passage>
    <passage>
      <offset>15001</offset>
      <text>Promoter enzyme immuno assay.</text>
    </passage>
    <passage>
      <offset>15031</offset>
      <text>As performed in the pull-down assay, HEK293T cells were transfected with RUNX1 or RUNX3 and subsequently lysed. Insoluble material was removed by centrifugation. 384-well plates, precoated with streptavidin (Thermo Fisher Scientific) were washed 3 times with washing buffer (PBS and 0.05% Tween 20). Biotinylated FOXP3 promoter/oligonucleotides probes containing the RUNX binding sites were added (1 pmol per well; 50 fmol/microl) and incubated for 1 h at room temperature. Either a combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used or single oligonucleotides were used in the assay. After 3 washing steps with washing buffer, the nuclear extract was added (concentration &gt; 0.2 microg/microl) and incubated overnight at 4degreesC. The lysates were incubated with 10 microg of poly-deoxyinosinic-deoxycytidylic acid (Sigma-Aldrich). The plate was washed with HKMG buffer and incubated with a 1:1000 dilution of rabbit anti-RUNX1 (ab11903, Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50, Santa Cruz Biotechnology, Inc.) at 4degreesC for 2 h. After three washing steps with HKMG buffer, a secondary antibody (anti-rabbit IgG-HRP, 1:3,000 in HKMG buffer, Cell Signaling Technology) was added, and the plate was incubated for 1 h at 4degreesC. The wells were washed 4 times with HKMG buffer before adding the substrate reagent (R&amp;D Systems). The colorimetric reaction was stopped by adding 2 M H2SO4. Absorbance at 450 nm was measured using a microplate reader (Berthold Technologies).</text>
      <annotation id="T175">
        <infon key="type">Protein</infon>
        <location offset="15068" length="15081"></location>
        <text>HEK293T cells</text>
      </annotation>
      <annotation id="T176">
        <infon key="type">Protein</infon>
        <location offset="15104" length="15109"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T177">
        <infon key="type">Protein</infon>
        <location offset="15113" length="15118"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T178">
        <infon key="type">Protein</infon>
        <location offset="15344" length="15349"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T179">
        <infon key="type">Protein</infon>
        <location offset="16008" length="16013"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T180">
        <infon key="type">Protein</infon>
        <location offset="16064" length="16069"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T274">
        <infon key="type">Gene_expression</infon>
        <location offset="15087" length="15098"></location>
        <text>transfected</text>
      </annotation>
    </passage>
    <passage>
      <offset>16573</offset>
      <text>ChIP.</text>
    </passage>
    <passage>
      <offset>16579</offset>
      <text>Human naive CD4+ T cells were cultured either with IL-2 only or with IL-2, anti-CD2/3/28, and TGF-beta for 72 h, and protein-DNA complexes were fixed by cross-linking with formaldehyde in a final concentration of 1.42% for 15 min. Formaldehyde was quenched with 125 mM glycine for 5 min, and cells were subsequently harvested. The ChIP assay was performed as described in the fast chromatin immunoprecipitation method (Nelson et al., 2006). Cells were lysed with immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, NP-40 [0.5% vol/vol]) containing phosphatase (Roche) and protease inhibitors cocktails (Roche), the nuclear pellet was washed, the chromatin was sheared by sonication and incubated with antibodies for RUNX1 (H-65 X; Santa Cruz Biotechnology, Inc.), RUNX3 (H-50 X; Santa Cruz Biotechnology, Inc.), CBFbeta (PEBP2beta; FL-182 X; Santa Cruz Biotechnology, Inc.), and as controls normal rabbit IgG (Santa Cruz Biotechnology, Inc.), anti-human RNA polymerase II antibody, and mouse control IgG (both from SA Biosciences). The cleared chromatin was incubated with protein A agarose beads and, after several washing steps, DNA was isolated with 10% (wt/vol) Chelex 100 resin. Samples were treated with proteinase K at 55degreesC for 30 min. The proteinase K was then inactivated by boiling the samples for 10 min. The purified DNA was used in a real-time PCR reaction. Specific primers for the FOXP3 promoter, spanning the region from -87 to -3, FOXP3 promoter forward primer 5'-AGAGGTCTGCGGCTTCCA-3', FOXP3 promoter reverse primer 5'-GGAAACTGTCACGTATCAAAAACAA-3', or control GAPDH primer (SA Biosciences) for the RNA polymerase II were used. A negative control PCR for each immunoprecipitation using IGX1A negative control primer targeting ORF-free intergenic DNA (SA Biosciences) was used. The fold enrichment in site occupancy was calculated incorporating IgG control values and input DNA values using the ChampionChIP qPCR data analysis file (SA Biosciences).</text>
      <annotation id="T181">
        <infon key="type">Protein</infon>
        <location offset="16591" length="16594"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T182">
        <infon key="type">Protein</infon>
        <location offset="16630" length="16634"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T183">
        <infon key="type">Protein</infon>
        <location offset="16648" length="16652"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T184">
        <infon key="type">Protein</infon>
        <location offset="16659" length="16662"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T185">
        <infon key="type">Protein</infon>
        <location offset="16663" length="16664"></location>
        <text>3</text>
      </annotation>
      <annotation id="T186">
        <infon key="type">Protein</infon>
        <location offset="16665" length="16667"></location>
        <text>28</text>
      </annotation>
      <annotation id="T187">
        <infon key="type">Protein</infon>
        <location offset="16673" length="16681"></location>
        <text>TGF-beta</text>
      </annotation>
      <annotation id="T188">
        <infon key="type">Protein</infon>
        <location offset="17319" length="17324"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T189">
        <infon key="type">Protein</infon>
        <location offset="17367" length="17372"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T190">
        <infon key="type">Protein</infon>
        <location offset="17415" length="17422"></location>
        <text>CBFbeta</text>
      </annotation>
      <annotation id="T191">
        <infon key="type">Protein</infon>
        <location offset="17557" length="17574"></location>
        <text>RNA polymerase II</text>
      </annotation>
      <annotation id="T192">
        <infon key="type">Protein</infon>
        <location offset="17676" length="17685"></location>
        <text>protein A</text>
      </annotation>
      <annotation id="T193">
        <infon key="type">Protein</infon>
        <location offset="17813" length="17825"></location>
        <text>proteinase K</text>
      </annotation>
      <annotation id="T194">
        <infon key="type">Protein</infon>
        <location offset="17856" length="17868"></location>
        <text>proteinase K</text>
      </annotation>
      <annotation id="T195">
        <infon key="type">Protein</infon>
        <location offset="18005" length="18010"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T196">
        <infon key="type">Protein</infon>
        <location offset="18057" length="18062"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T197">
        <infon key="type">Protein</infon>
        <location offset="18113" length="18118"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T198">
        <infon key="type">Protein</infon>
        <location offset="18187" length="18192"></location>
        <text>GAPDH</text>
      </annotation>
      <annotation id="T199">
        <infon key="type">Protein</infon>
        <location offset="18225" length="18242"></location>
        <text>RNA polymerase II</text>
      </annotation>
    </passage>
    <passage>
      <offset>18576</offset>
      <text>Quantification of cytokine levels.</text>
    </passage>
    <passage>
      <offset>18611</offset>
      <text>IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and IFN-gamma secretion was assessed using fluorescent bead-based technology. The Bio-Plex-hu Cytokine Panel, 17-Plex Group 1 was used according to the manufacturer's instructions (Bio-Rad Laboratories). Fluorescent signals were read and analyzed using the Bio-Plex 200 System (Bio-Rad Laboratories).</text>
      <annotation id="T200">
        <infon key="type">Protein</infon>
        <location offset="18611" length="18615"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T201">
        <infon key="type">Protein</infon>
        <location offset="18617" length="18621"></location>
        <text>IL-5</text>
      </annotation>
      <annotation id="T202">
        <infon key="type">Protein</infon>
        <location offset="18623" length="18627"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T203">
        <infon key="type">Protein</infon>
        <location offset="18629" length="18634"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T204">
        <infon key="type">Protein</infon>
        <location offset="18636" length="18641"></location>
        <text>IL-13</text>
      </annotation>
      <annotation id="T205">
        <infon key="type">Protein</infon>
        <location offset="18643" length="18648"></location>
        <text>IL-17</text>
      </annotation>
      <annotation id="T206">
        <infon key="type">Protein</infon>
        <location offset="18654" length="18663"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T275">
        <infon key="type">Localization</infon>
        <location offset="18664" length="18673"></location>
        <text>secretion</text>
      </annotation>
    </passage>
    <passage>
      <offset>18952</offset>
      <text>Online supplemental material.</text>
    </passage>
    <passage>
      <offset>18982</offset>
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  </document>
  <document>
    <id>PMC-2806624-09-Supplementary_Material</id>
    <passage>
      <offset>0</offset>
      <text>Supplementary Material</text>
    </passage>
    <passage>
      <offset>23</offset>
      <text>[Supplemental Material]</text>
    </passage>
  </document>
  <document>
    <id>PMC-2806624-10-caption-01</id>
    <passage>
      <offset>0</offset>
      <text>RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-beta stimulation in the presence of IL-2. Real-time PCR of human naive CD4+ T cells after 48 h of culture. Bars show the mean +/- SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-beta in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown. Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-beta and IL-2. Bars show the mean +/- SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3. Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d. Bars show the mean +/- SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown. Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-beta in the presence of IL-2. CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h. One of three independent experiments is shown. Statistical differences were verified by the paired Student's t test. *, P &lt; 0.05; **, P &lt; 0.01.</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E24">
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      <infon key="type">Event</infon>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T69" role="Trigger"></node>
      <node refid="T38" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T69" role="Trigger"></node>
      <node refid="T39" role="Theme"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T70" role="Trigger"></node>
      <node refid="T42" role="Theme"></node>
    </relation>
    <relation id="E30">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T71" role="Trigger"></node>
      <node refid="E29" role="Theme"></node>
      <node refid="E31" role="Cause"></node>
    </relation>
    <relation id="E32">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T71" role="Trigger"></node>
      <node refid="E29" role="Theme"></node>
      <node refid="E33" role="Cause"></node>
    </relation>
    <relation id="E34">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T72" role="Trigger"></node>
      <node refid="E31" role="Theme"></node>
    </relation>
    <relation id="E35">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T72" role="Trigger"></node>
      <node refid="E33" role="Theme"></node>
    </relation>
    <relation id="E31">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T73" role="Trigger"></node>
      <node refid="T43" role="Theme"></node>
    </relation>
    <relation id="E33">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T73" role="Trigger"></node>
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    </relation>
    <relation id="E36">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T74" role="Trigger"></node>
      <node refid="T46" role="Theme"></node>
    </relation>
    <relation id="E37">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T47" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2806624-11-caption-02</id>
    <passage>
      <offset>0</offset>
      <text>RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127-, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127-, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-beta, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25- cells. Bars show the mean +/- SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy. Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls. HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining. Data shown are representative from one of the three tissue samples with similar results. Bars, 5 microm. Statistical differences were verified by the paired Student's t test. *, P &lt; 0.05; **, P &lt; 0.01.</text>
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        <location offset="10" length="15"></location>
        <text>RUNX3</text>
      </annotation>
      <annotation id="T3">
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        <text>Foxp3</text>
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        <text>CD4</text>
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        <text>CD25</text>
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        <text>CD127</text>
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        <text>CD25</text>
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        <text>CD4</text>
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        <text>TGF-beta</text>
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    <relation id="E10">
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    <relation id="E12">
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    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMC-2806624-12-caption-03</id>
    <passage>
      <offset>0</offset>
      <text>Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide-transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean +/- SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFbeta to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-beta. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean +/- SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P &lt; 0.05</text>
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        <location offset="1057" length="1064"></location>
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        <text>FOXP3</text>
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        <location offset="1102" length="1105"></location>
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        <location offset="1139" length="1143"></location>
        <text>IL-2</text>
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        <location offset="1163" length="1166"></location>
        <text>CD2</text>
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        <location offset="1167" length="1168"></location>
        <text>3</text>
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        <location offset="1169" length="1171"></location>
        <text>28</text>
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        <location offset="1176" length="1184"></location>
        <text>TGF-beta</text>
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        <location offset="0" length="7"></location>
        <text>Binding</text>
      </annotation>
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        <infon key="type">Entity</infon>
        <location offset="53" length="66"></location>
        <text>binding sites</text>
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        <location offset="1009" length="1016"></location>
        <text>binding</text>
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        <text>promoter</text>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T3" role="Theme2"></node>
      <node refid="T18" role="Site2"></node>
    </relation>
    <relation id="E2">
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      <infon key="type">Event</infon>
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      <node refid="T2" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
      <node refid="T18" role="Site2"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
      <node refid="T20" role="Site2"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
      <node refid="T20" role="Site2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
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    </relation>
  </document>
  <document>
    <id>PMC-2806624-13-caption-04</id>
    <passage>
      <offset>0</offset>
      <text>Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector. Bars show the mean luciferase activity +/- SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 -511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene. Bars show the mean luciferase activity +/- SD measured as arbitrary light units of three independent experiments.</text>
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        <location offset="14" length="19"></location>
        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="41" length="46"></location>
        <text>RUNX1</text>
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        <infon key="type">Protein</infon>
        <location offset="51" length="56"></location>
        <text>RUNX3</text>
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        <infon key="type">Protein</infon>
        <location offset="76" length="79"></location>
        <text>CD4</text>
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        <location offset="184" length="189"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="207" length="212"></location>
        <text>FOXP3</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="237" length="247"></location>
        <text>luciferase</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="278" length="281"></location>
        <text>GFP</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="283" length="288"></location>
        <text>RUNX1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="293" length="298"></location>
        <text>RUNX3</text>
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      <annotation id="T11">
        <infon key="type">Protein</infon>
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        <text>luciferase</text>
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        <location offset="450" length="453"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="546" length="551"></location>
        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
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        <text>FOXP3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="599" length="609"></location>
        <text>luciferase</text>
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        <text>FOXP3</text>
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        <infon key="type">Protein</infon>
        <location offset="861" length="871"></location>
        <text>luciferase</text>
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        <text>luciferase</text>
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        <text>transfected</text>
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        <text>transfected</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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  </document>
  <document>
    <id>PMC-2806624-14-caption-05</id>
    <passage>
      <offset>0</offset>
      <text>Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, +/- 10 nM retinoic acid (RA), and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-gamma expression. One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 x 106 cells per transfer). 6 wk later, TCRbeta+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression. Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown. The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P &lt; 0.05 between groups.</text>
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        <text>CD4-cre</text>
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        <text>CD4</text>
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        <text>CD4</text>
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        <text>CD8</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>CD3</text>
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        <text>28</text>
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        <text>IL-2</text>
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      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="320" length="328"></location>
        <text>TGF-beta</text>
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        <text>Foxp3</text>
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        <text>IFN-gamma</text>
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        <text>CD4</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>Foxp3</text>
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        <text>GFP</text>
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        <text>Rag</text>
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        <text>Foxp3-GFP</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>deficient</text>
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        <text>expression</text>
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        <text>deficient</text>
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        <infon key="type">Gene_expression</infon>
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        <text>expression</text>
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    <relation id="E3">
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      <infon key="type">Event</infon>
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    <relation id="E4">
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      <infon key="type">Event</infon>
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      <node refid="T22" role="Theme"></node>
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    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T25" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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    </relation>
  </document>
  <document>
    <id>PMC-2806624-15-caption-06</id>
    <passage>
      <offset>0</offset>
      <text>CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8- T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-beta. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown.</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>CD4</text>
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        <text>RUNX1</text>
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        <text>RUNX3</text>
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        <text>8</text>
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        <text>Cbfb</text>
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        <text>CD4-cre</text>
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        <text>CD4-cre</text>
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        <text>Cd45.2</text>
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        <text>CD3</text>
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        <text>28</text>
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        <text>IL-2</text>
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        <text>TGF-beta</text>
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        <text>Foxp3-GFP</text>
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        <text>CD45.1</text>
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        <text>CD3</text>
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        <text>CD45.1</text>
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        <text>CD4</text>
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        <text>RUNX1</text>
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        <text>RUNX3</text>
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        <text>CD4</text>
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        <text>CD3</text>
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        <text>CD4</text>
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        <text>CD4</text>
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        <text>mediated</text>
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        <text>knock down</text>
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        <location offset="1172" length="1177"></location>
        <text>siRNA</text>
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      <infon key="type">Event</infon>
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    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E2">
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      <infon key="type">Event</infon>
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      <node refid="T4" role="Theme"></node>
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    <relation id="E4">
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      <infon key="type">Event</infon>
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      <node refid="T5" role="Theme"></node>
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    <relation id="E5">
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E6">
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      <infon key="type">Event</infon>
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  </document>
  <document>
    <id>PMC-2806624-16-caption-07</id>
    <passage>
      <offset>0</offset>
      <text>[Supplemental Material]</text>
    </passage>
  </document>
  <document>
    <id>PMID-10089566</id>
    <passage>
      <offset>0</offset>
      <text>Involvement of adenylate cyclase and p70(S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1.</text>
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        <text>IL-10</text>
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        <text>gp41</text>
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        <text>Involvement</text>
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        <text>activation</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="72" length="85"></location>
        <text>up-regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>171</offset>
      <text>Our previous results show that recombinant gp41 (aa565-647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-kappaB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-kappaB binding activity in as much as no NF-kappaB bound to the main NF-kappaB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70(S6)-kinase (rapamycin), and Gi protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-kappaB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive Gi/Go protein to effect p70(S6)-kinase activation.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="214" length="218"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="305" length="319"></location>
        <text>interleukin-10</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="321" length="326"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="455" length="459"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="468" length="473"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="583" length="587"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="729" length="733"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="742" length="747"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="786" length="790"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="915" length="920"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="948" length="952"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1134" length="1139"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1154" length="1158"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1337" length="1341"></location>
        <text>gp41</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1350" length="1355"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="294" length="304"></location>
        <text>stimulates</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="328" length="338"></location>
        <text>production</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="460" length="467"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="474" length="487"></location>
        <text>up-regulation</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="491" length="499"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Negative_regulation</infon>
        <location offset="718" length="728"></location>
        <text>suppressed</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="734" length="741"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Gene_expression</infon>
        <location offset="748" length="758"></location>
        <text>production</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Binding</infon>
        <location offset="865" length="870"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Entity</infon>
        <location offset="878" length="907"></location>
        <text>main NF-kappaB-binding site 2</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Negative_regulation</infon>
        <location offset="1111" length="1120"></location>
        <text>prevented</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1121" length="1130"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Gene_expression</infon>
        <location offset="1140" length="1150"></location>
        <text>production</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="1342" length="1349"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1356" length="1369"></location>
        <text>up-regulation</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Positive_regulation</infon>
        <location offset="1458" length="1465"></location>
        <text>operate</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E2" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="T7" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T31" role="Site"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10090947</id>
    <passage>
      <offset>0</offset>
      <text>Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells.</text>
    </passage>
    <passage>
      <offset>78</offset>
      <text>Human T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-kappaB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-kappaB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I-infected T-cell lines, display constitutive NF-kappaB DNA binding activity and increased degradation of IkappaBalpha (an inhibitor of NF-kappaB). Whereas the NF-kappaB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-kappaB activity, as well as enhanced degradation of IkappaBalpha, despite the lack of detectable Tax expression. Interestingly, the NF-kappaB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-kappaB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-kappaB subunit activation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="194" length="197"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="407" length="410"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="768" length="780"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="852" length="855"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="900" length="903"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="904" length="909"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="939" length="942"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="943" length="946"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="951" length="954"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="955" length="958"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1108" length="1120"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1153" length="1156"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1220" length="1223"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1224" length="1227"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1232" length="1235"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1236" length="1239"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1349" length="1352"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="419" length="428"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="743" length="752"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein_catabolism</infon>
        <location offset="753" length="764"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="1084" length="1092"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein_catabolism</infon>
        <location offset="1093" length="1104"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1157" length="1167"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T7" role="Event"></node>
      <node refid="T8" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T13" role="Event"></node>
      <node refid="T14" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10092076</id>
    <passage>
      <offset>0</offset>
      <text>Apoptosis-resistant T cells have a deficiency in NF-kappaB-mediated induction of Fas ligand transcription.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="81" length="91"></location>
        <text>Fas ligand</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Negative_regulation</infon>
        <location offset="35" length="45"></location>
        <text>deficiency</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="68" length="77"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="92" length="105"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>108</offset>
      <text>Apoptosis induced through the TCR in CD4+ T cells is mostly mediated by the inducible expression of Fas ligand (FasL) as a primary event leading to the commitment to death. To gain a better understanding of the transcriptional events that regulate this expression, we took advantage of our previously described mutant Jurkat cells. These cells are deficient in FasL expression and apoptosis induced upon TCR triggering, although their cytokine (IL-2 and IFN-gamma) production is normal. Here we show that both a FasL- and a consensus NF-kappaB- reporter construct are inefficiently induced in these cells compared to wild-type cells. In addition, we demonstrate that the inducible transcriptional activity of the FasL reporter is abolished by specific inhibitors of NF-kappaB activation. Thus, we could trace the deficit of the mutant cells to an inefficient NF-kappaB activation, evidencing a relevant role for NF-kappaB in the regulation of FasL expression in activated T cells. Furthermore, our results suggest that the induction of FasL versus cytokine gene expression is differentially sensitive to NF-kappaB deprivation.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="145" length="148"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="208" length="218"></location>
        <text>Fas ligand</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="220" length="224"></location>
        <text>FasL</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="469" length="473"></location>
        <text>FasL</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="553" length="557"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="562" length="571"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="620" length="624"></location>
        <text>FasL</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="821" length="825"></location>
        <text>FasL</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1051" length="1055"></location>
        <text>FasL</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1144" length="1148"></location>
        <text>FasL</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="184" length="193"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="194" length="204"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Negative_regulation</infon>
        <location offset="456" length="465"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="474" length="484"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="499" length="506"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="573" length="583"></location>
        <text>production</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Negative_regulation</infon>
        <location offset="587" length="593"></location>
        <text>normal</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Regulation</infon>
        <location offset="1011" length="1015"></location>
        <text>role</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Regulation</infon>
        <location offset="1037" length="1047"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="1056" length="1066"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="1131" length="1140"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Gene_expression</infon>
        <location offset="1170" length="1180"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10092091</id>
    <passage>
      <offset>0</offset>
      <text>NF-kappaB regulates Fas/APO-1/CD95- and TCR- mediated apoptosis of T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="20" length="23"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="24" length="29"></location>
        <text>APO-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="30" length="34"></location>
        <text>CD95</text>
      </annotation>
    </passage>
    <passage>
      <offset>83</offset>
      <text>The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-kappaB has been shown to play a role in protecting cells against death mediated by TNF We show here that NF-kappaB also has a role in regulating Fas/APO-1/CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-kappaB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-kappaB activation by a soluble peptide inhibitor or a dominant form of the NF-kappaB inhibitor, IkappaB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR-mediated apoptosis. These observations were corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IkappaB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-kappaB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF-kappaB, with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, our results provide compelling evidence that NF-kappaB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="375" length="378"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="379" length="384"></location>
        <text>APO-1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="385" length="389"></location>
        <text>CD95</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="507" length="510"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="515" length="518"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="546" length="549"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="742" length="745"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="950" length="953"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="958" length="961"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1075" length="1078"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1255" length="1258"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1493" length="1496"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1601" length="1604"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="450" length="462"></location>
        <text>Transfection</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="450" length="462"></location>
        <text>Transfection</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="934" length="946"></location>
        <text>transfection</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="934" length="946"></location>
        <text>transfection</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10092775</id>
    <passage>
      <offset>0</offset>
      <text>Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="58" length="62"></location>
        <text>CD34</text>
      </annotation>
    </passage>
    <passage>
      <offset>140</offset>
      <text>Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC--&gt;DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC--&gt;DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="428" length="432"></location>
        <text>CD34</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="548" length="557"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="559" length="563"></location>
        <text>CD34</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="726" length="730"></location>
        <text>CD34</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="833" length="837"></location>
        <text>CD34</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="924" length="950"></location>
        <text>granulocyte-macrophage CSF</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="956" length="965"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1027" length="1036"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1257" length="1261"></location>
        <text>CD83</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1263" length="1267"></location>
        <text>CD86</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1269" length="1273"></location>
        <text>CD14</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1302" length="1306"></location>
        <text>RelB</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1311" length="1317"></location>
        <text>DC-CK1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1548" length="1563"></location>
        <text>TNF receptors 1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1568" length="1569"></location>
        <text>2</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1571" length="1589"></location>
        <text>PKC isoforms alpha</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1591" length="1597"></location>
        <text>beta I</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1599" length="1606"></location>
        <text>beta II</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1612" length="1614"></location>
        <text>mu</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1620" length="1624"></location>
        <text>RelB</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1813" length="1817"></location>
        <text>CD34</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1286" length="1296"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="1323" length="1333"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="1534" length="1544"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="1672" length="1681"></location>
        <text>important</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T20" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T21" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T18" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T15" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T19" role="Cause"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E16" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E17" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
    <relation id="M8">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E19" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10092783</id>
    <passage>
      <offset>0</offset>
      <text>IL-2-mediated cell cycle progression and inhibition of apoptosis does not require NF-kappa B or activating protein-1 activation in primary human T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="4"></location>
        <text>IL-2</text>
      </annotation>
    </passage>
    <passage>
      <offset>155</offset>
      <text>The IL-2 growth hormone is the major growth factor of activated T lymphocytes during a developing immune response. IL-2 is required not only for cell cycle progression but also to protect Ag-activated T cells from programmed cell death. In several cell types, activation of NF-kappa B and/or activating protein-1 (AP-1) has been demonstrated to be extremely important in blocking apoptosis. To determine whether either or both of these transcription factors are involved in cell survival or cell cycle progression in response to IL-2, primary human T cells responsive to the growth factor were analyzed for NF-kappa B and AP-1 activation. The current study clearly demonstrates that IL-2 does not induce I kappa B alpha degradation or NF-kappa B activation in primary human T cells that respond to IL-2 by entering the cell cycle and avoiding apoptosis. Similarly, IL-2 neither activates JNK nor increases AP-1 binding activity to a consensus o-tetradecanoylphorbol 13-acetate (TPA) response element. On the other hand, the growth factor does induce the activation of STAT3 and STAT5 in these cells, as has been previously demonstrated. These data show that neither NF-kappa B nor AP-1 activation is required for IL-2-mediated survival or cell cycle progression in activated primary human T cells.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="159" length="163"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="270" length="274"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="684" length="688"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="838" length="842"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="859" length="874"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="953" length="957"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1020" length="1024"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1223" length="1228"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1368" length="1372"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="852" length="858"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein_catabolism</infon>
        <location offset="875" length="886"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="1198" length="1204"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="1209" length="1219"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10092801</id>
    <passage>
      <offset>0</offset>
      <text>Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation.</text>
    </passage>
    <passage>
      <offset>111</offset>
      <text>We introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="346" length="361"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="363" length="376"></location>
        <text>mut-I kappa B</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="598" length="602"></location>
        <text>CD44</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="603" length="607"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="610" length="613"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="869" length="883"></location>
        <text>TCR beta-chain</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="271" length="278"></location>
        <text>express</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Regulation</infon>
        <location offset="809" length="824"></location>
        <text>appeared normal</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Gene_expression</infon>
        <location offset="851" length="861"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T1" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10092805</id>
    <passage>
      <offset>0</offset>
      <text>Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="27" length="33"></location>
        <text>4E-BP1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="38" length="44"></location>
        <text>4E-BP2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="13" length="23"></location>
        <text>regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>132</offset>
      <text>Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="748" length="754"></location>
        <text>4E-BP1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="759" length="765"></location>
        <text>4E-BP2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="847" length="856"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="924" length="930"></location>
        <text>4E-BP1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="953" length="959"></location>
        <text>4E-BP1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1240" length="1246"></location>
        <text>4E-BP1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1315" length="1321"></location>
        <text>4E-BP2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Regulation</infon>
        <location offset="692" length="702"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="864" length="871"></location>
        <text>results</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="910" length="920"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Negative_regulation</infon>
        <location offset="1230" length="1239"></location>
        <text>decreases</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="1262" length="1271"></location>
        <text>affecting</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Phosphorylation</infon>
        <location offset="1276" length="1291"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="1305" length="1314"></location>
        <text>increases</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10096561</id>
    <passage>
      <offset>0</offset>
      <text>Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="15" length="19"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="11"></location>
        <text>Stimulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>138</offset>
      <text>Here, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="183" length="187"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="325" length="329"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="393" length="397"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="567" length="571"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="582" length="586"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="619" length="623"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="656" length="665"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="670" length="697"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="709" length="731"></location>
        <text>interleukin (IL)-1beta</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="735" length="739"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="752" length="756"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="775" length="780"></location>
        <text>CD40L</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="824" length="829"></location>
        <text>CD40L</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="878" length="911"></location>
        <text>intercellular adhesion molecule-1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="974" length="978"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="980" length="984"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="986" length="1009"></location>
        <text>tumor necrosis factor-a</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1015" length="1063"></location>
        <text>granulocyte macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1164" length="1168"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1183" length="1189"></location>
        <text>HLA-A2</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1192" length="1198"></location>
        <text>MelanA</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1199" length="1204"></location>
        <text>MART1</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1270" length="1276"></location>
        <text>HLA-A2</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1289" length="1295"></location>
        <text>MelanA</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1296" length="1302"></location>
        <text>MART-1</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1332" length="1336"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1421" length="1425"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="1426" length="1431"></location>
        <text>CD40L</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="1536" length="1540"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="169" length="179"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Gene_expression</infon>
        <location offset="338" length="348"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Negative_regulation</infon>
        <location offset="352" length="366"></location>
        <text>down-regulated</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Gene_expression</infon>
        <location offset="398" length="408"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Gene_expression</infon>
        <location offset="572" length="580"></location>
        <text>negative</text>
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        <text>enhanced</text>
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        <text>ligation</text>
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        <text>up-regulated</text>
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        <text>expression</text>
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        <text>secretion</text>
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        <text>ligation</text>
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        <text>ligation</text>
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        <infon key="type">Binding</infon>
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        <text>interactions</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <node refid="E12" role="Cause"></node>
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    <relation id="E17">
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      <infon key="type">Event</infon>
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      <node refid="E19" role="Theme"></node>
      <node refid="E12" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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    <relation id="E21">
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E22">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T28" role="Theme"></node>
      <node refid="T29" role="Theme2"></node>
    </relation>
    <relation id="M1">
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      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
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      <infon key="type">Event modification</infon>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
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    </relation>
  </document>
  <document>
    <id>PMID-10101001</id>
    <passage>
      <offset>0</offset>
      <text>Essential role of alveolar macrophages in intrapulmonary activation of NF-kappaB.</text>
    </passage>
    <passage>
      <offset>83</offset>
      <text>Acute inflammatory injury in rat lung induced by deposition of immunoglobulin G immune complexes requires expression of cytokines and chemokines as well as activation of the transcription factor nuclear factor (NF)-kappaB. There is little direct evidence regarding the role of alveolar macrophages in these activation events. In the present studies, rat lungs were depleted of alveolar macrophages by airway instillation of liposome-encapsulated dichloromethylene diphosphonate. These procedures, which greatly reduced the number of retrievable alveolar macrophages, suppressed activation of lung NF-kappaB in the inflammatory model. In addition, bronchoalveolar lavage levels of tumor necrosis factor-alpha (TNF-alpha) and the CXC chemokine, macrophage inflammatory protein-2, were substantially reduced. In parallel, upregulation of the lung vascular adhesion molecule, intercellular adhesion molecule-1, was greatly reduced by intrapulmonary instillation of phosphonate-containing liposomes. Neutrophil accumulation and development of lung injury were also substantially diminished. Lung instillation of TNF-alpha in alveolar macrophage-depleted rats restored the NF-kappaB activation response in whole lung. These data suggest that, in this inflammatory model, initial activation of NF-kappaB occurs in alveolar macrophages and the ensuing production of TNF-alpha may propagate NF-kappaB activation to other cell types in the lung.</text>
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        <text>tumor necrosis factor-alpha</text>
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        <text>TNF-alpha</text>
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        <text>TNF-alpha</text>
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        <text>reduced</text>
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        <text>upregulation</text>
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        <text>reduced</text>
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        <text>production</text>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <node refid="T6" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10196286</id>
    <passage>
      <offset>0</offset>
      <text>Role of cellular tumor necrosis factor receptor-associated factors in NF-kappaB activation and lymphocyte transformation by herpesvirus Saimiri STP.</text>
    </passage>
    <passage>
      <offset>150</offset>
      <text>The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-kappaB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10--&gt;R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10--&gt;R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts.</text>
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        <text>tumor necrosis factor receptor-associated factor (TRAF) 1</text>
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        <text>2</text>
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        <text>3</text>
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        <text>STP-A11</text>
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        <location offset="502" length="510"></location>
        <text>STP-C488</text>
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        <text>STP-C488</text>
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        <text>STP-C488</text>
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        <text>interleukin-2</text>
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        <text>STP-C488</text>
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        <text>TRAF2</text>
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        <text>STP-C488</text>
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        <text>associated</text>
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        <text>association</text>
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        <text>association</text>
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        <text>associate</text>
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        <text>transformation</text>
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  <document>
    <id>PMID-10229231</id>
    <passage>
      <offset>0</offset>
      <text>Regulation of Fas ligand expression and cell death by apoptosis-linked gene 4.</text>
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        <text>Fas ligand</text>
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        <text>apoptosis-linked gene 4</text>
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        <text>Regulation</text>
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        <location offset="25" length="35"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>80</offset>
      <text>Programmed cell death is a process required for the normal development of an organism. One of the best understood apoptotic pathways occurs in T lymphocytes and is mediated by Fas/Fas ligand (FasL) interaction. During studies of apoptosis induced by T cell-receptor engagement, we identified ALG-4F, a truncated transcript that prevents T cell-receptor-induced FasL upregulation and cell death. Overexpression of full-length ALG-4 induced transcription of FasL and, consequently, apoptosis. These results indicate that ALG-4 is necessary and sufficient for FasL expression. Fas/FasL interaction initiates cell death in many other systems, and its dysregulation is a mechanism by which several pathologic conditions arise. Understanding the molecular mechanisms of FasL regulation could be very useful in elucidating how these diseases develop and in identifying potential therapeutic targets.</text>
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        <text>Fas</text>
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        <text>Fas ligand</text>
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        <text>FasL</text>
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        <text>FasL</text>
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        <text>ALG-4</text>
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        <text>FasL</text>
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        <text>ALG-4</text>
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        <text>FasL</text>
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        <text>Fas</text>
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        <text>FasL</text>
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        <text>FasL</text>
      </annotation>
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        <infon key="type">Binding</infon>
        <location offset="278" length="289"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T17">
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        <text>prevents</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>induced</text>
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        <text>upregulation</text>
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        <text>Overexpression</text>
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        <text>Overexpression</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
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        <text>induced</text>
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        <infon key="type">Transcription</infon>
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        <text>transcription</text>
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      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="608" length="632"></location>
        <text>necessary and sufficient</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="642" length="652"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="663" length="674"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Regulation</infon>
        <location offset="727" length="740"></location>
        <text>dysregulation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T5" role="Theme2"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T12" role="Theme2"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10229275</id>
    <passage>
      <offset>0</offset>
      <text>High molecular weight dextran sulfate increases the activity of NF-kappaB-regulated promoter in monocyte-derived macrophages.</text>
    </passage>
    <passage>
      <offset>127</offset>
      <text>It is known that sulfated polysaccharides can mimic the action of common T-cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte-derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF-kappaB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF-kappaB. The activation of NF-kappaB correlated with the increased expression of B7.1 molecules. It was postulated that this NF-kappaB-regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV-1 in monocyte-derived macrophages.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="407" length="441"></location>
        <text>chloramphenicol acetyl transferase</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="443" length="446"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="682" length="685"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="816" length="820"></location>
        <text>B7.1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Positive_regulation</infon>
        <location offset="792" length="801"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Gene_expression</infon>
        <location offset="802" length="812"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10395645</id>
    <passage>
      <offset>0</offset>
      <text>Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3.</text>
    </passage>
    <passage>
      <offset>145</offset>
      <text>NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="1026" length="1036"></location>
        <text>Fas ligand</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="1038" length="1042"></location>
        <text>CD95</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1108" length="1117"></location>
        <text>caspase-1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1149" length="1158"></location>
        <text>caspase-3</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1165" length="1170"></location>
        <text>CPP32</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1171" length="1175"></location>
        <text>Yama</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1176" length="1183"></location>
        <text>apopain</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1190" length="1199"></location>
        <text>caspase-6</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1229" length="1238"></location>
        <text>caspase-3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1282" length="1309"></location>
        <text>DNA fragmentation factor-45</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="1354" length="1364"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T6" role="Event"></node>
      <node refid="T7" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10395652</id>
    <passage>
      <offset>0</offset>
      <text>Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="57" length="77"></location>
        <text>toll-like receptor 2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="47" length="56"></location>
        <text>activated</text>
      </annotation>
    </passage>
    <passage>
      <offset>80</offset>
      <text>Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="86" length="106"></location>
        <text>Toll-like receptor 2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="108" length="112"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="237" length="241"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="275" length="279"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="343" length="347"></location>
        <text>CD14</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="440" length="444"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="544" length="548"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="593" length="597"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="818" length="822"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="876" length="880"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="967" length="999"></location>
        <text>TNF receptor-associated factor 6</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1005" length="1030"></location>
        <text>NF-kappaB-inducing kinase</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1054" length="1058"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1069" length="1073"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1160" length="1164"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1232" length="1236"></location>
        <text>TLR2</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="143" length="151"></location>
        <text>responds</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="280" length="290"></location>
        <text>associates</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Binding</infon>
        <location offset="638" length="645"></location>
        <text>recruit</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Binding</infon>
        <location offset="788" length="802"></location>
        <text>form complexes</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1037" length="1048"></location>
        <text>coexpressed</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Negative_regulation</infon>
        <location offset="1218" length="1228"></location>
        <text>inhibition</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10395671</id>
    <passage>
      <offset>0</offset>
      <text>Activation of the Janus kinase 3-STAT5a pathway after CD40 triggering of human monocytes but not of resting B cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="18" length="32"></location>
        <text>Janus kinase 3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="33" length="39"></location>
        <text>STAT5a</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="54" length="58"></location>
        <text>CD40</text>
      </annotation>
    </passage>
    <passage>
      <offset>118</offset>
      <text>CD40/CD40 ligand interactions play a key role in the immune responses of B lymphocytes, monocytes, and dendritic cells. The signal transduction events triggered by cross-linking of the CD40 receptor have been widely studied in B cell lines, but little is known about signaling following CD40 stimulation of monocytes and resting tonsillar B cells. Therefore, we studied the CD40 pathway in highly purified human monocytes and resting B cells. After CD40 triggering, a similar activation of the NF-kappaB (but not of the AP-1) transcription factor complex occurred in both cell preparations. However, the components of the NF-kappaB complexes were different in monocytes and B cells, because p50 is part of the NF-kappaB complex induced by CD40 triggering in both monocytes and B cells, whereas p65 was only induced in B cells. In contrast, although the Janus kinase 3 tyrosine kinase was associated with CD40 molecules in both monocytes and resting B cells, Janus kinase 3 phosphorylation induction was observed only in CD40-activated monocytes, with subsequent induction of STAT5a DNA binding activity in the nucleus. These results suggest that the activation signals in human B cells and monocytes differ following CD40 stimulation. This observation is consistent with the detection of normal CD40-induced monocyte activation in patients with CD40 ligand+ hyper IgM syndrome in whom a defect in CD40-induced B cell activation has been reported.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="118" length="122"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="123" length="134"></location>
        <text>CD40 ligand</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="303" length="307"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="405" length="409"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="492" length="496"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="567" length="571"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="809" length="812"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="857" length="861"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="912" length="915"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="971" length="985"></location>
        <text>Janus kinase 3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1022" length="1026"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1076" length="1090"></location>
        <text>Janus kinase 3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1138" length="1142"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1193" length="1199"></location>
        <text>STAT5a</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1335" length="1339"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1413" length="1417"></location>
        <text>CD40</text>
      </annotation>
      <annotation id="T20">
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        <text>induction</text>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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  <document>
    <id>PMID-10402173</id>
    <passage>
      <offset>0</offset>
      <text>Suppression of NF-kappaB activation in normal T cells by supernatant fluid from human renal cell carcinomas.</text>
    </passage>
    <passage>
      <offset>110</offset>
      <text>T lymphocytes from patients with renal cell carcinoma (RCC) show reduced immune function and impaired activation of the transcription factor, NF-kappaB. We determined the mechanism of NF-kappaB suppression in T cells of RCC patient and determined whether supernatant fluid from RCC explants (RCC-S) induced the same phenotype of NF-kappaB suppression in normal T cells that is observed in patient T cells. The pattern of kappaB-binding activity in T cells of RCC patient was altered as compared to that seen in T cells obtained from normal volunteers. In some patients, no activation of RelA/NFkappaB1-binding activity was detectable, while in others kappaB-binding activity was modestly induced but the duration was reduced. IkappaBalpha was degraded normally following stimulation in both normal controls and T cells from RCC patients. RCC-S did not alter the cytoplasmic levels of RelA and NF-kappaB1 but did suppress their nuclear localization and inhibited the activation of RelA/NF-kappaB1 binding complexes. These results show that RCC-S can induce in normal T cells the same phenotype of impaired NF-kappaB activation that is detected in T cells of RCC patient. It also appears that NF-kappaB suppression by RCC-S may contribute to the immunosuppression of host immunity.</text>
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        <text>NFkappaB1</text>
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        <text>IkappaBalpha</text>
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        <text>RelA</text>
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        <text>NF-kappaB1</text>
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        <text>RelA</text>
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        <text>NF-kappaB1</text>
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        <text>binding</text>
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        <text>degraded</text>
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        <text>following</text>
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        <text>alter</text>
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        <text>suppress</text>
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        <text>nuclear</text>
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        <text>localization</text>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E9">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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      <node refid="T5" role="Theme"></node>
      <node refid="T14" role="ToLoc"></node>
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      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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      <node refid="T14" role="ToLoc"></node>
    </relation>
    <relation id="M1">
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      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
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      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
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  </document>
  <document>
    <id>PMID-10403270</id>
    <passage>
      <offset>0</offset>
      <text>Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels.</text>
    </passage>
    <passage>
      <offset>131</offset>
      <text>OBJECTIVE: To create monoclonal anti-endothelial cell antibodies (mAECA) from a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mechanism of EC activation. METHODS: A panel of mAECA was generated from peripheral blood lymphocytes of a patient with Takayasu arteritis, using Epstein-Barr virus transformation. Activity against macrovascular EC (HUVEC) and microvascular EC (human bone marrow EC immortalized by SV40) antigens was detected by enzyme-linked immunosorbent assay. Inhibition studies were used to select the monoclonal antibodies (mAECA) which share the same EC epitope binding specificity as the total IgG-AECA from the Takayasu arteritis patient. The binding of the mAECA to human aortic EC was studied by immunohistochemistry. The secretion levels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determined, to serve as markers for EC activation. The activated EC were examined for the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin. In addition, nuclear extracts of the mAECA-treated EC were analyzed for the induction of translocation of nuclear factor kappaB (NF-kappaB), using a specific NF-kappaB oligoprobe in an electrophoretic mobility shift assay. RESULTS: Six mAECA were selected, the mixture of which produced 100% inhibition of binding of the original IgG (from the patient with Takayasu arteritis) to HUVEC. All mAECA possessed high activity against macrovascular EC, but none had significant antimicrovascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA activated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA induced EC expression of adhesion molecules and increased adhesion of U937 monocytic cells to EC. In addition, these mAECA stimulated the nuclear translocation of the NF-kappaB transcription factor. CONCLUSION: Our findings suggest that AECA may directly stimulate EC in Takayasu arteritis through elevation of adhesion molecule expression associated with NF-kappaB activation and adhesion of monocytes, and may therefore play a pathogenic role in the development of the vasculopathy in Takayasu arteritis.</text>
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        <text>IgG</text>
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        <text>interleukin-6</text>
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        <infon key="type">Protein</infon>
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        <text>IL-6</text>
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        <infon key="type">Protein</infon>
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        <text>von Willebrand factor</text>
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        <infon key="type">Protein</infon>
        <location offset="1038" length="1041"></location>
        <text>vWF</text>
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        <infon key="type">Protein</infon>
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        <text>vascular cell adhesion molecule 1</text>
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        <text>intercellular adhesion molecule 1</text>
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        <text>E-selectin</text>
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        <text>IgG</text>
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        <text>IL-6</text>
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        <text>secretion</text>
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        <text>expression</text>
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        <text>increased</text>
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        <infon key="type">Localization</infon>
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        <text>secretion</text>
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    </relation>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E8">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
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      <infon key="modification type">Speculation</infon>
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    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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      <infon key="modification type">Speculation</infon>
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  </document>
  <document>
    <id>PMID-10409763</id>
    <passage>
      <offset>0</offset>
      <text>The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation.</text>
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        <text>latent membrane protein 1</text>
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        <location offset="124" length="129"></location>
        <text>TRADD</text>
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        <location offset="61" length="68"></location>
        <text>engages</text>
      </annotation>
    </passage>
    <passage>
      <offset>241</offset>
      <text>A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth.</text>
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        <text>tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein</text>
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        <text>TNFR1</text>
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        <location offset="629" length="634"></location>
        <text>TRADD</text>
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        <location offset="636" length="640"></location>
        <text>LMP1</text>
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        <text>TRADD</text>
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        <text>TNFR1</text>
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        <location offset="795" length="799"></location>
        <text>LMP1</text>
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        <location offset="817" length="822"></location>
        <text>TRADD</text>
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        <text>TNFR1</text>
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        <location offset="890" length="895"></location>
        <text>TRADD</text>
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        <location offset="917" length="921"></location>
        <text>LMP1</text>
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        <infon key="type">Protein</infon>
        <location offset="973" length="978"></location>
        <text>TRADD</text>
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        <location offset="1028" length="1033"></location>
        <text>TNFR1</text>
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        <location offset="1035" length="1039"></location>
        <text>LMP1</text>
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        <location offset="1193" length="1197"></location>
        <text>LMP1</text>
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        <location offset="1283" length="1288"></location>
        <text>TRADD</text>
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        <infon key="type">Protein</infon>
        <location offset="1297" length="1301"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1484" length="1488"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1490" length="1495"></location>
        <text>TRADD</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="326" length="336"></location>
        <text>associates</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Binding</infon>
        <location offset="1044" length="1052"></location>
        <text>interact</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Binding</infon>
        <location offset="1113" length="1123"></location>
        <text>associates</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Binding</infon>
        <location offset="1266" length="1277"></location>
        <text>association</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T2" role="Theme2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T5" role="Theme2"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
      <node refid="T25" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
      <node refid="T23" role="Theme2"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10411003</id>
    <passage>
      <offset>0</offset>
      <text>Regulatory effects of interleukin-11 during acute lung inflammatory injury.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="22" length="36"></location>
        <text>interleukin-11</text>
      </annotation>
    </passage>
    <passage>
      <offset>77</offset>
      <text>The role of interleukin-11 (IL-11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL-11 mRNA and protein were both up-regulated during the course of this inflammatory response. Exogenously administered IL-11 substantially reduced, in a dose-dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti-inflammatory effects of IL-11 were associated with reduced NF-kappaB activation in lung, reduced levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluids, and diminished up-regulation of lung vascular ICAM-1. It is interesting that IL-11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-inducible neutrophil chemoattractant (CINC); the presence of IL-11 did not affect these chemokines. However, BAL content of C5a was reduced by IL-11. These data indicate that IL-11 is a regulatory cytokine in the lung and that, like other members of this family, its anti-inflammatory properties appear to be linked to its suppression of NF-kappaB activation, diminished production of TNF-alpha, and reduced up-regulation of lung vascular ICAM-1.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="89" length="103"></location>
        <text>interleukin-11</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="105" length="110"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="188" length="193"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="308" length="313"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="494" length="499"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="577" length="604"></location>
        <text>tumor necrosis factor alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="606" length="615"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="703" length="709"></location>
        <text>ICAM-1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="734" length="739"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="796" length="829"></location>
        <text>macrophage inflammatory protein-2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="831" length="836"></location>
        <text>MIP-2</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="912" length="917"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="994" length="999"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1026" length="1031"></location>
        <text>IL-11</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1236" length="1245"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1290" length="1296"></location>
        <text>ICAM-1</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="221" length="233"></location>
        <text>up-regulated</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="559" length="573"></location>
        <text>reduced levels</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="661" length="671"></location>
        <text>diminished</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="672" length="685"></location>
        <text>up-regulation</text>
      </annotation>
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        <infon key="type">Regulation</infon>
        <location offset="748" length="754"></location>
        <text>affect</text>
      </annotation>
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        <infon key="type">Regulation</infon>
        <location offset="926" length="932"></location>
        <text>affect</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1211" length="1221"></location>
        <text>diminished</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="1222" length="1232"></location>
        <text>production</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1251" length="1258"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="1259" length="1272"></location>
        <text>up-regulation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T12" role="Event"></node>
      <node refid="T11" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T8" role="Event"></node>
      <node refid="T7" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T13" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10411007</id>
    <passage>
      <offset>0</offset>
      <text>ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="79" length="88"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="26" length="34"></location>
        <text>regulate</text>
      </annotation>
    </passage>
    <passage>
      <offset>91</offset>
      <text>We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="149" length="170"></location>
        <text>necrosis factor alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="172" length="181"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="252" length="261"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="381" length="390"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="439" length="442"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="840" length="844"></location>
        <text>GABP</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1011" length="1027"></location>
        <text>interferon-gamma</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1112" length="1121"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="282" length="290"></location>
        <text>activity</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="291" length="295"></location>
        <text>when</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="409" length="435"></location>
        <text>resulted in an enhancement</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Negative_regulation</infon>
        <location offset="473" length="483"></location>
        <text>eliminated</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="791" length="796"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="1028" length="1037"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Regulation</infon>
        <location offset="1056" length="1060"></location>
        <text>role</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="1098" length="1108"></location>
        <text>production</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10415075</id>
    <passage>
      <offset>0</offset>
      <text>Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="123" length="126"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="127" length="131"></location>
        <text>RelA</text>
      </annotation>
    </passage>
    <passage>
      <offset>153</offset>
      <text>Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="802" length="806"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1509" length="1512"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1616" length="1619"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1639" length="1642"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1703" length="1706"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Negative_regulation</infon>
        <location offset="1596" length="1615"></location>
        <text>decreased or absent</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Positive_regulation</infon>
        <location offset="1657" length="1681"></location>
        <text>transcriptionally active</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10415609</id>
    <passage>
      <offset>0</offset>
      <text>Possible differences in the mechanism(s) of action of different glucocorticoid hormone compounds.</text>
    </passage>
    <passage>
      <offset>99</offset>
      <text>Different glucocorticoid hormones (GCH) show differences in the intensity and in the kinetics of their immunomodulating activity. The mechanism(s) of action of GCH is under investigation, but is has been noted that they exert immune activity via the genomic pathway. We have studied the effects of prednisone (PDN), deflazacort (DFC), and dexamethasone (DXM) on the production of cytokines (IL-2, IL-6, TNF-alpha, IL-10) by peripheral T lymphocytes, and the effects on the inhibition of NF-kB DNA binding activity by activated Jurkat cell line. The data obtained show that the three GCH molecules exert an immunosuppression on cytokine production by T lymphocytes and a strong decrease in the nuclear translocation of NF-kB in Jurkat cells; moreover, (a) not all the cytokines investigated were affected, and not with the same intensity, by the three GCH and (b) DXM inhibited the binding activity of NF-kB less than that of DFC and PDN. These data are in agreement with the concept that different GCH compounds might differ in their binding and affinity properties, tissue-specific metabolism, and interaction with transcription factor.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="490" length="494"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="496" length="500"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="502" length="511"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="513" length="518"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Regulation</infon>
        <location offset="386" length="393"></location>
        <text>effects</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Gene_expression</infon>
        <location offset="465" length="475"></location>
        <text>production</text>
      </annotation>
    </passage>
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      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10416957</id>
    <passage>
      <offset>0</offset>
      <text>The p53 paradox in the pathogenesis of tumor progression.</text>
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        <infon key="type">Protein</infon>
        <location offset="4" length="7"></location>
        <text>p53</text>
      </annotation>
    </passage>
    <passage>
      <offset>59</offset>
      <text>Recent evidence suggests that the p53 molecule appears in two different forms: the mutant p53 that stimulates tumor progression, and wild type p53 that inhibits tumor progression. In addition, it has been established that tumor necrosis factor-alpha (TNF-alpha) can activate the expression of wild type p53 in concert with the nuclear transcription factor, NF-kappa B. Both TNF-alpha and NF-kappa B are also involved in the stimulation of the pathway that leads to the expression of major histocompatibility complex (MHC) class I molecules and, hence, antigen presentation to the T cells. In this paper we shall advance the hypothesis that: (i) TNF-alpha indirectly controls immune surveillance; and (ii) TNF-alpha controls DNA repair and tumor suppression through the regulation of wild type p53. Thus, it is hypothesized that elevated TNF-alpha is primarily responsible for promoting tumor progression.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="93" length="96"></location>
        <text>p53</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="149" length="152"></location>
        <text>p53</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="202" length="205"></location>
        <text>p53</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="281" length="308"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="310" length="319"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="362" length="365"></location>
        <text>p53</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="433" length="442"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="704" length="713"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="764" length="773"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="852" length="855"></location>
        <text>p53</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="896" length="905"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="325" length="333"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="338" length="348"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="828" length="838"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="887" length="895"></location>
        <text>elevated</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-10455128</id>
    <passage>
      <offset>0</offset>
      <text>Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis.</text>
    </passage>
    <passage>
      <offset>145</offset>
      <text>The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-beta activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimulation, which consisted of both IKK-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-alpha/beta heterodimer with NIK, IkappaB-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IkappaB-alpha and IkappaB-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate IkappaB-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappaB-dependent transcription more effectively than mutated IKK-alpha or -beta. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappaB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="488" length="497"></location>
        <text>IKK-alpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="499" length="507"></location>
        <text>IKK-beta</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="509" length="534"></location>
        <text>NF-kappaB-inducing kinase</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="536" length="539"></location>
        <text>NIK</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="542" length="551"></location>
        <text>IKK-gamma</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="589" length="619"></location>
        <text>IKK complex-associated protein</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="742" length="751"></location>
        <text>IKK-alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="760" length="768"></location>
        <text>IKK-beta</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="830" length="838"></location>
        <text>IKK-beta</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="992" length="1001"></location>
        <text>IKK-alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1006" length="1011"></location>
        <text>-beta</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1188" length="1191"></location>
        <text>NIK</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1193" length="1206"></location>
        <text>IkappaB-alpha</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1211" length="1219"></location>
        <text>-epsilon</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1298" length="1311"></location>
        <text>IkappaB-alpha</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1316" length="1331"></location>
        <text>IkappaB-epsilon</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1395" length="1404"></location>
        <text>IKK-alpha</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1406" length="1414"></location>
        <text>IKK-beta</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1420" length="1423"></location>
        <text>NIK</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1453" length="1456"></location>
        <text>NIK</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1478" length="1491"></location>
        <text>IkappaB-alpha</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1573" length="1576"></location>
        <text>NIK</text>
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      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1665" length="1674"></location>
        <text>IKK-alpha</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1678" length="1683"></location>
        <text>-beta</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Regulation</infon>
        <location offset="732" length="738"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="819" length="829"></location>
        <text>stimulated</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="862" length="871"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="955" length="966"></location>
        <text>stimulation</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Binding</infon>
        <location offset="1137" length="1148"></location>
        <text>association</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="1266" length="1269"></location>
        <text>led</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Binding</infon>
        <location offset="1332" length="1345"></location>
        <text>disappearance</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Binding</infon>
        <location offset="1355" length="1365"></location>
        <text>reassembly</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Regulation</infon>
        <location offset="1424" length="1432"></location>
        <text>remained</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="1441" length="1451"></location>
        <text>associated</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Phosphorylation</infon>
        <location offset="1464" length="1477"></location>
        <text>phosphorylate</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1464" length="1477"></location>
        <text>phosphorylate</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E24" role="Theme"></node>
      <node refid="T20" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E19" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E20" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E24" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E25" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1335418</id>
    <passage>
      <offset>0</offset>
      <text>Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="87" length="92"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="53" length="86"></location>
        <text>interact synergistically to raise</text>
      </annotation>
    </passage>
    <passage>
      <offset>129</offset>
      <text>We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="455" length="458"></location>
        <text>PKC</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="599" length="604"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="609" length="612"></location>
        <text>Jun</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="792" length="797"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="802" length="805"></location>
        <text>Jun</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="970" length="975"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="990" length="993"></location>
        <text>Jun</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1165" length="1168"></location>
        <text>PKC</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1271" length="1274"></location>
        <text>PKC</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="372" length="378"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="444" length="454"></location>
        <text>stimulates</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="589" length="595"></location>
        <text>levels</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="775" length="783"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Regulation</infon>
        <location offset="806" length="822"></location>
        <text>immunoreactivity</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="858" length="882"></location>
        <text>had only a slight effect</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="960" length="968"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="980" length="988"></location>
        <text>decrease</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="1257" length="1267"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1355356</id>
    <passage>
      <offset>0</offset>
      <text>Functional interaction between the two zinc finger domains of the v-erb A oncoprotein.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="66" length="73"></location>
        <text>v-erb A</text>
      </annotation>
    </passage>
    <passage>
      <offset>88</offset>
      <text>The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="92" length="99"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="268" length="275"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="401" length="423"></location>
        <text>retinoic acid receptor</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="429" length="436"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="536" length="543"></location>
        <text>c-erb A</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="602" length="609"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="832" length="839"></location>
        <text>c-erb A</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="916" length="923"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1027" length="1034"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1290" length="1297"></location>
        <text>v-erb A</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Negative_regulation</infon>
        <location offset="302" length="339"></location>
        <text>acts as a dominant negative inhibitor</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Regulation</infon>
        <location offset="588" length="597"></location>
        <text>important</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Regulation</infon>
        <location offset="752" length="756"></location>
        <text>role</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="894" length="910"></location>
        <text>restore function</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="932" length="950"></location>
        <text>rendered defective</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="1278" length="1285"></location>
        <text>crucial</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1675604</id>
    <passage>
      <offset>0</offset>
      <text>Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant.</text>
    </passage>
    <passage>
      <offset>112</offset>
      <text>The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="116" length="120"></location>
        <text>KBF1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="213" length="218"></location>
        <text>H-2Kb</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="250" length="253"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="399" length="403"></location>
        <text>KBF1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="404" length="407"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="531" length="536"></location>
        <text>c-rel</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="541" length="546"></location>
        <text>v-rel</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="592" length="596"></location>
        <text>KBF1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="597" length="600"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="659" length="663"></location>
        <text>KBF1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="664" length="667"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="874" length="878"></location>
        <text>KBF1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="879" length="882"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="884" length="886"></location>
        <text>c-</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="891" length="896"></location>
        <text>v-rel</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1169" length="1172"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1286" length="1291"></location>
        <text>H-2Kb</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="135" length="140"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Entity</infon>
        <location offset="148" length="158"></location>
        <text>enhancer A</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="415" length="435"></location>
        <text>binds as a homodimer</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Binding</infon>
        <location offset="449" length="466"></location>
        <text>form heterodimers</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Binding</infon>
        <location offset="690" length="694"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="714" length="724"></location>
        <text>form homo-</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Binding</infon>
        <location offset="728" length="740"></location>
        <text>heterodimers</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Negative_regulation</infon>
        <location offset="777" length="797"></location>
        <text>reduces or abolishes</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="815" length="822"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T2" role="Theme2"></node>
      <node refid="T19" role="Site2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T11" role="Theme2"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T11" role="Theme2"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1679576</id>
    <passage>
      <offset>0</offset>
      <text>NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="53" length="56"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="38" length="48"></location>
        <text>expressing</text>
      </annotation>
    </passage>
    <passage>
      <offset>109</offset>
      <text>The effect of constitutive Tax expression on the interaction of NF-kappa B with its recognition sequence and on NF-kappa B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-kappa B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-kappa B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="136" length="139"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="342" length="345"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="365" length="368"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="567" length="582"></location>
        <text>interferon beta</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="735" length="738"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="851" length="854"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="942" length="990"></location>
        <text>granulocyte-macrophage colony stimulating factor</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1186" length="1189"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="140" length="150"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="369" length="379"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="739" length="749"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="837" length="847"></location>
        <text>Expression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="877" length="885"></location>
        <text>resulted</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Transcription</infon>
        <location offset="889" length="902"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="932" length="941"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="1190" length="1200"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="E5" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1680914</id>
    <passage>
      <offset>0</offset>
      <text>Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="5" length="8"></location>
        <text>CD2</text>
      </annotation>
    </passage>
    <passage>
      <offset>86</offset>
      <text>The CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation, two processes that are involved in the spread of HIV infection. Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures. In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production. We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat. Furthermore, CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer. These studies suggest that interaction of CD2 with its natural ligand, LFA-3, may play a role in regulation of HIV expression.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="90" length="93"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="313" length="316"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="460" length="463"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="584" length="587"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="667" length="670"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="717" length="720"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="869" length="872"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="898" length="903"></location>
        <text>LFA-3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Binding</infon>
        <location offset="854" length="865"></location>
        <text>interaction</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T9" role="Theme2"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1682217</id>
    <passage>
      <offset>0</offset>
      <text>v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="6"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="48" length="54"></location>
        <text>c-erbA</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="137" length="141"></location>
        <text>CAII</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="7" length="21"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Negative_regulation</infon>
        <location offset="37" length="47"></location>
        <text>extinguish</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Regulation</infon>
        <location offset="102" length="112"></location>
        <text>regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>144</offset>
      <text>The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="148" length="154"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="260" length="266"></location>
        <text>c-erbA</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="477" length="483"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="488" length="494"></location>
        <text>c-erbA</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="570" length="591"></location>
        <text>carbonic anhydrase II</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="593" length="597"></location>
        <text>CAII</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="656" length="662"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="838" length="842"></location>
        <text>CAII</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="864" length="870"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="937" length="943"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="964" length="970"></location>
        <text>c-erbA</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1020" length="1026"></location>
        <text>c-erbA</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1053" length="1059"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1146" length="1152"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1209" length="1213"></location>
        <text>CAII</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1308" length="1312"></location>
        <text>CAII</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1345" length="1351"></location>
        <text>c-erbA</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1381" length="1385"></location>
        <text>CAII</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1454" length="1460"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1505" length="1511"></location>
        <text>v-erbA</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="495" length="499"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Transcription</infon>
        <location offset="613" length="626"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Negative_regulation</infon>
        <location offset="642" length="652"></location>
        <text>suppressed</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Regulation</infon>
        <location offset="798" length="804"></location>
        <text>target</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Negative_regulation</infon>
        <location offset="819" length="834"></location>
        <text>down-regulation</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Transcription</infon>
        <location offset="843" length="856"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Gene_expression</infon>
        <location offset="920" length="932"></location>
        <text>coexpressing</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="1199" length="1208"></location>
        <text>activated</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Transcription</infon>
        <location offset="1214" length="1227"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1487" length="1501"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Gene_expression</infon>
        <location offset="1487" length="1501"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Positive_regulation</infon>
        <location offset="1515" length="1523"></location>
        <text>required</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T9" role="Event"></node>
      <node refid="T8" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T9" role="Theme2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T9" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T12" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
      <node refid="E15" role="Cause"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1700011</id>
    <passage>
      <offset>0</offset>
      <text>NF-X2 that binds to the DRA X2-box is activator protein 1. Expression cloning of c-Jun.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="81" length="86"></location>
        <text>c-Jun</text>
      </annotation>
    </passage>
    <passage>
      <offset>89</offset>
      <text>Human class II MHC Ag are a family of cell surface glycoproteins. Their constitutive expression is limited to B lymphocytes and thymic epithelial cells. In many other cells their expression can be induced by IFN-gamma. Conserved upstream promoter sequences regulate this tissue-specific expression of class II genes. In the DRA promoter, one of these cis-acting regulatory motifs is the X2-box to which nuclear factor X2 (NF-X2) binds. Here, we present the isolation and characterization of the full-length cDNA clone encoding NF-X2. This cDNA clone was isolated by expression cDNA cloning, and encodes the human c-Jun protein, which together with c-Fos forms the heterodimeric activator protein-1 transcription complex. Whereas c-Fos/c-Jun heterodimers do not exist in B cells, they form and bind to the X2-box in class II nonexpressing cells. Thus, c-Fos/c-Jun heterodimers might contribute to the repression of DRA gene expression.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="297" length="306"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="702" length="707"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="737" length="742"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="818" length="823"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="824" length="829"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="940" length="945"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="946" length="951"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1003" length="1006"></location>
        <text>DRA</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Binding</infon>
        <location offset="801" length="808"></location>
        <text>complex</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="850" length="855"></location>
        <text>exist</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="873" length="877"></location>
        <text>form</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Binding</infon>
        <location offset="882" length="886"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Negative_regulation</infon>
        <location offset="989" length="999"></location>
        <text>repression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="1012" length="1022"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T4" role="Theme2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T7" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1712226</id>
    <passage>
      <offset>0</offset>
      <text>Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="14" length="17"></location>
        <text>jun</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="22" length="25"></location>
        <text>fos</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Regulation</infon>
        <location offset="0" length="10"></location>
        <text>Regulation</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="26" length="41"></location>
        <text>gene expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>107</offset>
      <text>The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M-CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="421" length="424"></location>
        <text>jun</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="429" length="432"></location>
        <text>fos</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="469" length="474"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="670" length="675"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="772" length="777"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="870" length="875"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="955" length="960"></location>
        <text>jun-B</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1034" length="1039"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1085" length="1090"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1223" length="1228"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1263" length="1268"></location>
        <text>fos-B</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1335" length="1340"></location>
        <text>fra-1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1380" length="1385"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1390" length="1395"></location>
        <text>fos-B</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="344" length="351"></location>
        <text>effects</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Regulation</infon>
        <location offset="393" length="402"></location>
        <text>involving</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="403" length="413"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Transcription</infon>
        <location offset="475" length="486"></location>
        <text>transcripts</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="492" length="502"></location>
        <text>detectable</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="657" length="666"></location>
        <text>increases</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Transcription</infon>
        <location offset="681" length="687"></location>
        <text>levels</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Regulation</infon>
        <location offset="762" length="771"></location>
        <text>regulates</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Gene_expression</infon>
        <location offset="778" length="788"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="800" length="808"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Transcription</infon>
        <location offset="812" length="825"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="837" length="866"></location>
        <text>prolongation in the half-life</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="938" length="947"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="976" length="986"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1004" length="1013"></location>
        <text>prolonged</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="1075" length="1084"></location>
        <text>increases</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="1206" length="1215"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1291" length="1298"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Gene_expression</infon>
        <location offset="1299" length="1309"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E30" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E30">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1719551</id>
    <passage>
      <offset>0</offset>
      <text>cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="46" length="50"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="55" length="59"></location>
        <text>JunB</text>
      </annotation>
    </passage>
    <passage>
      <offset>110</offset>
      <text>We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="130" length="134"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="391" length="395"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="499" length="503"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="603" length="607"></location>
        <text>JunB</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="652" length="656"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="673" length="677"></location>
        <text>JunB</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="737" length="742"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="792" length="796"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1168" length="1172"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="516" length="520"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Binding</infon>
        <location offset="691" length="695"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="718" length="736"></location>
        <text>in the presence of</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="758" length="765"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1740663</id>
    <passage>
      <offset>0</offset>
      <text>Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells.</text>
    </passage>
    <passage>
      <offset>142</offset>
      <text>The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="539" length="542"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="846" length="851"></location>
        <text>c-fms</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1208" length="1223"></location>
        <text>interferon beta</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1225" length="1233"></location>
        <text>IFN-beta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1398" length="1406"></location>
        <text>IFN-beta</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1411" length="1420"></location>
        <text>IFN-alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Positive_regulation</infon>
        <location offset="759" length="768"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Transcription</infon>
        <location offset="825" length="838"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Binding</infon>
        <location offset="1140" length="1150"></location>
        <text>interacted</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Entity</infon>
        <location offset="1173" length="1198"></location>
        <text>symmetric NF-kappa B site</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Transcription</infon>
        <location offset="1371" length="1383"></location>
        <text>Inducibility</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="1434" length="1443"></location>
        <text>increased</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T10" role="Site"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1740667</id>
    <passage>
      <offset>0</offset>
      <text>The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="122" length="135"></location>
        <text>interleukin 2</text>
      </annotation>
    </passage>
    <passage>
      <offset>147</offset>
      <text>Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="388" length="401"></location>
        <text>interleukin 2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="403" length="407"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="678" length="682"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="771" length="804"></location>
        <text>chloramphenicol acetyltransferase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="870" length="874"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="918" length="922"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="985" length="991"></location>
        <text>NFAT-1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1264" length="1268"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1382" length="1386"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1526" length="1531"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1536" length="1541"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1596" length="1600"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1765" length="1769"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="356" length="360"></location>
        <text>role</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="371" length="380"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Negative_regulation</infon>
        <location offset="655" length="664"></location>
        <text>abrogates</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Transcription</infon>
        <location offset="665" length="674"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="748" length="757"></location>
        <text>abolishes</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="758" length="767"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Regulation</infon>
        <location offset="1234" length="1240"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="1244" length="1256"></location>
        <text>inducibility</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Entity</infon>
        <location offset="1269" length="1277"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Negative_regulation</infon>
        <location offset="1356" length="1364"></location>
        <text>abrogate</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="1365" length="1374"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Entity</infon>
        <location offset="1387" length="1395"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Gene_expression</infon>
        <location offset="1542" length="1552"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="1571" length="1578"></location>
        <text>enhance</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="1579" length="1588"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Entity</infon>
        <location offset="1601" length="1609"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T23" role="Site"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
      <node refid="T26" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
      <node refid="E12" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T30" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1751404</id>
    <passage>
      <offset>0</offset>
      <text>The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation.</text>
    </passage>
    <passage>
      <offset>122</offset>
      <text>Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="476" length="481"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="1039" length="1044"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Gene_expression</infon>
        <location offset="482" length="492"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Gene_expression</infon>
        <location offset="1045" length="1055"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T3" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T4" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1763325</id>
    <passage>
      <offset>0</offset>
      <text>Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein.</text>
    </passage>
    <passage>
      <offset>96</offset>
      <text>Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="215" length="248"></location>
        <text>Hepatocyte nuclear factor-1 alpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="250" length="261"></location>
        <text>HNF-1 alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="328" length="364"></location>
        <text>dimerization cofactor of HNF-1 alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="366" length="370"></location>
        <text>DCoH</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="496" length="507"></location>
        <text>HNF-1 alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="553" length="557"></location>
        <text>DCoH</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="558" length="569"></location>
        <text>HNF-1 alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="621" length="632"></location>
        <text>HNF-1 alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="684" length="695"></location>
        <text>HNF-1 alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="749" length="753"></location>
        <text>DCoH</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="856" length="860"></location>
        <text>DCoH</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Localization</infon>
        <location offset="422" length="434"></location>
        <text>distribution</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Binding</infon>
        <location offset="447" length="451"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="485" length="495"></location>
        <text>stabilized</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="570" length="577"></location>
        <text>complex</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="585" length="593"></location>
        <text>required</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Regulation</infon>
        <location offset="642" length="648"></location>
        <text>change</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="657" length="664"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="E4" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-1990263</id>
    <passage>
      <offset>0</offset>
      <text>Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="33" length="46"></location>
        <text>proenkephalin</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Positive_regulation</infon>
        <location offset="23" length="32"></location>
        <text>activates</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Transcription</infon>
        <location offset="47" length="60"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>80</offset>
      <text>Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="201" length="214"></location>
        <text>proenkephalin</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="304" length="317"></location>
        <text>proenkephalin</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="622" length="635"></location>
        <text>proenkephalin</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="740" length="743"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="808" length="821"></location>
        <text>proenkephalin</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="874" length="877"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="888" length="891"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="974" length="987"></location>
        <text>proenkephalin</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="604" length="614"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Entity</infon>
        <location offset="636" length="644"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Negative_regulation</infon>
        <location offset="671" length="678"></location>
        <text>abolish</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="725" length="734"></location>
        <text>homodimer</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Binding</infon>
        <location offset="787" length="792"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Entity</infon>
        <location offset="797" length="804"></location>
        <text>B2 site</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="854" length="868"></location>
        <text>heterotetramer</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="956" length="966"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Entity</infon>
        <location offset="988" length="996"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="1000" length="1008"></location>
        <text>mediated</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
      <node refid="T13" role="Site"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T8" role="Theme2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T20" role="Site"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7478534</id>
    <passage>
      <offset>0</offset>
      <text>ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="4"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="30" length="36"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="5" length="19"></location>
        <text>transactivates</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Entity</infon>
        <location offset="37" length="45"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <passage>
      <offset>100</offset>
      <text>Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="275" length="323"></location>
        <text>Granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="325" length="331"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="542" length="548"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="733" length="737"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="742" length="746"></location>
        <text>ELF1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="885" length="891"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="933" length="937"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="947" length="951"></location>
        <text>ELF1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="971" length="977"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1254" length="1258"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1264" length="1270"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1308" length="1312"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1386" length="1390"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1478" length="1482"></location>
        <text>ETS1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1526" length="1532"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="360" length="369"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="370" length="380"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="401" length="410"></location>
        <text>increases</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Transcription</infon>
        <location offset="414" length="427"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="701" length="710"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Binding</infon>
        <location offset="797" length="805"></location>
        <text>interact</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="957" length="970"></location>
        <text>transactivate</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Entity</infon>
        <location offset="991" length="994"></location>
        <text>GM5</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Binding</infon>
        <location offset="1204" length="1211"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Negative_regulation</infon>
        <location offset="1241" length="1250"></location>
        <text>exception</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Entity</infon>
        <location offset="1271" length="1279"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Binding</infon>
        <location offset="1313" length="1321"></location>
        <text>specific</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Positive_regulation</infon>
        <location offset="1360" length="1369"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1391" length="1406"></location>
        <text>transactivation</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Regulation</infon>
        <location offset="1490" length="1498"></location>
        <text>involved</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="1502" length="1511"></location>
        <text>mediating</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1516" length="1525"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Gene_expression</infon>
        <location offset="1533" length="1543"></location>
        <text>production</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
      <node refid="T19" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E4" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
      <node refid="T27" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
      <node refid="T27" role="Site"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T14" role="Theme2"></node>
      <node refid="T30" role="Site"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T30" role="Site"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E17" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7478994</id>
    <passage>
      <offset>0</offset>
      <text>Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="41" length="56"></location>
        <text>15-lipoxygenase</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Regulation</infon>
        <location offset="16" length="26"></location>
        <text>regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>113</offset>
      <text>The 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="117" length="132"></location>
        <text>15-lipoxygenase</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="134" length="137"></location>
        <text>lox</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="334" length="340"></location>
        <text>15-lox</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="935" length="938"></location>
        <text>lox</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="147" length="156"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Negative_regulation</infon>
        <location offset="371" length="384"></location>
        <text>down-regulate</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7479915</id>
    <passage>
      <offset>0</offset>
      <text>Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="35" length="38"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="0" length="12"></location>
        <text>Coexpression</text>
      </annotation>
    </passage>
    <passage>
      <offset>137</offset>
      <text>Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="445" length="448"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="786" length="789"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1019" length="1022"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1142" length="1146"></location>
        <text>RelB</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1148" length="1151"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1157" length="1160"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1180" length="1183"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1285" length="1288"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="744" length="754"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="759" length="767"></location>
        <text>activity</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="1011" length="1018"></location>
        <text>express</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="1175" length="1179"></location>
        <text>lack</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="1254" length="1266"></location>
        <text>Coexpression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7486667</id>
    <passage>
      <offset>0</offset>
      <text>Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells.</text>
    </passage>
    <passage>
      <offset>95</offset>
      <text>The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="131" length="135"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="223" length="227"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="274" length="278"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="500" length="505"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1176" length="1181"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1483" length="1487"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1650" length="1655"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Transcription</infon>
        <location offset="99" length="123"></location>
        <text>transcriptional activity</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Positive_regulation</infon>
        <location offset="145" length="153"></location>
        <text>requires</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="259" length="270"></location>
        <text>participate</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Entity</infon>
        <location offset="279" length="287"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="288" length="298"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Phosphorylation</infon>
        <location offset="1131" length="1145"></location>
        <text>phosphorylates</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="1551" length="1557"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Phosphorylation</infon>
        <location offset="1656" length="1671"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T11" role="Site"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7488149</id>
    <passage>
      <offset>0</offset>
      <text>Cupric ion blocks NF kappa B activation through inhibiting the signal-induced phosphorylation of I kappa B alpha.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="97" length="112"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Negative_regulation</infon>
        <location offset="48" length="58"></location>
        <text>inhibiting</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Phosphorylation</infon>
        <location offset="78" length="93"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <passage>
      <offset>115</offset>
      <text>A transcription factor NF kappa B, which regulates expression of various cellular genes involved in immune responses and viral genes including HIV, is sequestered in the cytoplasm as a complex with an inhibitory protein I kappa B. Various extracellular signals induce phosphorylation and rapid degradation of I kappa B alpha to release NF kappa B. Cu2+ was found to inhibit the activation of NF kappa B induced by TNF-alpha, TPA, or H2O2. Deoxycholate treatment of the cytoplasmic extract prepared from cells stimulated by TNF-alpha in the presence of Cu2+ resulted in the release of NF kappa B from I kappa B alpha, indicating that Cu2+ interferes with the dissociation of the NF kappa B-I kappa B complex. Neither phosphorylation nor degradation of I kappa B alpha was observed upon TNF-alpha stimulation in the presence of Cu2+. These results indicate that Cu2+ inhibits the release of NF kappa B by blockade of a signal leading to the phosphorylation of I kappa B alpha.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="316" length="344"></location>
        <text>inhibitory protein I kappa B</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="424" length="439"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="529" length="538"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="638" length="647"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="715" length="730"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="804" length="813"></location>
        <text>I kappa B</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="866" length="881"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="900" length="909"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1073" length="1088"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Binding</infon>
        <location offset="300" length="307"></location>
        <text>complex</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="376" length="382"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Phosphorylation</infon>
        <location offset="383" length="398"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein_catabolism</infon>
        <location offset="409" length="420"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Phosphorylation</infon>
        <location offset="831" length="846"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein_catabolism</infon>
        <location offset="851" length="862"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="922" length="940"></location>
        <text>in the presence of</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Negative_regulation</infon>
        <location offset="1018" length="1026"></location>
        <text>blockade</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="1039" length="1046"></location>
        <text>leading</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Phosphorylation</infon>
        <location offset="1054" length="1069"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="E4" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7492771</id>
    <passage>
      <offset>0</offset>
      <text>Salicylates inhibit lipopolysaccharide-induced transcriptional activation of the tissue factor gene in human monocytic cells.</text>
    </passage>
    <passage>
      <offset>127</offset>
      <text>Binding of plasma Factor VII/VIIa to the tissue factor (TF) receptor initiates the coagulation protease cascades. TF expression by circulating monocytes is associated with thrombotic and inflammatory complications in a variety of diseases. Transcriptional activation of the human TF gene in monocytic cells exposed to bacterial lipopolysaccharide (LPS) is mediated by binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter. Here, we report that a family of anti-inflammatory agents, known as the salicylates, inhibited LPS induction of TF activity and TF gene transcription in human monocytes and monocytic THP-1 cells at clinically relevant doses. Furthermore, sodium salicylate blocked the LPS-induced proteolytic degradation of I kappa B alpha, which prevented the nuclear translocation of c-Rel/p65 heterodimers. In contrast, two other nonsteroidal anti-inflammatory drugs, ibuprofen and indomethacin, did not inhibit LPS induction of the TF gene. These results indicated that salicylates inhibited LPS induction of TF gene transcription in monocytic cells by preventing nuclear translocation of c-Rel/p65 heterodimers. The clinical benefits of salicylates in the treatment of several diseases, including atherosclerosis and rheumatoid arthritis, may be related to their ability to reduce monocyte gene expression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="145" length="160"></location>
        <text>Factor VII/VIIa</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="168" length="195"></location>
        <text>tissue factor (TF) receptor</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="241" length="243"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="407" length="409"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="506" length="511"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="512" length="515"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="554" length="556"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="679" length="681"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="695" length="697"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="874" length="889"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="936" length="941"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="942" length="945"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1086" length="1088"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1163" length="1165"></location>
        <text>TF</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1243" length="1248"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1249" length="1252"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Binding</infon>
        <location offset="127" length="134"></location>
        <text>Binding</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="244" length="254"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="367" length="393"></location>
        <text>Transcriptional activation</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="483" length="491"></location>
        <text>mediated</text>
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        <infon key="type">Binding</infon>
        <location offset="495" length="502"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Entity</infon>
        <location offset="557" length="565"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Negative_regulation</infon>
        <location offset="652" length="661"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="666" length="675"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Transcription</infon>
        <location offset="703" length="716"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Negative_regulation</infon>
        <location offset="823" length="830"></location>
        <text>blocked</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="839" length="846"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein_catabolism</infon>
        <location offset="847" length="870"></location>
        <text>proteolytic degradation</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Negative_regulation</infon>
        <location offset="897" length="906"></location>
        <text>prevented</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Entity</infon>
        <location offset="911" length="918"></location>
        <text>nuclear</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Localization</infon>
        <location offset="919" length="932"></location>
        <text>translocation</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Negative_regulation</infon>
        <location offset="1057" length="1064"></location>
        <text>inhibit</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1069" length="1078"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Negative_regulation</infon>
        <location offset="1136" length="1145"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="1150" length="1159"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Transcription</infon>
        <location offset="1171" length="1184"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Negative_regulation</infon>
        <location offset="1207" length="1217"></location>
        <text>preventing</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Entity</infon>
        <location offset="1218" length="1225"></location>
        <text>nuclear</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Localization</infon>
        <location offset="1226" length="1239"></location>
        <text>translocation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T2" role="Theme2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E6" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
      <node refid="T22" role="Site2"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
      <node refid="T22" role="Site2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T30" role="ToLoc"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T30" role="ToLoc"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
      <node refid="E24" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
      <node refid="E25" role="Cause"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E27" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E26" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
      <node refid="T38" role="ToLoc"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T38" role="ToLoc"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7495759</id>
    <passage>
      <offset>0</offset>
      <text>CIITA activates the expression of MHC class II genes in mouse T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>CIITA</text>
      </annotation>
    </passage>
    <passage>
      <offset>72</offset>
      <text>It has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="294" length="321"></location>
        <text>MHC class II transactivator</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="323" length="328"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="519" length="524"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="713" length="718"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="768" length="773"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Transcription</infon>
        <location offset="514" length="518"></location>
        <text>have</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Gene_expression</infon>
        <location offset="685" length="697"></location>
        <text>transfection</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Positive_regulation</infon>
        <location offset="685" length="697"></location>
        <text>transfection</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="754" length="764"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7499266</id>
    <passage>
      <offset>0</offset>
      <text>Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="21" length="36"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="144" length="159"></location>
        <text>I kappa B alpha</text>
      </annotation>
    </passage>
    <passage>
      <offset>162</offset>
      <text>Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex.</text>
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  </document>
  <document>
    <id>PMID-7499267</id>
    <passage>
      <offset>0</offset>
      <text>Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and Sp1 transcription factors.</text>
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        <text>involves cooperation</text>
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    <passage>
      <offset>219</offset>
      <text>We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma.</text>
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        <text>IFN-gamma</text>
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        <text>interleukin-6</text>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
      <node refid="T24" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T24" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T28" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
      <node refid="T28" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7505113</id>
    <passage>
      <offset>0</offset>
      <text>The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="49" length="52"></location>
        <text>tax</text>
      </annotation>
    </passage>
    <passage>
      <offset>102</offset>
      <text>The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="139" length="142"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="207" length="215"></location>
        <text>TNF-beta</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="313" length="316"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="459" length="462"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="520" length="523"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="614" length="617"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="708" length="711"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="796" length="799"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="889" length="892"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="905" length="908"></location>
        <text>fos</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="909" length="912"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="974" length="977"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1060" length="1063"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1088" length="1091"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1290" length="1293"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="222" length="231"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="449" length="458"></location>
        <text>expresses</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="529" length="537"></location>
        <text>inactive</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="566" length="576"></location>
        <text>importance</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="602" length="612"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="697" length="707"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="803" length="814"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="803" length="814"></location>
        <text>transfected</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7506531</id>
    <passage>
      <offset>0</offset>
      <text>Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases.</text>
    </passage>
    <passage>
      <offset>130</offset>
      <text>Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes. The regulatory role of protein tyrosine phosphatases (PTPases) in this process was explored by studying the effects of a powerful PTPase inhibitor, vanadate peroxide (pervanadate), on the activation cascade of Jurkat human leukaemic T-cells. Pervanadate induced activation of the tyrosine kinases lck and fyn (4- and 3-fold respectively) and a dramatic increase in tyrosine phosphorylation of cellular proteins, notably phospholipase C gamma 1. After this event, we observed a rise in intracellular Ca2+ concentration, corresponding to an influx. This effect required surface expression of the CD45 PTPase and was not observed in CD45-deficient variants of Jurkat cells. In the CD45-negative variant, the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing. Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain (CD25). Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production. Pervanadate activated NF-kappa B, as shown by an increase in DNA-binding activity of this transcription factor. We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation. Moreover, induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="562" length="565"></location>
        <text>lck</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="570" length="573"></location>
        <text>fyn</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="685" length="708"></location>
        <text>phospholipase C gamma 1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="859" length="863"></location>
        <text>CD45</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="895" length="899"></location>
        <text>CD45</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="943" length="947"></location>
        <text>CD45</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1154" length="1159"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1290" length="1294"></location>
        <text>CD69</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1311" length="1345"></location>
        <text>interleukin 2 receptor alpha-chain</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1347" length="1351"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1470" length="1483"></location>
        <text>interleukin 2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="527" length="537"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="618" length="626"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Entity</infon>
        <location offset="630" length="638"></location>
        <text>tyrosine</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Phosphorylation</infon>
        <location offset="639" length="654"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="841" length="851"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="1122" length="1132"></location>
        <text>stimulated</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Transcription</infon>
        <location offset="1133" length="1146"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="1169" length="1181"></location>
        <text>accumulation</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="1272" length="1282"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T10" role="Event"></node>
      <node refid="T9" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
      <node refid="T14" role="Site"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7510691</id>
    <passage>
      <offset>0</offset>
      <text>The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="4" length="17"></location>
        <text>interleukin-8</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Entity</infon>
        <location offset="18" length="22"></location>
        <text>AP-1</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Entity</infon>
        <location offset="27" length="39"></location>
        <text>kappa B-like</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Regulation</infon>
        <location offset="62" length="69"></location>
        <text>targets</text>
      </annotation>
    </passage>
    <passage>
      <offset>135</offset>
      <text>FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13-acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the IL-8 promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (c-Rel, p65, p50, and p49). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which NF-kappa B binding was also decreased by FK506, indicating that both IL-8 kappa B-like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="306" length="319"></location>
        <text>interleukin-8</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="321" length="325"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="497" length="501"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="944" length="948"></location>
        <text>JunD</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="952" length="957"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1167" length="1172"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1174" length="1177"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1179" length="1182"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1188" length="1191"></location>
        <text>p49</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1437" length="1441"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1464" length="1474"></location>
        <text>Ig kappa B</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1595" length="1599"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1600" length="1605"></location>
        <text>NF-AT</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1610" length="1617"></location>
        <text>NFIL-2A</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1628" length="1638"></location>
        <text>Ig kappa B</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1658" length="1662"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Negative_regulation</infon>
        <location offset="250" length="260"></location>
        <text>suppressed</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Transcription</infon>
        <location offset="265" length="278"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Negative_regulation</infon>
        <location offset="617" length="627"></location>
        <text>suppressed</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="647" length="654"></location>
        <text>through</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="686" length="693"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="746" length="753"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="923" length="933"></location>
        <text>recognized</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Entity</infon>
        <location offset="1663" length="1667"></location>
        <text>AP-1</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Entity</infon>
        <location offset="1672" length="1684"></location>
        <text>kappa B-like</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Regulation</infon>
        <location offset="1695" length="1704"></location>
        <text>terminals</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T19" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T18" role="Site"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T28" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7537762</id>
    <passage>
      <offset>0</offset>
      <text>Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation [see comments]</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="22" length="32"></location>
        <text>P-selectin</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="43" length="73"></location>
        <text>monocyte chemotactic protein-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="78" length="105"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Regulation</infon>
        <location offset="33" length="42"></location>
        <text>regulates</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Localization</infon>
        <location offset="106" length="115"></location>
        <text>secretion</text>
      </annotation>
    </passage>
    <passage>
      <offset>181</offset>
      <text>Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="430" length="440"></location>
        <text>P-selectin</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="526" length="556"></location>
        <text>monocyte chemotactic protein-1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="558" length="563"></location>
        <text>MCP-1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="569" length="596"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="598" length="607"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="757" length="767"></location>
        <text>P-selectin</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="786" length="796"></location>
        <text>P-selectin</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="825" length="857"></location>
        <text>P-selectin glycoprotein ligand-1</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="900" length="910"></location>
        <text>P-selectin</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1045" length="1050"></location>
        <text>MCP-1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1052" length="1061"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1127" length="1137"></location>
        <text>P-selectin</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="459" length="468"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="499" length="508"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Localization</infon>
        <location offset="513" length="522"></location>
        <text>secretion</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="627" length="631"></location>
        <text>when</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="777" length="785"></location>
        <text>prevents</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="802" length="809"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="1018" length="1026"></location>
        <text>required</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="1031" length="1041"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T8" role="Event"></node>
      <node refid="T7" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
      <node refid="T11" role="Theme2"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7575565</id>
    <passage>
      <offset>0</offset>
      <text>HMG-I binds to GATA motifs: implications for an HPFH syndrome.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Binding</infon>
        <location offset="6" length="11"></location>
        <text>binds</text>
      </annotation>
    </passage>
    <passage>
      <offset>64</offset>
      <text>We have examined binding of the nuclear protein HMG-I to the human gamma-globin promoter. We find that HMG-I binds preferentially to the more 3' of a pair of GATA motifs in the gamma-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (-175 T-C) in the area bound by HMG-I results in overexpression of gamma-globin in adult red blood cells (HPFH) and up-regulation of the gamma-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="112" length="117"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="167" length="172"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="315" length="321"></location>
        <text>GATA-1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="386" length="391"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="544" length="549"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="657" length="662"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="713" length="718"></location>
        <text>HMG-I</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Binding</infon>
        <location offset="81" length="88"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Binding</infon>
        <location offset="173" length="178"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="285" length="290"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Binding</infon>
        <location offset="377" length="382"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Binding</infon>
        <location offset="559" length="563"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="663" length="670"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7605990</id>
    <passage>
      <offset>0</offset>
      <text>Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="36" length="84"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Negative_regulation</infon>
        <location offset="13" length="32"></location>
        <text>negative regulation</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="22" length="32"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Entity</infon>
        <location offset="85" length="93"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <passage>
      <offset>149</offset>
      <text>The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM-CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13-acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="153" length="201"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="203" length="209"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="495" length="499"></location>
        <text>AML1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="587" length="595"></location>
        <text>alpha A1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="626" length="634"></location>
        <text>alpha B1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="639" length="647"></location>
        <text>alpha B2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="703" length="709"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="720" length="734"></location>
        <text>PEBP2 alpha A1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="736" length="744"></location>
        <text>alpha B1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="750" length="758"></location>
        <text>alpha B2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="806" length="812"></location>
        <text>GM-CSF</text>
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      <annotation id="T13">
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        <text>PEBP2 alpha A1</text>
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        <location offset="842" length="850"></location>
        <text>alpha B1</text>
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        <text>Coexpression</text>
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        <text>regulating</text>
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      <infon key="event type">Regulation</infon>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T24" role="Theme"></node>
      <node refid="T38" role="Site"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
      <node refid="T38" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
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    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7744623</id>
    <passage>
      <offset>0</offset>
      <text>Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy.</text>
    </passage>
    <passage>
      <offset>102</offset>
      <text>Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription. (ABSTRACT TRUNCATED AT 250 WORDS)</text>
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        <text>CD4</text>
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        <location offset="613" length="617"></location>
        <text>IL-2</text>
      </annotation>
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        <location offset="657" length="661"></location>
        <text>IL-2</text>
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        <location offset="1069" length="1073"></location>
        <text>IL-2</text>
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        <location offset="1137" length="1142"></location>
        <text>TCF-1</text>
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        <text>CD28RC</text>
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        <infon key="type">Protein</infon>
        <location offset="1525" length="1529"></location>
        <text>IL-2</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="580" length="591"></location>
        <text>up-regulate</text>
      </annotation>
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        <infon key="type">Transcription</infon>
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        <text>transcription</text>
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        <text>in consequence</text>
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        <text>produce</text>
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        <text>regulate</text>
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        <text>expression</text>
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        <text>binding</text>
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        <text>expressed</text>
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        <text>regulates</text>
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        <infon key="type">Transcription</infon>
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        <text>transcription</text>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
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      <node refid="T7" role="Theme"></node>
    </relation>
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      <infon key="modification type">Negation</infon>
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  </document>
  <document>
    <id>PMID-7747417</id>
    <passage>
      <offset>0</offset>
      <text>The retinoblastoma gene product negatively regulates transcriptional activation mediated by the human cytomegalovirus IE2 protein.</text>
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        <location offset="118" length="121"></location>
        <text>IE2</text>
      </annotation>
    </passage>
    <passage>
      <offset>132</offset>
      <text>The IE2 gene product of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately upon infection of the host cell. It is a potent transcriptional activator of many viral and cellular promoters. We found that the retinoblastoma susceptibility gene product (Rb) dramatically suppressed this IE2 transactivation of various promoters. However, unlike another tumor suppressor protein, p53, Rb did not have any significant effect on basal levels of transcription, suggesting that Rb specifically interacts with IE2 rather than other cellular factors involved in the general transcription machinery. We found by protein-affinity chromatography that Rb in nuclear extracts or produced by in vitro translation directly bound to IE2. Our results suggest that Rb may regulate the life cycle of HCMV, which is endemic in the human population. Furthermore, these data may provide new insights into the slow rate of HCMV DNA replication in cells and the possible involvement of HCMV in tumorigenesis.</text>
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        <infon key="type">Protein</infon>
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        <text>IE2</text>
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        <location offset="423" length="425"></location>
        <text>Rb</text>
      </annotation>
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        <location offset="456" length="459"></location>
        <text>IE2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="548" length="551"></location>
        <text>p53</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="553" length="555"></location>
        <text>Rb</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="642" length="644"></location>
        <text>Rb</text>
      </annotation>
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        <location offset="673" length="676"></location>
        <text>IE2</text>
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        <location offset="810" length="812"></location>
        <text>Rb</text>
      </annotation>
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        <location offset="887" length="890"></location>
        <text>IE2</text>
      </annotation>
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        <location offset="917" length="919"></location>
        <text>Rb</text>
      </annotation>
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        <location offset="227" length="236"></location>
        <text>expressed</text>
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        <text>upon</text>
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        <infon key="type">Binding</infon>
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        <text>interacts</text>
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      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="878" length="883"></location>
        <text>bound</text>
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    </passage>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
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    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E3">
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      <infon key="type">Event</infon>
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      <node refid="T8" role="Theme2"></node>
    </relation>
    <relation id="E4">
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      <infon key="type">Event</infon>
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      <node refid="T10" role="Theme2"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7747440</id>
    <passage>
      <offset>0</offset>
      <text>Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of BZLF-1 and its repressor RAZ.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="129" length="135"></location>
        <text>BZLF-1</text>
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        <infon key="type">Protein</infon>
        <location offset="154" length="157"></location>
        <text>RAZ</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="115" length="125"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>160</offset>
      <text>Epstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the BamHI ZLF-1 gene product, ZEBRA, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (RAZ), which comprised regions of the BZLF-1 locus and the adjacent BRLF-1 locus, was detected by RT-PCR. ZEBRA protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the EBNA-1 gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding gp350/220, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="595" length="606"></location>
        <text>BamHI ZLF-1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="621" length="626"></location>
        <text>ZEBRA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="723" length="726"></location>
        <text>RAZ</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="760" length="766"></location>
        <text>BZLF-1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="790" length="796"></location>
        <text>BRLF-1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="828" length="833"></location>
        <text>ZEBRA</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="947" length="953"></location>
        <text>EBNA-1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1121" length="1130"></location>
        <text>gp350/220</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1239" length="1245"></location>
        <text>LMP-2A</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1249" length="1255"></location>
        <text>EBER-1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="577" length="587"></location>
        <text>appearance</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="682" length="692"></location>
        <text>appearance</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="851" length="861"></location>
        <text>identified</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Transcription</infon>
        <location offset="986" length="997"></location>
        <text>transcribed</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="998" length="1002"></location>
        <text>from</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="1020" length="1031"></location>
        <text>rather than</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Transcription</infon>
        <location offset="1100" length="1111"></location>
        <text>Transcripts</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Transcription</infon>
        <location offset="1216" length="1229"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7747447</id>
    <passage>
      <offset>0</offset>
      <text>Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="13" length="16"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="9"></location>
        <text>Induction</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Phosphorylation</infon>
        <location offset="17" length="32"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <passage>
      <offset>135</offset>
      <text>In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="566" length="569"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="768" length="771"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="851" length="854"></location>
        <text>PHA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="859" length="872"></location>
        <text>interleukin 2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="942" length="945"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1030" length="1033"></location>
        <text>SP1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1124" length="1128"></location>
        <text>PP2A</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1228" length="1231"></location>
        <text>Tat</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1237" length="1240"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="704" length="711"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Phosphorylation</infon>
        <location offset="720" length="735"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="946" length="956"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Phosphorylation</infon>
        <location offset="1034" length="1049"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="1050" length="1057"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1130" length="1141"></location>
        <text>facilitates</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Binding</infon>
        <location offset="1175" length="1182"></location>
        <text>complex</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7749985</id>
    <passage>
      <offset>0</offset>
      <text>Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="75" length="80"></location>
        <text>CIITA</text>
      </annotation>
    </passage>
    <passage>
      <offset>153</offset>
      <text>Type II major histocompatibility complex combined immune deficiency (type II MHC CID or bare lymphocyte syndrome) is a congenital immunodeficiency disease characterized by absent MHC class II expression. Four distinct complementation groups have been identified. Recently, the defective gene in group II type II MHC CID has been isolated and termed CIITA. Here, we demonstrate that CIITA is an MHC class II gene-specific transcription activator. The transcription activation function is provided by the N-terminal acidic domain (amino acids 26-137), which is experimentally exchangeable with a heterologous viral transcription-activating domain. The specificity of CIITA for three major MHC class II genes, DR, DQ and DP, is mediated by its remaining C-terminal residues (amino acids 317-1130). The transactivation of multiple cis elements, especially S and X2, of the DR alpha proximal promoter in group II CID cells is CIITA dependent. Since CIITA overexpression in normal cells did not increase class II expression, we propose that initiation of CIITA expression serves as the on-off switch, while availability of downstream interactor(s) limits transcription.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="502" length="507"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="535" length="540"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="818" length="823"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1022" length="1030"></location>
        <text>DR alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1074" length="1079"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1097" length="1102"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1202" length="1207"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Positive_regulation</infon>
        <location offset="952" length="967"></location>
        <text>transactivation</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Entity</infon>
        <location offset="1005" length="1006"></location>
        <text>S</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Entity</infon>
        <location offset="1011" length="1013"></location>
        <text>X2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="1080" length="1089"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="1103" length="1117"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="1103" length="1117"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="1188" length="1198"></location>
        <text>initiation</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="1208" length="1218"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T10" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T11" role="Site"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7759875</id>
    <passage>
      <offset>0</offset>
      <text>Abnormal regulation of the IL-2 promoter in lpr CD4-CD8- T lymphocytes results in constitutive expression of a novel nuclear factor of activated T cells-binding factor.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="27" length="31"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="48" length="51"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="0" length="19"></location>
        <text>Abnormal regulation</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Entity</infon>
        <location offset="32" length="40"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <passage>
      <offset>170</offset>
      <text>The inert quality of MRL-Ipr/Ipr (Ipr) peripheral CD4-CD8- (CD4-8-) T cells manifests primarily as an inability to proliferate or produce IL-2 in response to TCR or mitogenic stimulation. Yet these same cells do initiate early TCR-mediated signaling events, such as generation of inositol phosphates and increased intracellular calcium. They also display constitutively high levels of p59fyn and CD3 zeta tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the IL-2 gene. We, therefore, compared the activation state of the IL-2 gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, NF-kappa B (p50-p65 heterodimer), and AP-1 transcriptional factors are constitutively elevated in freshly isolated Ipr CD4-8- T cells, consistent with the activated phenotype of these cells. Upon stimulation with mitogens, formation of the transactivating complex, nuclear factor of activated T cells (NF-AT), occurs with normal kinetics in Ipr CD4-8- T cells. Yet, the levels of the activating NF-AT complex never reach those observed in similarly stimulated normal T cells. Furthermore, nuclear extracts from Ipr CD4-8- T cells display high levels of a novel specific binding activity at the NF-AT site, which is present at much lower levels in freshly isolated normal T lymphocytes. Upon mitogenic stimulation, the binding activity of the novel NF-AT-binding factor is rapidly down-regulated in normal T cells, but persists at high levels in Ipr CD4-8- T cells. These two abnormalities at the NF-AT site provide a potential mechanism to account for the defect in IL-2 production from Ipr CD4-8- T cells.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="220" length="223"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="308" length="312"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="555" length="561"></location>
        <text>p59fyn</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="566" length="574"></location>
        <text>CD3 zeta</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="737" length="741"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="800" length="804"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="950" length="953"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="954" length="957"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1904" length="1908"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="272" length="281"></location>
        <text>inability</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="300" length="307"></location>
        <text>produce</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="540" length="551"></location>
        <text>high levels</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Entity</infon>
        <location offset="575" length="583"></location>
        <text>tyrosine</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Phosphorylation</infon>
        <location offset="584" length="599"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="692" length="699"></location>
        <text>results</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="703" length="729"></location>
        <text>transcriptional activation</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="776" length="786"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="1878" length="1885"></location>
        <text>account</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Negative_regulation</infon>
        <location offset="1894" length="1900"></location>
        <text>defect</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="1909" length="1919"></location>
        <text>production</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T13" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7759956</id>
    <passage>
      <offset>0</offset>
      <text>GM-CSF and IL-2 share common control mechanisms in response to costimulatory signals in T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="6"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="11" length="15"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Regulation</infon>
        <location offset="48" length="62"></location>
        <text>in response to</text>
      </annotation>
    </passage>
    <passage>
      <offset>98</offset>
      <text>Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="616" length="664"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="979" length="992"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="994" length="998"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1004" length="1008"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1089" length="1093"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1180" length="1228"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1230" length="1236"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1457" length="1463"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1530" length="1533"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1570" length="1576"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1581" length="1585"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1687" length="1690"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1705" length="1711"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1902" length="1905"></location>
        <text>tax</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1971" length="1977"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="2019" length="2023"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="2039" length="2043"></location>
        <text>IL-3</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="2132" length="2138"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="2143" length="2147"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="665" length="673"></location>
        <text>requires</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Regulation</infon>
        <location offset="755" length="762"></location>
        <text>respond</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Transcription</infon>
        <location offset="1009" length="1027"></location>
        <text>gene transcription</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="1071" length="1081"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="1136" length="1146"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Entity</infon>
        <location offset="1238" length="1246"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="1448" length="1456"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Gene_expression</infon>
        <location offset="1464" length="1474"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="1535" length="1565"></location>
        <text>acts as a costimulatory signal</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Transcription</infon>
        <location offset="1586" length="1604"></location>
        <text>gene transcription</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1691" length="1700"></location>
        <text>activates</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Entity</infon>
        <location offset="1712" length="1720"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Regulation</infon>
        <location offset="2148" length="2155"></location>
        <text>respond</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T8" role="Event"></node>
      <node refid="T9" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
      <node refid="T28" role="Site"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T14" role="Cause"></node>
      <node refid="T34" role="Site"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7760807</id>
    <passage>
      <offset>0</offset>
      <text>MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="143" length="154"></location>
        <text>MIP-1 alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="104" length="111"></location>
        <text>crucial</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="116" length="129"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>162</offset>
      <text>Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="169" length="208"></location>
        <text>macrophage inflammatory protein 1 alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="210" length="221"></location>
        <text>MIP-1 alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="249" length="254"></location>
        <text>GOS19</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="256" length="260"></location>
        <text>LD78</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="265" length="270"></location>
        <text>AT464</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="408" length="419"></location>
        <text>MIP-1 alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="420" length="425"></location>
        <text>GOS19</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="716" length="727"></location>
        <text>MIP-1 alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="728" length="733"></location>
        <text>GOS19</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="861" length="866"></location>
        <text>GOS19</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="889" length="895"></location>
        <text>ICK-1A</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Localization</infon>
        <location offset="339" length="347"></location>
        <text>Secreted</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="400" length="407"></location>
        <text>derived</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Regulation</infon>
        <location offset="690" length="697"></location>
        <text>control</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="702" length="712"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="850" length="856"></location>
        <text>affect</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Entity</infon>
        <location offset="867" length="875"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="1000" length="1009"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
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      <node refid="T8" role="Event"></node>
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    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
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    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T9" role="Event"></node>
      <node refid="T10" role="Event"></node>
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    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
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    <relation id="E3">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T7" role="Theme"></node>
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    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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  </document>
  <document>
    <id>PMID-7760810</id>
    <passage>
      <offset>0</offset>
      <text>Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="66" length="72"></location>
        <text>GATA-1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="80" length="97"></location>
        <text>estrogen receptor</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="17" length="27"></location>
        <text>repression</text>
      </annotation>
    </passage>
    <passage>
      <offset>100</offset>
      <text>High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. (ABSTRACT TRUNCATED AT 250 WORDS)</text>
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        <text>alpha-</text>
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        <infon key="type">Protein</infon>
        <location offset="388" length="399"></location>
        <text>beta-globin</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="459" length="461"></location>
        <text>H5</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="693" length="699"></location>
        <text>GATA-1</text>
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        <infon key="type">Protein</infon>
        <location offset="926" length="932"></location>
        <text>GATA-1</text>
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        <infon key="type">Protein</infon>
        <location offset="962" length="979"></location>
        <text>estrogen receptor</text>
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        <infon key="type">Protein</infon>
        <location offset="981" length="983"></location>
        <text>ER</text>
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        <location offset="1092" length="1094"></location>
        <text>ER</text>
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        <location offset="1118" length="1124"></location>
        <text>GATA-1</text>
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        <infon key="type">Protein</infon>
        <location offset="1285" length="1291"></location>
        <text>GATA-1</text>
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        <location offset="1293" length="1299"></location>
        <text>GATA-1</text>
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        <location offset="1304" length="1306"></location>
        <text>ER</text>
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        <location offset="1424" length="1430"></location>
        <text>GATA-1</text>
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        <location offset="1435" length="1437"></location>
        <text>ER</text>
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        <infon key="type">Protein</infon>
        <location offset="1512" length="1518"></location>
        <text>GATA-1</text>
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        <location offset="1527" length="1529"></location>
        <text>ER</text>
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        <location offset="1630" length="1636"></location>
        <text>GATA-1</text>
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        <location offset="1712" length="1718"></location>
        <text>GATA-1</text>
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        <location offset="1773" length="1775"></location>
        <text>ER</text>
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        <text>down-regulation</text>
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        <infon key="type">Regulation</infon>
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        <text>effects</text>
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        <text>bind</text>
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        <infon key="type">Binding</infon>
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        <text>associate</text>
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        <text>dependent</text>
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        <text>contacts</text>
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        <text>finger region</text>
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        <infon key="type">Entity</infon>
        <location offset="1598" length="1626"></location>
        <text>N-terminal activation domain</text>
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        <text>modulating</text>
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        <infon key="type">Binding</infon>
        <location offset="1752" length="1763"></location>
        <text>interaction</text>
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      <infon key="type">Equiv</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T14" role="Theme2"></node>
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    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T15" role="Theme"></node>
      <node refid="T16" role="Theme2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T18" role="Theme2"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T18" role="Theme2"></node>
      <node refid="T30" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T21" role="Theme2"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-7769834</id>
    <passage>
      <offset>0</offset>
      <text>Activation of pp90rsk and early growth response-1 gene expression by pokeweed mitogen in human B cells.</text>
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        <infon key="type">Protein</infon>
        <location offset="26" length="49"></location>
        <text>early growth response-1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="10"></location>
        <text>Activation</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="55" length="65"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>105</offset>
      <text>The present studies have examined the effects of pokeweed mitogen (PWM) on the induction of early growth response-1 gene (EGR-1) in normal human B cells. PWM regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene demonstrated that PWM induced EGR-1 transcription is conferred by the CArG motif (C C[AT]6GG) in the EGR-1 promoter. The results further demonstrated the activation of S6 kinase (pp90rsk), evidenced by phosphorylation of S6 and serum response factor (SRF) peptides, in PWM treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells.</text>
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        <infon key="type">Protein</infon>
        <location offset="197" length="220"></location>
        <text>early growth response-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="227" length="232"></location>
        <text>EGR-1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="273" length="278"></location>
        <text>EGR-1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="391" length="396"></location>
        <text>EGR-1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="430" length="463"></location>
        <text>chloramphenicol acetyltransferase</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="465" length="468"></location>
        <text>CAT</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="505" length="510"></location>
        <text>EGR-1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="576" length="581"></location>
        <text>EGR-1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="703" length="724"></location>
        <text>serum response factor</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="726" length="729"></location>
        <text>SRF</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="876" length="881"></location>
        <text>EGR-1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="143" length="150"></location>
        <text>effects</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="184" length="193"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Regulation</infon>
        <location offset="263" length="272"></location>
        <text>regulates</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="284" length="294"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="497" length="504"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Transcription</infon>
        <location offset="511" length="524"></location>
        <text>transcription</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="528" length="537"></location>
        <text>conferred</text>
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        <infon key="type">Entity</infon>
        <location offset="545" length="555"></location>
        <text>CArG motif</text>
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        <location offset="677" length="692"></location>
        <text>phosphorylation</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="882" length="891"></location>
        <text>induction</text>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E2">
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      <infon key="type">Event</infon>
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      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
      <node refid="T22" role="CSite"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8096091</id>
    <passage>
      <offset>0</offset>
      <text>NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="44" length="59"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="11" length="19"></location>
        <text>controls</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="20" length="30"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>112</offset>
      <text>The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="382" length="397"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="417" length="444"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="530" length="545"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="568" length="583"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="640" length="655"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="782" length="797"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="803" length="806"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1000" length="1015"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein_catabolism</infon>
        <location offset="505" length="516"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="554" length="564"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="589" length="599"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="677" length="684"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Binding</infon>
        <location offset="745" length="756"></location>
        <text>Association</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Regulation</infon>
        <location offset="973" length="981"></location>
        <text>controls</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="986" length="996"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T8" role="Theme2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8098618</id>
    <passage>
      <offset>0</offset>
      <text>Activation of primary human T-lymphocytes through CD2 plus CD28 adhesion molecules induces long-term nuclear expression of NF-kappa B.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="50" length="53"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="59" length="63"></location>
        <text>CD28</text>
      </annotation>
    </passage>
    <passage>
      <offset>136</offset>
      <text>Stimulation of highly purified human T-cells via CD2 and CD28 adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent interleukin 2 (IL-2)-dependent activation does not require monocytes or accessory cells. Long-lasting IL-2 receptivity is associated with high-level expression of the inducible IL-2 receptor alpha chain (IL-2R alpha) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of IL-2R alpha gene transcription involves the enhanced binding of the transcription factor NF-kappa B to its consensus sequence in the 5'-regulatory region of the IL-2R alpha gene. To dissect the molecular basis for the unusually persistent transcription of the IL-2R alpha gene, we analyzed nuclear NF-kappa B binding to a radiolabeled IL-2R alpha kappa B-specific oligonucleotide probe during the time course of CD2 + CD28 activation. Resting T-cell nuclear extracts contained KBF1/p50 homodimer. After stimulation, two new kappa B-specific complexes were identified as NF-kappa B p50-p65 heterodimer and putative c-Rel homodimer or c-Rel-p65 heterodimer. Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, CD2 + CD28 activation triggered a significant intracellular thiol decrease, suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules. Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of NF-kappa B in T-lymphocytes, also inhibited IL-2 secretion, IL-2R alpha cell surface expression, and T-cell proliferation. Together, these results suggest that NF-kappa B plays an important role in long-term activation of human primary T-lymphocytes via CD2 + CD28.</text>
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        <infon key="type">Protein</infon>
        <location offset="185" length="188"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="193" length="197"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="288" length="301"></location>
        <text>interleukin 2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="303" length="307"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="390" length="394"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="465" length="490"></location>
        <text>IL-2 receptor alpha chain</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="492" length="503"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="596" length="607"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="757" length="768"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="856" length="867"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="931" length="942"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1008" length="1011"></location>
        <text>CD2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1014" length="1018"></location>
        <text>CD28</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1073" length="1077"></location>
        <text>KBF1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1078" length="1081"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1177" length="1180"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1181" length="1184"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1210" length="1215"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1229" length="1234"></location>
        <text>c-Rel</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1235" length="1238"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1397" length="1400"></location>
        <text>CD2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1403" length="1407"></location>
        <text>CD28</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1749" length="1753"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1765" length="1776"></location>
        <text>IL-2R alpha</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1959" length="1962"></location>
        <text>CD2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1965" length="1969"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Gene_expression</infon>
        <location offset="437" length="447"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Regulation</infon>
        <location offset="518" length="527"></location>
        <text>regulated</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="584" length="592"></location>
        <text>Increase</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Transcription</infon>
        <location offset="613" length="626"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="640" length="648"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="649" length="656"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Transcription</infon>
        <location offset="835" length="848"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1019" length="1029"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Positive_regulation</infon>
        <location offset="1152" length="1162"></location>
        <text>identified</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Binding</infon>
        <location offset="1185" length="1196"></location>
        <text>heterodimer</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Binding</infon>
        <location offset="1216" length="1225"></location>
        <text>homodimer</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Binding</infon>
        <location offset="1239" length="1250"></location>
        <text>heterodimer</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Positive_regulation</infon>
        <location offset="1408" length="1418"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Negative_regulation</infon>
        <location offset="1739" length="1748"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Localization</infon>
        <location offset="1754" length="1763"></location>
        <text>secretion</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Gene_expression</infon>
        <location offset="1790" length="1800"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T6" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T16" role="Event"></node>
      <node refid="T17" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T9" role="Event"></node>
      <node refid="T8" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
      <node refid="T19" role="Theme2"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
      <node refid="T22" role="Theme2"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T25" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8098881</id>
    <passage>
      <offset>0</offset>
      <text>Synergism between the CD3 antigen- and CD2 antigen-derived signals. Exploration at the level of induction of DNA-binding proteins and characterization of the inhibitory activity of cyclosporine.</text>
    </passage>
    <passage>
      <offset>196</offset>
      <text>We have demonstrated earlier that the crosslinkage of the CD3/TCR complex with the CD2 antigen results in the proliferation of normal human T cells. The effect of this synergism was perceptible at the level of induction of the IL-2 gene, a process critical for T cell growth. To further understand the molecular and nuclear basis for this synergism, we have explored the induction of DNA-binding proteins in highly purified normal human T cells signaled via the CD3 and/or CD2 proteins. The effect of transmembrane signaling of T cells with ionomycin, and/or sn-1,2 dioctanoyl glycerol, was also determined. The emergence of nuclear binding proteins was investigated using interleukin-2 sequence specific oligonucleotide probes in the electrophoretic mobility shift assay. Our studies demonstrate for the first time that CD3 antigen-derived signals and CD2 antigen-derived signals are synergistic in inducing the emergence of transcription factors that bind to the NF-AT1, AP-1, and NF-kB sites located in the promoter/enhancer region of the IL-2 gene. Moreover, cyclosporine, at concentrations readily accomplished in clinical practice, was found to inhibit the emergence of these DNA-binding proteins in normal human T cells signaled via cell surface proteins implicated in antigen-dependent T cell activation and in T cells stimulated by mobilization of cellular calcium and activation of protein kinase C.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="423" length="427"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="869" length="882"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1238" length="1242"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Gene_expression</infon>
        <location offset="406" length="415"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T4" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8101106</id>
    <passage>
      <offset>0</offset>
      <text>Antisense oligonucleotides to the p65 subunit of NF-kappa B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="34" length="37"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="66" length="71"></location>
        <text>CD11b</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Negative_regulation</infon>
        <location offset="60" length="65"></location>
        <text>block</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="72" length="82"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>152</offset>
      <text>NF-kappa B is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of NF-kappa B to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the p50 and p65 subunits of the NF-kappa B complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of p65 was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in CD11b integrin expression on the surface of treated cells. Furthermore, the p65 antisense oligomer effectively abolished an upregulation of CD11b that was produced by formyl-met-leu-phe and TPA. However, the p65 antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to p65 can be used to define the role of NF-kappa B in the activation pathways of neutrophils.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="531" length="534"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="539" length="542"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="721" length="724"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1056" length="1061"></location>
        <text>CD11b</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1132" length="1135"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1196" length="1201"></location>
        <text>CD11b</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1264" length="1267"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1480" length="1483"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Negative_regulation</infon>
        <location offset="690" length="699"></location>
        <text>reduction</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="707" length="717"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Negative_regulation</infon>
        <location offset="1043" length="1052"></location>
        <text>reduction</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="1071" length="1081"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Negative_regulation</infon>
        <location offset="1167" length="1176"></location>
        <text>abolished</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1180" length="1192"></location>
        <text>upregulation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8106512</id>
    <passage>
      <offset>0</offset>
      <text>Human interferon regulatory factor 2 gene. Intron-exon organization and functional analysis of 5'-flanking region.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="6" length="36"></location>
        <text>interferon regulatory factor 2</text>
      </annotation>
    </passage>
    <passage>
      <offset>116</offset>
      <text>Interferon regulatory factor 2 (IRF-2) is a transcriptional regulatory protein that terminates interferon beta expression initiated by interferon regulatory factor 1. In this study, we isolated the genomic DNA for human IRF-2 gene, determined the intron-exon structure of the human IRF-2 gene, mapped the major transcription initiation site, identified a number of potential regulatory elements in the 5'-flanking region, and localized the IRF-2 gene on human chromosome 4. The IRF-2 promoter region contains a CpG island, with several GC boxes, a putative NF-kappa B-binding site, and a CAAT box, but no TATA box. When the promoter region was linked with a heterologous reporter gene, we found that the promoter region is inducible by both interferons (interferon-alpha and -gamma) and interferon regulatory factor 1. The region which induced these inductions was identified as being confined to 40 nucleotides 5' to the major transcriptional initiation site by testing a series of clones with truncated promoter of IRF-2. This region contains elements which are shared with the transcriptional enhancers of other genes including interferon regulatory factor 1, interferon beta, and interferon-inducible genes. These data suggest that interferon regulatory factor 1 not only triggers the activation of the interferon signal transduction pathway, but also may play a role in limiting the duration of this response by activating the transcription of IRF-2.</text>
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        <infon key="type">Protein</infon>
        <location offset="116" length="146"></location>
        <text>Interferon regulatory factor 2</text>
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      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="148" length="153"></location>
        <text>IRF-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="211" length="226"></location>
        <text>interferon beta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="251" length="281"></location>
        <text>interferon regulatory factor 1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="336" length="341"></location>
        <text>IRF-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="398" length="403"></location>
        <text>IRF-2</text>
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      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="556" length="561"></location>
        <text>IRF-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="594" length="599"></location>
        <text>IRF-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="870" length="886"></location>
        <text>interferon-alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="891" length="897"></location>
        <text>-gamma</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="903" length="933"></location>
        <text>interferon regulatory factor 1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1133" length="1138"></location>
        <text>IRF-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1247" length="1277"></location>
        <text>interferon regulatory factor 1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1279" length="1294"></location>
        <text>interferon beta</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1352" length="1382"></location>
        <text>interferon regulatory factor 1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1565" length="1570"></location>
        <text>IRF-2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Negative_regulation</infon>
        <location offset="200" length="210"></location>
        <text>terminates</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="227" length="237"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="238" length="247"></location>
        <text>initiated</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Entity</infon>
        <location offset="600" length="615"></location>
        <text>promoter region</text>
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      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="839" length="848"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="1533" length="1543"></location>
        <text>activating</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Transcription</infon>
        <location offset="1548" length="1561"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
      <node refid="T21" role="Site"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
      <node refid="T21" role="Site"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T12" role="Cause"></node>
      <node refid="T21" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8108127</id>
    <passage>
      <offset>0</offset>
      <text>Overproduction of NFKB2 (lyt-10) and c-Rel: a mechanism for HTLV-I Tax-mediated trans-activation via the NF-kappa B signalling pathway.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="18" length="23"></location>
        <text>NFKB2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="25" length="31"></location>
        <text>lyt-10</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="37" length="42"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="67" length="70"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Gene_expression</infon>
        <location offset="0" length="14"></location>
        <text>Overproduction</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="14"></location>
        <text>Overproduction</text>
      </annotation>
    </passage>
    <passage>
      <offset>137</offset>
      <text>Molecular, biochemical and epidemiological evidence implicate HTLV-I as an etiologic agent of adult T cell leukemia (ATL). The Tax protein of HTLV-I, a positive transcriptional activator of HTLV-I gene expression, is a viral oncogene that also increases transcription of cellular genes including GM-CSF, IL-2R alpha and IL-2. One of the cellular targets of the trans-activating effects of Tax is the NF-kappa B/Rel family of transcription factors, pleiotropic regulators of immunoregulatory, cytokine and viral gene expression. In this report, we demonstrate that NFKB2 (lyt-10) and c-Rel are overexpressed in HTLV-I infected and Tax-expressing cells and, together, account for the majority of the constitutive NF-kappa B binding activity in these cells before and after PMA stimulation. Most importantly, we show a Tax-dependent correlation between expression of NFKB2(p100) and processing to the DNA binding NFKB2(p52) form, induction of c-Rel, and trans-activation of NF-kappa B-mediated gene expression. Furthermore, the NFKB2 precursor is physically associated with c-Rel and with Tax in HTLV-I infected cells. We propose that NFKB2 synthesis and processing allows continuous nuclear expression of an otherwise cytoplasmic protein and, in conjunction with overexpression of c-Rel, NFKB2 alters the NF-kappa B signalling pathway and contributes to leukemic transformation of T cells by HTLV-I.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="264" length="267"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="433" length="439"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="441" length="452"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="457" length="461"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="526" length="529"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="701" length="706"></location>
        <text>NFKB2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="708" length="714"></location>
        <text>lyt-10</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="720" length="725"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="767" length="770"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="953" length="956"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1001" length="1006"></location>
        <text>NFKB2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1007" length="1011"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1047" length="1052"></location>
        <text>NFKB2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1053" length="1056"></location>
        <text>p52</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1077" length="1082"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1162" length="1177"></location>
        <text>NFKB2 precursor</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1208" length="1213"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1223" length="1226"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1269" length="1274"></location>
        <text>NFKB2</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1416" length="1421"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1423" length="1428"></location>
        <text>NFKB2</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="381" length="390"></location>
        <text>increases</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Transcription</infon>
        <location offset="391" length="404"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Gene_expression</infon>
        <location offset="730" length="743"></location>
        <text>overexpressed</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="730" length="743"></location>
        <text>overexpressed</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="987" length="997"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1064" length="1073"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="1192" length="1202"></location>
        <text>associated</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Gene_expression</infon>
        <location offset="1275" length="1284"></location>
        <text>synthesis</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1300" length="1306"></location>
        <text>allows</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Entity</infon>
        <location offset="1318" length="1325"></location>
        <text>nuclear</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Localization</infon>
        <location offset="1326" length="1336"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Gene_expression</infon>
        <location offset="1398" length="1412"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Positive_regulation</infon>
        <location offset="1398" length="1412"></location>
        <text>overexpression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T10" role="Event"></node>
      <node refid="T11" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
      <node refid="T22" role="Theme2"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
      <node refid="T21" role="Theme2"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="E19" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
      <node refid="T37" role="AtLoc"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8108414</id>
    <passage>
      <offset>0</offset>
      <text>Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="48" length="52"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="54" length="57"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Regulation</infon>
        <location offset="0" length="14"></location>
        <text>Autoregulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>122</offset>
      <text>RelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-kappa B complex and contains a high-affinity binding site for its cytoplasmic inhibitor, I kappa B alpha. After cellular activation, I kappa B alpha is rapidly degraded in concert with the induced nuclear translocation of NF-kappa B. The present study demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B alpha in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on I kappa B alpha result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with I kappa B alpha but also with other ankyrin motif-rich proteins including the products of the NF-kappa B2 (p100) and NF-kappa B1 (p105) genes; (ii) in contrast to RelA-I kappa B alpha, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor alpha activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for I kappa B alpha binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with I kappa B alpha, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-kappa B.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="122" length="126"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="128" length="131"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="206" length="209"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="210" length="213"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="306" length="321"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="350" length="365"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="487" length="514"></location>
        <text>tumor necrosis factor alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="538" length="553"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="647" length="662"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="747" length="751"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="807" length="811"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="847" length="862"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="941" length="952"></location>
        <text>NF-kappa B2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="954" length="958"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="964" length="975"></location>
        <text>NF-kappa B1</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="977" length="981"></location>
        <text>p105</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1010" length="1014"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1015" length="1030"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1032" length="1036"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1037" length="1041"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1094" length="1121"></location>
        <text>tumor necrosis factor alpha</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1140" length="1144"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1180" length="1184"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1241" length="1245"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1250" length="1254"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1358" length="1362"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="1386" length="1401"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="1415" length="1419"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="1434" length="1438"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="1534" length="1538"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="1566" length="1581"></location>
        <text>I kappa B alpha</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="1591" length="1595"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="1607" length="1611"></location>
        <text>p100</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Positive_regulation</infon>
        <location offset="323" length="328"></location>
        <text>After</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Protein_catabolism</infon>
        <location offset="377" length="385"></location>
        <text>degraded</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Positive_regulation</infon>
        <location offset="515" length="522"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Protein_catabolism</infon>
        <location offset="523" length="534"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Phosphorylation</infon>
        <location offset="596" length="611"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Positive_regulation</infon>
        <location offset="663" length="672"></location>
        <text>result in</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Entity</infon>
        <location offset="673" length="680"></location>
        <text>nuclear</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Localization</infon>
        <location offset="681" length="693"></location>
        <text>mobilization</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Binding</infon>
        <location offset="822" length="832"></location>
        <text>associated</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Binding</infon>
        <location offset="1226" length="1237"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Entity</infon>
        <location offset="1327" length="1354"></location>
        <text>nuclear localization signal</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Positive_regulation</infon>
        <location offset="1373" length="1381"></location>
        <text>required</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Binding</infon>
        <location offset="1402" length="1409"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Negative_regulation</infon>
        <location offset="1420" length="1430"></location>
        <text>inhibition</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Positive_regulation</infon>
        <location offset="1448" length="1456"></location>
        <text>requires</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Positive_regulation</infon>
        <location offset="1500" length="1508"></location>
        <text>mediates</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Entity</infon>
        <location offset="1509" length="1520"></location>
        <text>cytoplasmic</text>
      </annotation>
      <annotation id="T54">
        <infon key="type">Localization</infon>
        <location offset="1521" length="1530"></location>
        <text>retention</text>
      </annotation>
      <annotation id="T55">
        <infon key="type">Positive_regulation</infon>
        <location offset="1596" length="1606"></location>
        <text>stimulates</text>
      </annotation>
      <annotation id="T56">
        <infon key="type">Transcription</infon>
        <location offset="1629" length="1639"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T57">
        <infon key="type">Gene_expression</infon>
        <location offset="1629" length="1639"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T17" role="Event"></node>
      <node refid="T18" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T15" role="Event"></node>
      <node refid="T16" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="E4" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T43" role="ToLoc"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T17" role="Theme2"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T14" role="Theme2"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T15" role="Theme2"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T26" role="Theme"></node>
      <node refid="T27" role="Theme2"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
      <node refid="T28" role="Cause"></node>
      <node refid="T47" role="CSite"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T28" role="Theme"></node>
      <node refid="T29" role="Theme2"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
      <node refid="T30" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T51" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T52" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T54" role="Trigger"></node>
      <node refid="T32" role="Theme"></node>
      <node refid="T53" role="AtLoc"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T55" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T34" role="Cause"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T55" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
      <node refid="T34" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T56" role="Trigger"></node>
      <node refid="T35" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T57" role="Trigger"></node>
      <node refid="T35" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8112299</id>
    <passage>
      <offset>0</offset>
      <text>Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="61" length="65"></location>
        <text>MAD3</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Negative_regulation</infon>
        <location offset="40" length="50"></location>
        <text>inactivate</text>
      </annotation>
    </passage>
    <passage>
      <offset>96</offset>
      <text>The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK-506). Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506- and CsA-sensitive enzyme required for TcR mediated activation of the IL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-1) and IL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-kappa B-dependent element. Calcineurin stimulates the NF-kappa B element by enhancing inactivation of I kappa B/MAD3, an inhibitor of NF-kappa B, thereby increasing the amount of nuclear NF-kappa B DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate I kappa B, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="100" length="113"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="115" length="119"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="492" length="496"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="638" length="642"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="692" length="695"></location>
        <text>OAP</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="700" length="705"></location>
        <text>Oct-1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="930" length="934"></location>
        <text>MAD3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Entity</infon>
        <location offset="121" length="129"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="209" length="218"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Negative_regulation</infon>
        <location offset="263" length="274"></location>
        <text>inhibitable</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="448" length="456"></location>
        <text>required</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="474" length="484"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Entity</infon>
        <location offset="497" length="505"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="625" length="633"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Entity</infon>
        <location offset="661" length="666"></location>
        <text>IL-2A</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="674" length="679"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="894" length="903"></location>
        <text>enhancing</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Negative_regulation</infon>
        <location offset="904" length="916"></location>
        <text>inactivation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T10" role="Site"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
      <node refid="T15" role="Site"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8134378</id>
    <passage>
      <offset>0</offset>
      <text>Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B [published erratum appears in Proc Natl Acad Sci U S A 1995 Apr 11;92(8):3632]</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="18" length="31"></location>
        <text>interleukin 6</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="120" length="126"></location>
        <text>NF-IL6</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="10"></location>
        <text>Activation</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="92" length="100"></location>
        <text>mediated</text>
      </annotation>
    </passage>
    <passage>
      <offset>222</offset>
      <text>The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guerin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner. LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes. Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay. Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements.</text>
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        <infon key="type">Protein</infon>
        <location offset="590" length="603"></location>
        <text>interleukin 6</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="605" length="609"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="894" length="898"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="962" length="966"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1042" length="1046"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1146" length="1179"></location>
        <text>chloramphenicol acetyltransferase</text>
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      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1206" length="1212"></location>
        <text>NF-IL6</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1419" length="1423"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1621" length="1625"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1686" length="1692"></location>
        <text>NF-IL6</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1722" length="1726"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="883" length="885"></location>
        <text>to</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Localization</infon>
        <location offset="886" length="893"></location>
        <text>release</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="950" length="958"></location>
        <text>inducers</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="972" length="982"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="1032" length="1041"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Entity</infon>
        <location offset="1047" length="1055"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="1595" length="1604"></location>
        <text>abolished</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="1605" length="1617"></location>
        <text>inducibility</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Entity</infon>
        <location offset="1626" length="1634"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="1714" length="1721"></location>
        <text>mediate</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="1727" length="1736"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T21" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T24" role="Site"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8135780</id>
    <passage>
      <offset>0</offset>
      <text>Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="45" length="51"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="12" length="21"></location>
        <text>activates</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Transcription</infon>
        <location offset="22" length="35"></location>
        <text>transcription</text>
      </annotation>
    </passage>
    <passage>
      <offset>133</offset>
      <text>Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)- expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+- signaling pathways downstream of T cell activation.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="192" length="240"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="242" length="248"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="678" length="684"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="721" length="724"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="725" length="728"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="740" length="745"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="746" length="751"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="833" length="839"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="890" length="896"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1024" length="1030"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1158" length="1203"></location>
        <text>Ca2+/calmodulin-dependent protein phosphatase</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1395" length="1401"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1537" length="1543"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Entity</infon>
        <location offset="157" length="174"></location>
        <text>GM-kappa B/GC-box</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Entity</infon>
        <location offset="179" length="183"></location>
        <text>CLE0</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="259" length="267"></location>
        <text>required</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="280" length="289"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="648" length="656"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Transcription</infon>
        <location offset="657" length="670"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="691" length="705"></location>
        <text>Cotransfection</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="691" length="705"></location>
        <text>Cotransfection</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="1507" length="1515"></location>
        <text>required</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="1520" length="1529"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
      <node refid="T18" role="CSite"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
      <node refid="T17" role="CSite"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8388998</id>
    <passage>
      <offset>0</offset>
      <text>Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription.</text>
    </passage>
    <passage>
      <offset>116</offset>
      <text>Interaction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential.</text>
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        <infon key="type">Protein</infon>
        <location offset="164" length="187"></location>
        <text>glucocorticoid receptor</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="189" length="191"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="330" length="332"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="634" length="636"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="686" length="688"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="805" length="839"></location>
        <text>chloramphenicol acetyl-transferase</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="881" length="883"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>c-jun</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1071" length="1076"></location>
        <text>jun-B</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
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        <text>jun-D</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1198" length="1232"></location>
        <text>chloramphenicol acetyl-transferase</text>
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        <infon key="type">Protein</infon>
        <location offset="1373" length="1375"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1431" length="1436"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1438" length="1443"></location>
        <text>jun-B</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1448" length="1453"></location>
        <text>jun-D</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1490" length="1492"></location>
        <text>GR</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1558" length="1563"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1568" length="1573"></location>
        <text>jun-B</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1595" length="1597"></location>
        <text>GR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1653" length="1658"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1687" length="1689"></location>
        <text>GR</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1797" length="1799"></location>
        <text>GR</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1826" length="1831"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
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        <text>c-fos</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="2012" length="2014"></location>
        <text>GR</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="2109" length="2111"></location>
        <text>GR</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="2300" length="2305"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="1054" length="1063"></location>
        <text>Increased</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Gene_expression</infon>
        <location offset="1088" length="1098"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Negative_regulation</infon>
        <location offset="1659" length="1683"></location>
        <text>had a negative influence</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Negative_regulation</infon>
        <location offset="1752" length="1773"></location>
        <text>antagonistic activity</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Positive_regulation</infon>
        <location offset="1765" length="1773"></location>
        <text>activity</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1801" length="1805"></location>
        <text>high</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Gene_expression</infon>
        <location offset="1812" length="1822"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Gene_expression</infon>
        <location offset="1950" length="1964"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1950" length="1964"></location>
        <text>overexpression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
      <node refid="T20" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8389757</id>
    <passage>
      <offset>0</offset>
      <text>Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation.</text>
    </passage>
    <passage>
      <offset>158</offset>
      <text>Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="325" length="330"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="694" length="699"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="903" length="908"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="961" length="966"></location>
        <text>c-fms</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1204" length="1209"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1236" length="1241"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1344" length="1349"></location>
        <text>jun-B</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1354" length="1359"></location>
        <text>fra-1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1436" length="1441"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1854" length="1859"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="308" length="317"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="682" length="690"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="895" length="902"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="909" length="922"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="943" length="953"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="1160" length="1169"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="1210" length="1220"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1242" length="1251"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="1370" length="1377"></location>
        <text>induced</text>
      </annotation>
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        <location offset="1442" length="1452"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Negative_regulation</infon>
        <location offset="1456" length="1466"></location>
        <text>attenuated</text>
      </annotation>
    </passage>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8524232</id>
    <passage>
      <offset>0</offset>
      <text>Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected] [published erratum appears in Mol Cell Biol 1996 Feb;16(2):735]</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="28"></location>
        <text>Initiation binding repressor</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="94" length="104"></location>
        <text>histone h5</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="44" length="49"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Entity</infon>
        <location offset="57" length="86"></location>
        <text>transcription initiation site</text>
      </annotation>
    </passage>
    <passage>
      <offset>251</offset>
      <text>Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="251" length="279"></location>
        <text>Initiation binding repressor</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="293" length="296"></location>
        <text>IBR</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="448" length="458"></location>
        <text>histone h5</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="577" length="580"></location>
        <text>IBR</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="609" length="612"></location>
        <text>IBF</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="653" length="656"></location>
        <text>IBR</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="683" length="686"></location>
        <text>IBR</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="724" length="727"></location>
        <text>IBR</text>
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        <infon key="type">Protein</infon>
        <location offset="732" length="735"></location>
        <text>IBF</text>
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        <infon key="type">Protein</infon>
        <location offset="737" length="740"></location>
        <text>IBR</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="837" length="842"></location>
        <text>NRF-1</text>
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        <infon key="type">Protein</infon>
        <location offset="843" length="859"></location>
        <text>alpha-Pal factor</text>
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        <infon key="type">Protein</infon>
        <location offset="921" length="925"></location>
        <text>P3A2</text>
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      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="930" length="947"></location>
        <text>erected wing gene</text>
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      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="957" length="960"></location>
        <text>EWG</text>
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        <infon key="type">Protein</infon>
        <location offset="988" length="991"></location>
        <text>IBR</text>
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        <infon key="type">Protein</infon>
        <location offset="996" length="999"></location>
        <text>IBF</text>
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        <infon key="type">Protein</infon>
        <location offset="1126" length="1129"></location>
        <text>IBR</text>
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        <infon key="type">Protein</infon>
        <location offset="1130" length="1131"></location>
        <text>F</text>
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        <infon key="type">Protein</infon>
        <location offset="1294" length="1297"></location>
        <text>IBR</text>
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        <infon key="type">Protein</infon>
        <location offset="1298" length="1299"></location>
        <text>F</text>
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        <infon key="type">Protein</infon>
        <location offset="1631" length="1634"></location>
        <text>IBR</text>
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        <infon key="type">Protein</infon>
        <location offset="1635" length="1636"></location>
        <text>F</text>
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      <annotation id="T28">
        <infon key="type">Binding</infon>
        <location offset="375" length="380"></location>
        <text>binds</text>
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        <infon key="type">Entity</infon>
        <location offset="411" length="440"></location>
        <text>transcription initiation site</text>
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      <annotation id="T30">
        <infon key="type">Negative_regulation</infon>
        <location offset="465" length="475"></location>
        <text>repressing</text>
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      <annotation id="T31">
        <infon key="type">Transcription</infon>
        <location offset="480" length="493"></location>
        <text>transcription</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="572" length="576"></location>
        <text>have</text>
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      <annotation id="T33">
        <infon key="type">Binding</infon>
        <location offset="633" length="643"></location>
        <text>recognizes</text>
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        <infon key="type">Binding</infon>
        <location offset="1158" length="1189"></location>
        <text>associates as stable homodimers</text>
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        <infon key="type">Binding</infon>
        <location offset="1229" length="1236"></location>
        <text>binding</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1498" length="1507"></location>
        <text>necessary</text>
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      <annotation id="T37">
        <infon key="type">Entity</infon>
        <location offset="1512" length="1519"></location>
        <text>nuclear</text>
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      <annotation id="T38">
        <infon key="type">Localization</infon>
        <location offset="1520" length="1529"></location>
        <text>targeting</text>
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      <infon key="type">Equiv</infon>
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      <infon key="type">Event</infon>
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      <node refid="T2" role="Theme2"></node>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
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      <node refid="T5" role="Theme2"></node>
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    </relation>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E2" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
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      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
      <node refid="T37" role="AtLoc"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
      <node refid="T37" role="AtLoc"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8561779</id>
    <passage>
      <offset>0</offset>
      <text>Inhibition of NF-AT signal transduction events by a dominant-negative form of calcineurin.</text>
    </passage>
    <passage>
      <offset>92</offset>
      <text>An inhibitory, "dominant-negative," form of the calcineurin catalytic (A) subunit was prepared, which lacks the calmodulin-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block calcineurin-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleukin (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced NF-AT activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt calcineurin-mediated signal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits.</text>
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        <infon key="type">Protein</infon>
        <location offset="140" length="173"></location>
        <text>calcineurin catalytic (A) subunit</text>
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        <infon key="type">Protein</infon>
        <location offset="204" length="214"></location>
        <text>calmodulin</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="302" length="324"></location>
        <text>regulatory (B) subunit</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="568" length="586"></location>
        <text>interleukin (IL)-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="701" length="721"></location>
        <text>endogenous B subunit</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="818" length="827"></location>
        <text>B subunit</text>
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      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="961" length="970"></location>
        <text>B subunit</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1223" length="1236"></location>
        <text>calcineurin A</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1241" length="1251"></location>
        <text>B subunits</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="547" length="563"></location>
        <text>promoter element</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="592" length="601"></location>
        <text>induction</text>
      </annotation>
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        <infon key="type">Binding</infon>
        <location offset="667" length="695"></location>
        <text>formation of a tight complex</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="758" length="778"></location>
        <text>inhibitory mechanism</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="784" length="792"></location>
        <text>involves</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Localization</infon>
        <location offset="797" length="810"></location>
        <text>sequestration</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="931" length="945"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="931" length="945"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Negative_regulation</infon>
        <location offset="988" length="997"></location>
        <text>depletion</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="1167" length="1175"></location>
        <text>blocking</text>
      </annotation>
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        <infon key="type">Binding</infon>
        <location offset="1176" length="1219"></location>
        <text>formation of the catalytically active dimer</text>
      </annotation>
    </passage>
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      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="E6" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T9" role="Theme2"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8617979</id>
    <passage>
      <offset>0</offset>
      <text>Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells.</text>
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        <infon key="type">Protein</infon>
        <location offset="25" length="34"></location>
        <text>IL-1 beta</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="39" length="63"></location>
        <text>IL-1 receptor antagonist</text>
      </annotation>
    </passage>
    <passage>
      <offset>152</offset>
      <text>A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells.</text>
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        <infon key="type">Protein</infon>
        <location offset="795" length="843"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
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      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="845" length="851"></location>
        <text>GM-CSF</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="894" length="900"></location>
        <text>GM-CSF</text>
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        <infon key="type">Protein</infon>
        <location offset="1200" length="1206"></location>
        <text>GM-CSF</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1260" length="1264"></location>
        <text>IL-8</text>
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        <infon key="type">Protein</infon>
        <location offset="1266" length="1275"></location>
        <text>IL-1 beta</text>
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        <infon key="type">Protein</infon>
        <location offset="1280" length="1284"></location>
        <text>IL-6</text>
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        <infon key="type">Protein</infon>
        <location offset="1294" length="1303"></location>
        <text>TNF-alpha</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1305" length="1334"></location>
        <text>IL-1 receptor antagonist (ra)</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1382" length="1388"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1549" length="1555"></location>
        <text>GM-CSF</text>
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        <infon key="type">Protein</infon>
        <location offset="1637" length="1646"></location>
        <text>IL-1 beta</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1708" length="1714"></location>
        <text>IL-1ra</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1793" length="1802"></location>
        <text>IL-1 beta</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1823" length="1829"></location>
        <text>IL-1ra</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1207" length="1214"></location>
        <text>induced</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="1219" length="1229"></location>
        <text>production</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1623" length="1633"></location>
        <text>inhibition</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Localization</infon>
        <location offset="1647" length="1654"></location>
        <text>release</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1680" length="1693"></location>
        <text>up-regulation</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1715" length="1725"></location>
        <text>production</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Negative_regulation</infon>
        <location offset="1737" length="1744"></location>
        <text>balance</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="1769" length="1777"></location>
        <text>produced</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E1" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8621390</id>
    <passage>
      <offset>0</offset>
      <text>Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="34" length="47"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Regulation</infon>
        <location offset="0" length="26"></location>
        <text>Transcriptional regulation</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Transcription</infon>
        <location offset="0" length="26"></location>
        <text>Transcriptional regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>175</offset>
      <text>To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="293" length="306"></location>
        <text>Interleukin-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="308" length="312"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="501" length="505"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="630" length="648"></location>
        <text>phytohemagglutinin</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="984" length="988"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1155" length="1159"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="989" length="1015"></location>
        <text>transcriptional regulation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8621480</id>
    <passage>
      <offset>0</offset>
      <text>A hydrophobic domain of Ca2+-modulating cyclophilin ligand modulates calcium influx signaling in T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="24" length="58"></location>
        <text>Ca2+-modulating cyclophilin ligand</text>
      </annotation>
    </passage>
    <passage>
      <offset>113</offset>
      <text>Ca2+-modulating cyclophilin ligand (CAML) was originally described as a cyclophilin B-binding protein whose overexpression in T cells causes a rise in intracellular calcium, thus activating transcription factors responsible for the early immune response. As reported here, structure-function analysis of the CAML gene in Jurkat T cells indicates that two of CAML's putative membrane-spanning domains are necessary and sufficient for the modulation of intracellular calcium. We propose that the hydrophobic C-terminal tail of CAML forms its effector domain, thus implicating the N-terminal hydrophilic domain in a regulatory role. These findings define a novel protein motif that functions in intracellular calcium signaling.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="113" length="147"></location>
        <text>Ca2+-modulating cyclophilin ligand</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="149" length="153"></location>
        <text>CAML</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="185" length="198"></location>
        <text>cyclophilin B</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="421" length="425"></location>
        <text>CAML</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="471" length="475"></location>
        <text>CAML</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="638" length="642"></location>
        <text>CAML</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Positive_regulation</infon>
        <location offset="221" length="235"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Gene_expression</infon>
        <location offset="221" length="235"></location>
        <text>overexpression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8621538</id>
    <passage>
      <offset>0</offset>
      <text>Interactions of a transcriptional activator in the env gene of the mouse mammary tumor virus with activation-dependent, T cell-specific transacting factors.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="51" length="54"></location>
        <text>env</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Binding</infon>
        <location offset="0" length="12"></location>
        <text>Interactions</text>
      </annotation>
    </passage>
    <passage>
      <offset>158</offset>
      <text>The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="188" length="191"></location>
        <text>env</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="515" length="522"></location>
        <text>DNase I</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1101" length="1105"></location>
        <text>SV40</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1226" length="1230"></location>
        <text>SV40</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8621773</id>
    <passage>
      <offset>0</offset>
      <text>Monocytic cell type-specific transcriptional induction of collagenase.</text>
    </passage>
    <passage>
      <offset>72</offset>
      <text>Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="98" length="103"></location>
        <text>MMP-1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Gene_expression</infon>
        <location offset="126" length="134"></location>
        <text>produced</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T2" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8622636</id>
    <passage>
      <offset>0</offset>
      <text>Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor-alpha generation, on activation of nuclear factor-kappa B.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="72" length="99"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Negative_regulation</infon>
        <location offset="63" length="71"></location>
        <text>suppress</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="100" length="110"></location>
        <text>generation</text>
      </annotation>
    </passage>
    <passage>
      <offset>154</offset>
      <text>(2E)-3-[5-(2,3-Dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid (E3330), is a novel agent with hepatoprotective activity. We report the effect of E3330 on transcriptional activation of tumor necrosis factor (TNF)-alpha gene and on nuclear factor (NF)-kappa B activation. Nuclear run-on experiments showed that E3330 decreases transcriptional activation of TNF-alpha gene induced by lipopolysaccharide (LPS) stimulation in human peripheral monocytes. To investigate the inhibitory mechanisms, we constructed a secreted-type placental alkaline phosphatase (PLAP) reporter gene whose transcription is controlled by a 1.4-kb human TNF-alpha promoter. A stable transformant of the PLAP reporter gene derived from human monocytic cell line showed very little activity on the promoter before stimulation, whereas LPS stimulation led to a dramatic increase in PLAP activity. E3330 inhibited this induced promoter activity in a dose-dependent manner. There are four putative NF-kappa B binding sites (kappa B-1, kappa B-2, kappa B-3, kappa B-4) in human TNF-alpha promoter. By using mutated promoter-PLAP plasmids, we established that these NF-kappa B sites were necessary for induction of TNF-alpha transcription on stimulation with LPS. A gel retardation experiment with synthetic double-stranded oligonucleotides showed that activated NF-kappa B consisting of p50/p65 heterodimer bound to all four putative NF-kappa B DNA probes, suggesting that all four putative NF-kappa B recognition sites play an important role in inducible TNF-alpha expression. E3330 decreased activated NF-kappa B in nuclei, suggesting that E3330 inhibits NF-kappa B activation and/or translocation of the nuclei. Western blotting analysis with anti-I kappa B-alpha antibody indicated that E3330 inhibited degradation of I kappa B-alpha, which is an inhibitory protein of NF-kappa B, in LPS-stimulated monocytes. E3330 may suppress the production of active oxygen species serving as common messengers to activate NF-kappa B.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="692" length="722"></location>
        <text>placental alkaline phosphatase</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="724" length="728"></location>
        <text>PLAP</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="796" length="805"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="845" length="849"></location>
        <text>PLAP</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1021" length="1025"></location>
        <text>PLAP</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1214" length="1223"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1260" length="1264"></location>
        <text>PLAP</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1350" length="1359"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1523" length="1526"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1527" length="1530"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1692" length="1701"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1887" length="1902"></location>
        <text>I kappa B-alpha</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1958" length="1973"></location>
        <text>I kappa B-alpha</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Transcription</infon>
        <location offset="750" length="763"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Regulation</infon>
        <location offset="767" length="777"></location>
        <text>controlled</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Entity</infon>
        <location offset="806" length="814"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="1323" length="1332"></location>
        <text>necessary</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="1337" length="1346"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Transcription</infon>
        <location offset="1360" length="1373"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="1543" length="1548"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="1656" length="1678"></location>
        <text>play an important role</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="1702" length="1712"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Negative_regulation</infon>
        <location offset="1933" length="1942"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein_catabolism</infon>
        <location offset="1943" length="1954"></location>
        <text>degradation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
      <node refid="T19" role="CSite"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8622883</id>
    <passage>
      <offset>0</offset>
      <text>The role of p16 in the E2F-dependent thymidine kinase regulation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="12" length="15"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="37" length="53"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Regulation</infon>
        <location offset="4" length="8"></location>
        <text>role</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Regulation</infon>
        <location offset="54" length="64"></location>
        <text>regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>67</offset>
      <text>The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="98" length="102"></location>
        <text>MTS1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="159" length="162"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="181" length="206"></location>
        <text>cyclin-dependent-kinase-4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="211" length="212"></location>
        <text>6</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="272" length="294"></location>
        <text>retinoblastoma protein</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="298" length="324"></location>
        <text>cyclin-dependent kinases 4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="329" length="330"></location>
        <text>6</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="470" length="486"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="507" length="510"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="689" length="705"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="709" length="727"></location>
        <text>phytohemagglutinin</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="775" length="778"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="950" length="966"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1032" length="1048"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1090" length="1093"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1250" length="1272"></location>
        <text>retinoblastoma protein</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1347" length="1350"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1390" length="1406"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1469" length="1491"></location>
        <text>retinoblastoma protein</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1543" length="1559"></location>
        <text>thymidine kinase</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1598" length="1601"></location>
        <text>p16</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1706" length="1728"></location>
        <text>retinoblastoma protein</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Regulation</infon>
        <location offset="79" length="90"></location>
        <text>alterations</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Negative_regulation</infon>
        <location offset="168" length="177"></location>
        <text>inhibitor</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Phosphorylation</infon>
        <location offset="249" length="264"></location>
        <text>Phosphorylation</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="249" length="264"></location>
        <text>Phosphorylation</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Negative_regulation</infon>
        <location offset="331" length="339"></location>
        <text>prevents</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Binding</infon>
        <location offset="344" length="355"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="410" length="418"></location>
        <text>promotes</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Gene_expression</infon>
        <location offset="423" length="433"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Regulation</infon>
        <location offset="499" length="503"></location>
        <text>role</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Negative_regulation</infon>
        <location offset="579" length="583"></location>
        <text>loss</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Gene_expression</infon>
        <location offset="779" length="787"></location>
        <text>negative</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Positive_regulation</infon>
        <location offset="936" length="946"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Transcription</infon>
        <location offset="1054" length="1064"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Gene_expression</infon>
        <location offset="1076" length="1086"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Gene_expression</infon>
        <location offset="1231" length="1238"></location>
        <text>express</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Positive_regulation</infon>
        <location offset="1329" length="1343"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Gene_expression</infon>
        <location offset="1329" length="1343"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Positive_regulation</infon>
        <location offset="1351" length="1364"></location>
        <text>reestablished</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Regulation</infon>
        <location offset="1376" length="1386"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Positive_regulation</infon>
        <location offset="1425" length="1433"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Negative_regulation</infon>
        <location offset="1441" length="1465"></location>
        <text>underphosphorylated form</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Phosphorylation</infon>
        <location offset="1441" length="1465"></location>
        <text>underphosphorylated form</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Negative_regulation</infon>
        <location offset="1496" length="1504"></location>
        <text>decrease</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Binding</infon>
        <location offset="1517" length="1522"></location>
        <text>bound</text>
      </annotation>
      <annotation id="T51">
        <infon key="type">Entity</infon>
        <location offset="1526" length="1535"></location>
        <text>its motif</text>
      </annotation>
      <annotation id="T52">
        <infon key="type">Positive_regulation</infon>
        <location offset="1602" length="1621"></location>
        <text>causes upregulation</text>
      </annotation>
      <annotation id="T53">
        <infon key="type">Regulation</infon>
        <location offset="1602" length="1608"></location>
        <text>causes</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="E6" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="E9" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T11" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="E20" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="E28" role="Theme"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
      <node refid="T51" role="Site"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T52" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E30">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T53" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
      <node refid="T23" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8622948</id>
    <passage>
      <offset>0</offset>
      <text>Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="3"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="55" length="68"></location>
        <text>interleukin 3</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="73" length="121"></location>
        <text>granulocyte-macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Negative_regulation</infon>
        <location offset="4" length="13"></location>
        <text>deficient</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Negative_regulation</infon>
        <location offset="30" length="37"></location>
        <text>defects</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Gene_expression</infon>
        <location offset="41" length="51"></location>
        <text>production</text>
      </annotation>
    </passage>
    <passage>
      <offset>124</offset>
      <text>The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.</text>
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        <infon key="type">Protein</infon>
        <location offset="128" length="133"></location>
        <text>c-rel</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="235" length="238"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="466" length="469"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="584" length="587"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="653" length="701"></location>
        <text>interleukin 2 receptor alpha (IL-2R alpha) chain</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="703" length="707"></location>
        <text>CD25</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="710" length="714"></location>
        <text>CD69</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="719" length="729"></location>
        <text>L-selectin</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="731" length="735"></location>
        <text>CD62</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="768" length="771"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="824" length="827"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="932" length="936"></location>
        <text>IL-3</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="938" length="942"></location>
        <text>IL-5</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="944" length="993"></location>
        <text>granulocyte- macrophage colony-stimulating factor</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="995" length="1001"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1004" length="1031"></location>
        <text>tumor necrosis factor alpha</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1033" length="1042"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1049" length="1065"></location>
        <text>gamma interferon</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1067" length="1076"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1168" length="1172"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1184" length="1187"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1277" length="1281"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1353" length="1357"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1405" length="1409"></location>
        <text>IL-5</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Protein</infon>
        <location offset="1411" length="1420"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein</infon>
        <location offset="1426" length="1435"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Protein</infon>
        <location offset="1445" length="1449"></location>
        <text>IL-3</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Protein</infon>
        <location offset="1454" length="1460"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Protein</infon>
        <location offset="1537" length="1540"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Protein</infon>
        <location offset="1583" length="1586"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Protein</infon>
        <location offset="1639" length="1645"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Protein</infon>
        <location offset="1677" length="1680"></location>
        <text>Rel</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Protein</infon>
        <location offset="1793" length="1797"></location>
        <text>IL-3</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Protein</infon>
        <location offset="1802" length="1808"></location>
        <text>GM-CSF</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Negative_regulation</infon>
        <location offset="227" length="234"></location>
        <text>lacking</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Negative_regulation</infon>
        <location offset="458" length="462"></location>
        <text>loss</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Gene_expression</infon>
        <location offset="604" length="614"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Negative_regulation</infon>
        <location offset="1101" length="1106"></location>
        <text>lower</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Positive_regulation</infon>
        <location offset="1382" length="1390"></location>
        <text>restores</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Gene_expression</infon>
        <location offset="1391" length="1401"></location>
        <text>production</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Gene_expression</infon>
        <location offset="1461" length="1471"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Positive_regulation</infon>
        <location offset="1602" length="1616"></location>
        <text>produce higher</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Positive_regulation</infon>
        <location offset="1749" length="1757"></location>
        <text>required</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Gene_expression</infon>
        <location offset="1779" length="1789"></location>
        <text>production</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T12" role="Event"></node>
      <node refid="T11" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T9" role="Event"></node>
      <node refid="T8" role="Event"></node>
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    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T18" role="Event"></node>
      <node refid="T17" role="Event"></node>
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    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T20" role="Event"></node>
      <node refid="T19" role="Event"></node>
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    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T22" role="Event"></node>
      <node refid="T21" role="Event"></node>
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    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E24" role="Theme"></node>
      <node refid="T25" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T28" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T27" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T29" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T31" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E26">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T34" role="Theme"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T35" role="Cause"></node>
    </relation>
    <relation id="E28">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T35" role="Cause"></node>
    </relation>
    <relation id="E29">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E20" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8623933</id>
    <passage>
      <offset>0</offset>
      <text>Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="121" length="131"></location>
        <text>galectin-3</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="62" length="69"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="70" length="80"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>134</offset>
      <text>Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L-29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor-kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I.</text>
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        <infon key="type">Protein</infon>
        <location offset="249" length="259"></location>
        <text>Galectin-3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="322" length="332"></location>
        <text>epsilon BP</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="334" length="353"></location>
        <text>IgE-binding protein</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="356" length="361"></location>
        <text>CBP35</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="363" length="368"></location>
        <text>Mac-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="370" length="374"></location>
        <text>L-29</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="380" length="384"></location>
        <text>L-34</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="541" length="551"></location>
        <text>Galectin-3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="715" length="725"></location>
        <text>galectin-3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="983" length="993"></location>
        <text>galectin-3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1132" length="1142"></location>
        <text>galectin-3</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1248" length="1258"></location>
        <text>galectin-3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1388" length="1398"></location>
        <text>galectin-3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1480" length="1483"></location>
        <text>Tax</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1548" length="1551"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1575" length="1585"></location>
        <text>galectin-3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1874" length="1877"></location>
        <text>Tax</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1904" length="1914"></location>
        <text>galectin-3</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1959" length="1969"></location>
        <text>galectin-3</text>
      </annotation>
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        <text>interleukin-2</text>
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        <text>Tax</text>
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        <infon key="type">Protein</infon>
        <location offset="2178" length="2188"></location>
        <text>galectin-3</text>
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        <text>expression</text>
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        <text>distributed</text>
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        <location offset="615" length="624"></location>
        <text>expressed</text>
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        <location offset="740" length="749"></location>
        <text>expressed</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="887" length="896"></location>
        <text>expressed</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1013" length="1022"></location>
        <text>increased</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1115" length="1128"></location>
        <text>up-regulation</text>
      </annotation>
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        <text>expression</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Gene_expression</infon>
        <location offset="1213" length="1225"></location>
        <text>undetectable</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="1234" length="1244"></location>
        <text>low levels</text>
      </annotation>
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        <infon key="type">Entity</infon>
        <location offset="1399" length="1407"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Positive_regulation</infon>
        <location offset="1426" length="1438"></location>
        <text>up-regulated</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Entity</infon>
        <location offset="1586" length="1594"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Positive_regulation</infon>
        <location offset="1595" length="1604"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Positive_regulation</infon>
        <location offset="1608" length="1617"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Positive_regulation</infon>
        <location offset="1812" length="1824"></location>
        <text>Transfection</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Gene_expression</infon>
        <location offset="1812" length="1824"></location>
        <text>Transfection</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Positive_regulation</infon>
        <location offset="1896" length="1903"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Gene_expression</infon>
        <location offset="1915" length="1925"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Positive_regulation</infon>
        <location offset="1983" length="1991"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Gene_expression</infon>
        <location offset="2006" length="2016"></location>
        <text>production</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Gene_expression</infon>
        <location offset="2099" length="2109"></location>
        <text>production</text>
      </annotation>
      <annotation id="T48">
        <infon key="type">Positive_regulation</infon>
        <location offset="2110" length="2117"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T49">
        <infon key="type">Transcription</infon>
        <location offset="2122" length="2135"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T50">
        <infon key="type">Localization</infon>
        <location offset="2165" length="2174"></location>
        <text>secretion</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
      <node refid="T15" role="Cause"></node>
      <node refid="T36" role="Site"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T38" role="Site"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
      <node refid="E16" role="Cause"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
      <node refid="T20" role="Cause"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T46" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E26" role="Theme"></node>
      <node refid="E22" role="Cause"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T48" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
      <node refid="E22" role="Cause"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T49" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T50" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E23" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E24" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8626528</id>
    <passage>
      <offset>0</offset>
      <text>Nuclear appearance of a factor that binds the CD28 response element within the interleukin-2 enhancer correlates with interleukin-2 production.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="46" length="50"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="79" length="92"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="118" length="131"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="132" length="142"></location>
        <text>production</text>
      </annotation>
    </passage>
    <passage>
      <offset>145</offset>
      <text>Activation of T lymphocytes requires the combined signaling of the T cell receptor and costimulatory molecules such as CD28. The ability of T cells to produce interleukin-2 (IL-2) is a critical control point in T lymphocyte activation. The IL-2 enhancer contains a functional motif named CD28 response element (CD28RE) that serves a role as a target for mitogenic T cell activation signals. The CD28RE sequence reveals similarity to the consensus kappaB binding motif. Here we demonstrate that CD28RE binds an inducible protein with a molecular mass of approximately 35 kDa called nuclear factor of mitogenic-activated T cells (NF-MATp35) that is clearly different from the known NF- kappaB/Rel family members. Induction of NF-MATp35 was shown to depend on de novo protein synthesis and was restricted to T cells that received a mitogenic combination of T cell stimuli, not necessarily including CD28 signaling. Nonmitogenic T cell stimulation did not result in appearance of NF-MATp35. These results indicate that mitogenic combinations of T cell activation signals are integrated at the level of NF-MATp35 induction. Similar to its effect on IL-2 production, cyclosporin A inhibited the induction of NF-MATp35. Taken together, these data demonstrate that the nuclear appearance of NF-MATp35 shows excellent correlation with IL-2 production, which is a unique characteristic among nuclear factors implicated in the control of IL-2 gene expression.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="264" length="268"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="304" length="317"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="319" length="323"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="385" length="389"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="433" length="437"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1041" length="1045"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1289" length="1293"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1471" length="1475"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1572" length="1576"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="296" length="303"></location>
        <text>produce</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="1294" length="1304"></location>
        <text>production</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="1320" length="1329"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="1476" length="1486"></location>
        <text>production</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Regulation</infon>
        <location offset="1561" length="1568"></location>
        <text>control</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="1582" length="1592"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8626752</id>
    <passage>
      <offset>0</offset>
      <text>Preassociation of STAT1 with STAT2 and STAT3 in separate signalling complexes prior to cytokine stimulation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="18" length="23"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="29" length="34"></location>
        <text>STAT2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="39" length="44"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Regulation</infon>
        <location offset="48" length="56"></location>
        <text>separate</text>
      </annotation>
    </passage>
    <passage>
      <offset>110</offset>
      <text>A variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="671" length="676"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="680" length="685"></location>
        <text>STAT2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="777" length="782"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="783" length="788"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="850" length="855"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="860" length="865"></location>
        <text>STAT2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="869" length="874"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="879" length="884"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1137" length="1142"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1143" length="1148"></location>
        <text>STAT2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1185" length="1190"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1223" length="1228"></location>
        <text>STAT2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1233" length="1238"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1257" length="1262"></location>
        <text>STAT2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1267" length="1272"></location>
        <text>STAT3</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1312" length="1317"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1352" length="1357"></location>
        <text>STAT1</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Binding</infon>
        <location offset="713" length="738"></location>
        <text>yields coimmunoadsorption</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="789" length="825"></location>
        <text>heterocomplexes are coimmunoadsorbed</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Binding</infon>
        <location offset="933" length="953"></location>
        <text>form heterocomplexes</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="954" length="958"></location>
        <text>when</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="1054" length="1061"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Entity</infon>
        <location offset="1062" length="1070"></location>
        <text>tyrosine</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Phosphorylation</infon>
        <location offset="1071" length="1086"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="1087" length="1110"></location>
        <text>increases the stability</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Binding</infon>
        <location offset="1191" length="1196"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Binding</infon>
        <location offset="1273" length="1306"></location>
        <text>exist in separate heterocomplexes</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T7" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T9" role="Theme2"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
      <node refid="T11" role="Theme2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T27" role="Site"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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  <document>
    <id>PMID-8649779</id>
    <passage>
      <offset>0</offset>
      <text>Inhibition of p105 processing by NF-kappaB proteins in transiently transfected cells.</text>
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        <infon key="type">Protein</infon>
        <location offset="14" length="18"></location>
        <text>p105</text>
      </annotation>
    </passage>
    <passage>
      <offset>87</offset>
      <text>Regulation of the transcription factor NF-kappaB involves proteasome-mediated processing of the NF-kappaB1 p105 precursor protein, which generates the p50 subunit of NF-kappaB. The processing of p105 occurs constitutively in vivo but can be markedly enhanced by various cellular activation agents, although the underlying regulatory mechanism is not yet clear. In the present study, we demonstrate that signal-mediated induction of p105 processing in human T cells is associated with de novo synthesis of this precursor protein. Transient transfection studies performed in COS7 cells revealed that the newly synthesized p105 protein appears to be more rapidly processed compared to its accumulated form that is already associated with the processed product p50. Interestingly, the processing rate of p105 is markedly inhibited in cells co-transfected with p50 or other NF-kappaB subunits, including RelA and c-Rel, that physically interact with p105. These findings suggest that the processing of p105 is subject to negative regulation by the various NF-kappaB subunits. We further demonstrate that p105 undergoes degradation in lipopolysaccharide-stimulated human monocytic cells. However, the inducible degradation of p105 is not coupled with the generation of p50. Together, these studies demonstrate that the processing and inducible degradation of p105 are differentially regulated.</text>
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        <infon key="type">Protein</infon>
        <location offset="183" length="193"></location>
        <text>NF-kappaB1</text>
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        <location offset="194" length="198"></location>
        <text>p105</text>
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        <location offset="238" length="241"></location>
        <text>p50</text>
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        <location offset="282" length="286"></location>
        <text>p105</text>
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        <location offset="519" length="523"></location>
        <text>p105</text>
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        <location offset="707" length="711"></location>
        <text>p105</text>
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        <infon key="type">Protein</infon>
        <location offset="844" length="847"></location>
        <text>p50</text>
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        <location offset="887" length="891"></location>
        <text>p105</text>
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        <location offset="943" length="946"></location>
        <text>p50</text>
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        <location offset="986" length="990"></location>
        <text>RelA</text>
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        <location offset="995" length="1000"></location>
        <text>c-Rel</text>
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        <location offset="1032" length="1036"></location>
        <text>p105</text>
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        <location offset="1084" length="1088"></location>
        <text>p105</text>
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        <location offset="1186" length="1190"></location>
        <text>p105</text>
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        <location offset="1307" length="1311"></location>
        <text>p105</text>
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        <location offset="1350" length="1353"></location>
        <text>p50</text>
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        <location offset="1440" length="1444"></location>
        <text>p105</text>
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        <location offset="224" length="233"></location>
        <text>generates</text>
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        <location offset="806" length="816"></location>
        <text>associated</text>
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        <location offset="1018" length="1026"></location>
        <text>interact</text>
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        <location offset="1191" length="1200"></location>
        <text>undergoes</text>
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        <location offset="1201" length="1212"></location>
        <text>degradation</text>
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        <location offset="1292" length="1303"></location>
        <text>degradation</text>
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        <location offset="1336" length="1346"></location>
        <text>generation</text>
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        <location offset="1425" length="1436"></location>
        <text>degradation</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Regulation</infon>
        <location offset="1449" length="1473"></location>
        <text>differentially regulated</text>
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      <infon key="type">Equiv</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E3">
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
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      <node refid="T13" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8662960</id>
    <passage>
      <offset>0</offset>
      <text>Globin gene switching. In vivo protein-DNA interactions of the human beta-globin locus in erythroid cells expressing the fetal or the adult globin gene program.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="69" length="80"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="43" length="55"></location>
        <text>interactions</text>
      </annotation>
    </passage>
    <passage>
      <offset>162</offset>
      <text>To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene.</text>
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        <location offset="260" length="271"></location>
        <text>beta-globin</text>
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        <infon key="type">Protein</infon>
        <location offset="370" length="375"></location>
        <text>fetal</text>
      </annotation>
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        <location offset="383" length="395"></location>
        <text>adult globin</text>
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        <infon key="type">Protein</infon>
        <location offset="460" length="471"></location>
        <text>beta-globin</text>
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        <infon key="type">Protein</infon>
        <location offset="521" length="527"></location>
        <text>GATA-1</text>
      </annotation>
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        <location offset="579" length="584"></location>
        <text>fetal</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="592" length="610"></location>
        <text>adult globin genes</text>
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        <infon key="type">Protein</infon>
        <location offset="721" length="726"></location>
        <text>fetal</text>
      </annotation>
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        <location offset="734" length="746"></location>
        <text>adult globin</text>
      </annotation>
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        <location offset="1070" length="1077"></location>
        <text>Agamma-</text>
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        <infon key="type">Protein</infon>
        <location offset="1086" length="1097"></location>
        <text>beta-globin</text>
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        <location offset="1147" length="1158"></location>
        <text>beta-globin</text>
      </annotation>
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        <location offset="1314" length="1320"></location>
        <text>gamma-</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1325" length="1336"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1379" length="1390"></location>
        <text>beta-globin</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1458" length="1469"></location>
        <text>beta-globin</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1572" length="1577"></location>
        <text>fetal</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1585" length="1597"></location>
        <text>adult globin</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="207" length="216"></location>
        <text>important</text>
      </annotation>
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        <infon key="type">Regulation</infon>
        <location offset="239" length="246"></location>
        <text>control</text>
      </annotation>
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        <location offset="348" length="358"></location>
        <text>expressing</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="615" length="624"></location>
        <text>expressed</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="706" length="716"></location>
        <text>expressing</text>
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        <location offset="962" length="976"></location>
        <text>more extensive</text>
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        <infon key="type">Binding</infon>
        <location offset="977" length="987"></location>
        <text>footprints</text>
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        <infon key="type">Entity</infon>
        <location offset="1034" length="1057"></location>
        <text>3' regulatory sequences</text>
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        <location offset="1118" length="1122"></location>
        <text>role</text>
      </annotation>
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        <location offset="1164" length="1174"></location>
        <text>expression</text>
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        <location offset="1405" length="1416"></location>
        <text>play a role</text>
      </annotation>
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        <infon key="type">Regulation</infon>
        <location offset="1438" length="1448"></location>
        <text>regulation</text>
      </annotation>
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        <location offset="1475" length="1485"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="1498" length="1508"></location>
        <text>increasing</text>
      </annotation>
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        <infon key="type">Binding</infon>
        <location offset="1528" length="1539"></location>
        <text>interaction</text>
      </annotation>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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      <node refid="E3" role="Theme"></node>
    </relation>
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      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T28" role="Site"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T28" role="Site"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E20" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E21" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E16" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E17" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8675228</id>
    <passage>
      <offset>0</offset>
      <text>Interleukin 10 induced c-fos expression in human B cells by activation of divergent protein kinases.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="14"></location>
        <text>Interleukin 10</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="23" length="28"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="15" length="22"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Gene_expression</infon>
        <location offset="29" length="39"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>102</offset>
      <text>IL-10 is a potent mediator of human B cell growth and plasma cell formation. However, signal transduction of IL-10 in B cells is poorly understood. In this study the effect of IL-10 on the expression of the protooncogene c-fos was investigated, because Fos plays a potential role in the regulation of B cell proliferation and differentiation. B cells were purified from buffy coat preparations of healthy blood donors by positive selection using an anti CD20 monoclonal antibody and a MiniMACS separation unit. B cells were prestimulated with SAC for 48 hrs. Then, cells were incubated with medium or IL-10 (100 ng/ml) for 10 to 120 min. RNA was extracted by phenol/chloroform and c-fos expression was analyzed by PCR assisted mRNA assay. A significant 2-4 fold increase of c-fos expression was observed within 30 min of stimulation with IL-10 (p &lt; 0.01). After 2 hrs c-fos expression declined to basal levels. The effect of IL-10 was dose-dependent with a maximum stimulation using 100 ng/ml of IL-10. The IL-10 effect on c-fos expression was not blocked by polymyxin B. Using the tyrosine kinase inhibitor genistein (10 microM) a complete inhibition of IL-10 induced c-fos expression was observed. In addition, H-7 (10 microM), a specific inhibitor of serine/threonine kinases, significantly blocked IL-10 mediated c-fos expression (p &lt; 0.05). In conclusion, these data show that IL-10 induces c-fos expression in human B-cells by activation of tyrosine and serine/threonine kinases. Since this is the first report on IL-10 induced signal transduction, these data may help to identify the intracellular mechanisms by which IL-10 stimulates human B-cells.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="102" length="107"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="211" length="216"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="278" length="283"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="323" length="328"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="556" length="560"></location>
        <text>CD20</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="703" length="708"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="783" length="788"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="876" length="881"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="940" length="945"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="970" length="975"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1027" length="1032"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1098" length="1103"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1109" length="1114"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1125" length="1130"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1257" length="1262"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1271" length="1276"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1404" length="1409"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1419" length="1424"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1484" length="1489"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1498" length="1503"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1622" length="1627"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1727" length="1732"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Regulation</infon>
        <location offset="268" length="274"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Gene_expression</infon>
        <location offset="291" length="301"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Gene_expression</infon>
        <location offset="789" length="799"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="864" length="872"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Gene_expression</infon>
        <location offset="882" length="892"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="976" length="986"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Negative_regulation</infon>
        <location offset="987" length="995"></location>
        <text>declined</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Regulation</infon>
        <location offset="1115" length="1121"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Gene_expression</infon>
        <location offset="1131" length="1141"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Negative_regulation</infon>
        <location offset="1150" length="1157"></location>
        <text>blocked</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Negative_regulation</infon>
        <location offset="1243" length="1253"></location>
        <text>inhibition</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Positive_regulation</infon>
        <location offset="1263" length="1270"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Gene_expression</infon>
        <location offset="1277" length="1287"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Negative_regulation</infon>
        <location offset="1396" length="1403"></location>
        <text>blocked</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Positive_regulation</infon>
        <location offset="1410" length="1418"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Gene_expression</infon>
        <location offset="1425" length="1435"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Positive_regulation</infon>
        <location offset="1490" length="1497"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Gene_expression</infon>
        <location offset="1504" length="1514"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="T15" role="Cause"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="T19" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8690900</id>
    <passage>
      <offset>0</offset>
      <text>IL-10 inhibits nuclear factor-kappa B/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>IL-10</text>
      </annotation>
    </passage>
    <passage>
      <offset>110</offset>
      <text>IL-10 markedly reduces nuclear factor (NF)-kappa B/Rel nuclear activity induced in PBMC by stimulation with the anti-CD3 mAb OKT3. The inhibition is exerted specifically on the NF-kappa B/Rel activation induced by mAb OKT3, and not that produced by PMA. As judged by supershifting the DNA-protein complexes with Abs recognizing specific components of the NF-kappa B/Rel protein family, the p50/p65 (Rel A) heterodimeric form of NF-kappa B is primarily affected. The maximal effect is observed at the IL-10 concentration of 20 U/ml. IL-10 inhibitory activity is exerted on T lymphocytes and is mediated by monocytes. Indeed, monocytes pretreated with IL-10 are able so inhibit NF-kappa B nuclear activity in purified T lymphocytes stimulated with OKT3. Soluble factors do not appear to be involved in the mechanism of inhibition. On the other hand, the up-regulation of CD80 Ag, found on monocytes obtained from PBMC incubated with OKT3, is not detected after addition of IL-10, and the anti-CD28 mAb CLB-CD28/1 restores the NF-kappa B/Rel nuclear activity in IL-10-inhibited lymphocytes. Therefore, the NF-kappa B/Rel inhibition might be ascribed to a lack of cooperation between accessory cells and T lymphocytes, resulting from down-regulation of a costimulatory molecule, such as CD80, produced by IL-10 on activated monocytes. Our results demonstrate that IL-10 can inhibit the induction of NF-kappa B/Rel nuclear activity in CD3-stimulated T lymphocytes. Since inappropriate activation of kappa B-driven genes has a physiopathologic role in a number of diseases, such as HIV infection, our findings support the possibility of using this cytokine to suppress an undesirable activation of these transcription factors.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="110" length="115"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="500" length="503"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="504" length="507"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="509" length="514"></location>
        <text>Rel A</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="610" length="615"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="642" length="647"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="760" length="765"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="979" length="986"></location>
        <text>CD80 Ag</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1081" length="1086"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1101" length="1105"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1169" length="1174"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1393" length="1397"></location>
        <text>CD80</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1411" length="1416"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1470" length="1475"></location>
        <text>IL-10</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="562" length="570"></location>
        <text>affected</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="962" length="975"></location>
        <text>up-regulation</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1054" length="1062"></location>
        <text>detected</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="1340" length="1355"></location>
        <text>down-regulation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="T10" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T14" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8877104</id>
    <passage>
      <offset>0</offset>
      <text>Vitamin D3- and retinoic acid-induced monocytic differentiation: interactions between the endogenous vitamin D3 receptor, retinoic acid receptors, and retinoid X receptors in U-937 cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="101" length="120"></location>
        <text>vitamin D3 receptor</text>
      </annotation>
    </passage>
    <passage>
      <offset>189</offset>
      <text>Retinoic acid (RA) and 1,25 alpha-dihydroxycholecalciferol (VitD3) are potent regulators of hematopoletic differentiation. Yet, little is known as to how the RA and VitD3 receptor network operates in hematopoietic cells, and whether receptor interactions can explain the interplay between the RA- and VitD3-signaling pathways during differentiation. Therefore, we analyzed the expression, DNA binding, and transcriptional activity of the endogenous RA and VitD3 receptors [retinoic acid receptors (RARs), retinoid X receptors (RXRs), and VitD3 receptor (VDR)] in the U-937 cell line, in which RA and VitD3 induce distinct monocytic differentiation pathways. VitD3 induction resulted in the formation of VDR/RXR DNA-binding complexes on both VitD3 response elements and RA response elements (RAREs). However, transcriptional activation was only observed from a VitD3 response element-driven reporter construct. Several DNA-binding complexes were detected on RAREs in undifferentiated cells. Stimulation by RA resulted in increased RAR beta/RXR DNA binding, activated RARE-dependent transcription, and increased expression of RAR-beta. Concomitant stimulation by VitD3 inhibited the RA-stimulated formation of RAR beta/RXR heterodimers, favoring VDR/RXR binding to the RARE. Also, VitD3 inhibited the expression of CD23 and CD49f, characteristic markers of retinoid-induced U-937 cell differentiation. In contrast, neither the RA-stimulated, RARE-mediated transcription nor the induced RAR-beta expression was suppressed by VitD3, suggesting that VitD3 selectively inhibited the retinoid-induced differentiation program but not the RARE-mediated signal. These results demonstrate a complex role for VitD3 in modifying the retinoid differentiation pathway and may have implications for differentiation-inducing therapy of hematopoietic tumors.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="727" length="741"></location>
        <text>VitD3 receptor</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="743" length="746"></location>
        <text>VDR</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="892" length="895"></location>
        <text>VDR</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1219" length="1227"></location>
        <text>RAR beta</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1313" length="1321"></location>
        <text>RAR-beta</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1397" length="1405"></location>
        <text>RAR beta</text>
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      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1433" length="1436"></location>
        <text>VDR</text>
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      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1502" length="1506"></location>
        <text>CD23</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1511" length="1516"></location>
        <text>CD49f</text>
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      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1673" length="1681"></location>
        <text>RAR-beta</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="566" length="576"></location>
        <text>expression</text>
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      <annotation id="T13">
        <infon key="type">Binding</infon>
        <location offset="582" length="589"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="863" length="871"></location>
        <text>resulted</text>
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      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="879" length="888"></location>
        <text>formation</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="1197" length="1218"></location>
        <text>resulted in increased</text>
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      <annotation id="T17">
        <infon key="type">Binding</infon>
        <location offset="1236" length="1243"></location>
        <text>binding</text>
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      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1289" length="1298"></location>
        <text>increased</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="1299" length="1309"></location>
        <text>expression</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1356" length="1365"></location>
        <text>inhibited</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1373" length="1383"></location>
        <text>stimulated</text>
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      <annotation id="T22">
        <infon key="type">Binding</infon>
        <location offset="1410" length="1422"></location>
        <text>heterodimers</text>
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      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="1424" length="1432"></location>
        <text>favoring</text>
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        <infon key="type">Binding</infon>
        <location offset="1441" length="1448"></location>
        <text>binding</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1474" length="1483"></location>
        <text>inhibited</text>
      </annotation>
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        <location offset="1488" length="1498"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="1665" length="1672"></location>
        <text>induced</text>
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      <annotation id="T28">
        <infon key="type">Gene_expression</infon>
        <location offset="1682" length="1692"></location>
        <text>expression</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1697" length="1707"></location>
        <text>suppressed</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
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    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
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      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E20" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8887687</id>
    <passage>
      <offset>0</offset>
      <text>Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="91" length="100"></location>
        <text>p21Ha-ras</text>
      </annotation>
    </passage>
    <passage>
      <offset>103</offset>
      <text>The induction of immediate-early (IE) response genes, such as egr-1, c-fos, and c-jun, occurs rapidly after the activation of T lymphocytes. The process of activation involves calcium mobilization, activation of protein kinase C (PKC), and phosphorylation of tyrosine kinases. p21(ras), a guanine nucleotide binding factor, mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways. The involvement of p21(ras) in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT. We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells (in the presence of activated p21(ras)) and their correlated consequences. The expression of activated p21(ras) negatively regulated the induction of IE genes by calcium ionophore. This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin in this regulation. A later result of inhibition of this activation pathway by p21(ras) was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production. These results suggest that p2l(ras) is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations.</text>
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        <infon key="type">Protein</infon>
        <location offset="165" length="170"></location>
        <text>egr-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="172" length="177"></location>
        <text>c-fos</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="183" length="188"></location>
        <text>c-jun</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="380" length="388"></location>
        <text>p21(ras)</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="534" length="542"></location>
        <text>p21(ras)</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="795" length="803"></location>
        <text>p21(ras)</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="868" length="876"></location>
        <text>p21(ras)</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1168" length="1175"></location>
        <text>p21(ras</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1286" length="1290"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1358" length="1366"></location>
        <text>p2l(ras)</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="107" length="116"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="190" length="196"></location>
        <text>occurs</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="785" length="794"></location>
        <text>activated</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="844" length="867"></location>
        <text>expression of activated</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="1272" length="1282"></location>
        <text>reductions</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Transcription</infon>
        <location offset="1291" length="1306"></location>
        <text>gene expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8892614</id>
    <passage>
      <offset>0</offset>
      <text>Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="73" length="78"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="83" length="91"></location>
        <text>IL-1beta</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="160" length="163"></location>
        <text>Fas</text>
      </annotation>
    </passage>
    <passage>
      <offset>188</offset>
      <text>Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses.</text>
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        <infon key="type">Protein</infon>
        <location offset="188" length="191"></location>
        <text>Fas</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="336" length="339"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="344" length="354"></location>
        <text>Fas ligand</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="436" length="441"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="517" length="520"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="601" length="606"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="672" length="677"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="780" length="785"></location>
        <text>Bcl-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="833" length="836"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="954" length="960"></location>
        <text>Bcl-xL</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="976" length="983"></location>
        <text>E1B 19K</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1012" length="1015"></location>
        <text>Fas</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1130" length="1133"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1275" length="1280"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1285" length="1302"></location>
        <text>interleukin-1beta</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1380" length="1385"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1390" length="1396"></location>
        <text>Bcl-xL</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1444" length="1447"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="323" length="335"></location>
        <text>up-regulates</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="355" length="365"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Binding</infon>
        <location offset="383" length="394"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="418" length="432"></location>
        <text>Overexpression</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Gene_expression</infon>
        <location offset="418" length="432"></location>
        <text>Overexpression</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="655" length="666"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Gene_expression</infon>
        <location offset="655" length="666"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Gene_expression</infon>
        <location offset="762" length="776"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="762" length="776"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Gene_expression</infon>
        <location offset="940" length="950"></location>
        <text>Expression</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="1372" length="1379"></location>
        <text>express</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1397" length="1406"></location>
        <text>following</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8895544</id>
    <passage>
      <offset>0</offset>
      <text>Interferons induce normal and aberrant retinoic-acid receptors type alpha in acute promyelocytic leukemia cells: potentiation of the induction of retinoid-dependent differentiation markers.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="39" length="73"></location>
        <text>retinoic-acid receptors type alpha</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="12" length="18"></location>
        <text>induce</text>
      </annotation>
    </passage>
    <passage>
      <offset>191</offset>
      <text>Treatment of the acute promyelocytic (APL) cell line NB4 with interferon alpha (IFN(alpha)), as well as IFN(beta) and gamma, results in an increased expression of the transcripts coding for retinoic-acid receptor type alpha (RAR(alpha)) and the leukemia-specific retinoic acid receptor PML-RAR. Transcriptional induction of the RAR(alpha) and PML-RAR mRNAs is rapid and it is parallelled by an increase in the corresponding proteins. Up-regulation of RAR(alpha) and PML-RAR gene expression by IFN(alpha) is accompanied by a strong potentiation in the induction of 2 retinoid-dependent granulocytic markers, i.e., granulocyte-colony-stimulating factor receptor mRNA and leukocyte alkaline phosphatase. However, IFN(alpha) does not have any effects on the retinoid-dependent regulation of the myeloid surface markers CD11b and CD33. The IFN-dependent increase in RAR(alpha) levels and the enhancing effect of the cytokine on retinoid-dependent granulocytic markers expression may be a characteristic of PML-RAR positive cells, since the phenomena are not observed in HL-60 promyelocytes. Interferons as well as retinoids inhibit the growth of NB4 cells, although the 2 classes of compounds do not significantly interact in terms of anti-proliferative activity. These results suggest the possible use of combinations between IFNs and retinoic acid in the cyto-differentiating treatment of APL patients.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="381" length="414"></location>
        <text>retinoic-acid receptor type alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="416" length="426"></location>
        <text>RAR(alpha)</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="436" length="476"></location>
        <text>leukemia-specific retinoic acid receptor</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="477" length="484"></location>
        <text>PML-RAR</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="519" length="529"></location>
        <text>RAR(alpha)</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="534" length="541"></location>
        <text>PML-RAR</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="642" length="652"></location>
        <text>RAR(alpha)</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="657" length="664"></location>
        <text>PML-RAR</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="804" length="850"></location>
        <text>granulocyte-colony-stimulating factor receptor</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="860" length="890"></location>
        <text>leukocyte alkaline phosphatase</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1006" length="1011"></location>
        <text>CD11b</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1016" length="1020"></location>
        <text>CD33</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1052" length="1062"></location>
        <text>RAR(alpha)</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1192" length="1199"></location>
        <text>PML-RAR</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="316" length="339"></location>
        <text>results in an increased</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Transcription</infon>
        <location offset="340" length="350"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="486" length="511"></location>
        <text>Transcriptional induction</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="567" length="578"></location>
        <text>parallelled</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="585" length="593"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="625" length="638"></location>
        <text>Up-regulation</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="670" length="680"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="742" length="751"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="766" length="775"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Regulation</infon>
        <location offset="930" length="937"></location>
        <text>effects</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Regulation</infon>
        <location offset="964" length="974"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="1040" length="1048"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Gene_expression</infon>
        <location offset="1063" length="1069"></location>
        <text>levels</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="1244" length="1252"></location>
        <text>observed</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="E6" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E20" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E21" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E26" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8896456</id>
    <passage>
      <offset>0</offset>
      <text>An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="3" length="7"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="38" length="63"></location>
        <text>IL-2 receptor alpha chain</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="107" length="112"></location>
        <text>Stat5</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="114" length="119"></location>
        <text>Elf-1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="121" length="129"></location>
        <text>HMG-I(Y)</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="101" length="106"></location>
        <text>binds</text>
      </annotation>
    </passage>
    <passage>
      <offset>158</offset>
      <text>Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.</text>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="182" length="223"></location>
        <text>interleukin-2 (IL-2) receptor alpha chain</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="272" length="276"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="331" length="335"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="648" length="652"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="715" length="719"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="790" length="795"></location>
        <text>Stat5</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="797" length="802"></location>
        <text>Elf-1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="804" length="812"></location>
        <text>HMG-I(Y)</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="850" length="854"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1093" length="1104"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1121" length="1125"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1134" length="1145"></location>
        <text>IL-2R alpha</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="158" length="168"></location>
        <text>Expression</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="241" length="252"></location>
        <text>upregulated</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Binding</infon>
        <location offset="838" length="842"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="1063" length="1082"></location>
        <text>negatively regulate</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Entity</infon>
        <location offset="1105" length="1113"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="1126" length="1133"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Entity</infon>
        <location offset="1146" length="1154"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="1164" length="1172"></location>
        <text>requires</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T7" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T23" role="Site"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
      <node refid="T25" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8898960</id>
    <passage>
      <offset>0</offset>
      <text>Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="26" length="49"></location>
        <text>class II transactivator</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Positive_regulation</infon>
        <location offset="0" length="6"></location>
        <text>Active</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Negative_regulation</infon>
        <location offset="7" length="18"></location>
        <text>suppression</text>
      </annotation>
    </passage>
    <passage>
      <offset>223</offset>
      <text>In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism.</text>
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        <infon key="type">Protein</infon>
        <location offset="788" length="811"></location>
        <text>class II transactivator</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="813" length="818"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1043" length="1048"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1081" length="1086"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1153" length="1158"></location>
        <text>CIITA</text>
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      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1556" length="1561"></location>
        <text>CIITA</text>
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        <infon key="type">Protein</infon>
        <location offset="1706" length="1718"></location>
        <text>HLA-DQ alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1722" length="1730"></location>
        <text>-DQ beta</text>
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      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1791" length="1796"></location>
        <text>CIITA</text>
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        <infon key="type">Protein</infon>
        <location offset="2048" length="2053"></location>
        <text>CIITA</text>
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        <infon key="type">Negative_regulation</infon>
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        <text>act</text>
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        <infon key="type">Transcription</infon>
        <location offset="736" length="749"></location>
        <text>transcription</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>express</text>
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        <infon key="type">Transcription</infon>
        <location offset="1096" length="1107"></location>
        <text>transcripts</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="1109" length="1121"></location>
        <text>Transfection</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1109" length="1121"></location>
        <text>Transfection</text>
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        <infon key="type">Regulation</infon>
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        <text>under the control</text>
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        <location offset="1562" length="1573"></location>
        <text>transfected</text>
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        <text>transfected</text>
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        <infon key="type">Regulation</infon>
        <location offset="1682" length="1694"></location>
        <text>attributable</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1698" length="1702"></location>
        <text>lack</text>
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        <infon key="type">Transcription</infon>
        <location offset="1731" length="1744"></location>
        <text>transcription</text>
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        <text>present</text>
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        <infon key="type">Positive_regulation</infon>
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        <text>transfected</text>
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        <location offset="1797" length="1808"></location>
        <text>transfected</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1830" length="1844"></location>
        <text>reduced levels</text>
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    </passage>
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      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T2" role="Event"></node>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
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    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
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    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
      <node refid="E20" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
      <node refid="E20" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8901569</id>
    <passage>
      <offset>0</offset>
      <text>Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="19" length="31"></location>
        <text>fetal globin</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="118" length="122"></location>
        <text>EKLF</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="0" length="9"></location>
        <text>Silencing</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="32" length="42"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Negative_regulation</infon>
        <location offset="46" length="54"></location>
        <text>impaired</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Negative_regulation</infon>
        <location offset="62" length="69"></location>
        <text>absence</text>
      </annotation>
    </passage>
    <passage>
      <offset>125</offset>
      <text>Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.</text>
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        <infon key="type">Protein</infon>
        <location offset="228" length="248"></location>
        <text>human fetal (gamma)-</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="251" length="270"></location>
        <text>adult (beta)-globin</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="431" length="460"></location>
        <text>erythroid Kruppel-like factor</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="462" length="466"></location>
        <text>EKLF</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="503" length="514"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="643" length="647"></location>
        <text>EKLF</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="693" length="705"></location>
        <text>gamma-globin</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="724" length="728"></location>
        <text>EKLF</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="777" length="788"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="859" length="863"></location>
        <text>EKLF</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="877" length="888"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="925" length="937"></location>
        <text>gamma-globin</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1007" length="1019"></location>
        <text>gamma-globin</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1042" length="1046"></location>
        <text>EKLF</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1117" length="1129"></location>
        <text>gamma-globin</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1133" length="1144"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1228" length="1232"></location>
        <text>EKLF</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1290" length="1301"></location>
        <text>beta-globin</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1363" length="1375"></location>
        <text>gamma-globin</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Negative_regulation</infon>
        <location offset="216" length="222"></location>
        <text>switch</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="216" length="222"></location>
        <text>switch</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Gene_expression</infon>
        <location offset="271" length="281"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="472" length="480"></location>
        <text>required</text>
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      <annotation id="T30">
        <infon key="type">Gene_expression</infon>
        <location offset="485" length="495"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="540" length="547"></location>
        <text>through</text>
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      <annotation id="T32">
        <infon key="type">Binding</infon>
        <location offset="548" length="559"></location>
        <text>interaction</text>
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      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="635" length="639"></location>
        <text>role</text>
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      <annotation id="T34">
        <infon key="type">Regulation</infon>
        <location offset="669" length="679"></location>
        <text>regulation</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Gene_expression</infon>
        <location offset="889" length="899"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Negative_regulation</infon>
        <location offset="916" length="923"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Positive_regulation</infon>
        <location offset="954" length="962"></location>
        <text>elevated</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Negative_regulation</infon>
        <location offset="985" length="993"></location>
        <text>Impaired</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Negative_regulation</infon>
        <location offset="994" length="1003"></location>
        <text>silencing</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Gene_expression</infon>
        <location offset="1020" length="1030"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Positive_regulation</infon>
        <location offset="1081" length="1094"></location>
        <text>participating</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Negative_regulation</infon>
        <location offset="1145" length="1151"></location>
        <text>switch</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Positive_regulation</infon>
        <location offset="1145" length="1151"></location>
        <text>switch</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Positive_regulation</infon>
        <location offset="1233" length="1241"></location>
        <text>mediates</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Binding</infon>
        <location offset="1266" length="1277"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T46">
        <infon key="type">Entity</infon>
        <location offset="1307" length="1315"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T47">
        <infon key="type">Negative_regulation</infon>
        <location offset="1350" length="1358"></location>
        <text>excludes</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="E4" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="E12" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="E24" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="E26" role="Theme"></node>
      <node refid="T19" role="Cause"></node>
    </relation>
    <relation id="E26">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
      <node refid="T46" role="Site"></node>
    </relation>
    <relation id="E27">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T47" role="Trigger"></node>
      <node refid="T21" role="Theme"></node>
      <node refid="E25" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8903467</id>
    <passage>
      <offset>0</offset>
      <text>Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin's disease.</text>
    </passage>
    <passage>
      <offset>133</offset>
      <text>Nasal T/NK-cell lymphomas can be further separated into those of natural killer (NK) cell lineage or of T-cell lineage, with differences in cellular phenotype, T-cell receptor (TcR) gene rearrangement and TcR transcript expression. Both NK- and T-cell subtypes are closely associated with Epstein-Barr virus (EBV). In this study, EBV gene expression was determined in 23 cases of nasal lymphoma (NL) by in situ hybridisation (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IH). Of the 23 cases, 19 were classified as NK-cell and 4 as T-cell tumours. ISH for EBV-encoded small non-polyadenylated RNAs showed that all cases, whether NK or T, harboured EBV in virtually all tumour cells. RT-PCR demonstrated that NL of both subtypes expressed EBNAI of the QUK splice pattern, the latent membrane proteins, LMP1 and 2 and the BamHI A rightward transcripts in the absence of EBNA2 mRNAs, compatible with the latency type II pattern. In addition, analysis of EBV protein expression by IH revealed a heterogeneous pattern of EBV gene expression at the single-cell level consisting of both LMP1+ and LMP1- tumour cells, suggesting a mixture of latency I and II. Although 2 early lytic transcripts, BZLF1 and BHRF1, were also detected in 13 and 10 cases, respectively, the lack of ZEBRA staining in any case indicates that these lytic transcripts are most likely expressed by rare cells in the biopsies entering lytic cycle. The viral transcriptional pattern similar to that of nasopharyngeal carcinoma and Hodgkin's disease suggests that EBV can exploit common regulatory mechanisms for gene transcription in diverse host cell types. Down-regulation of immunogenic proteins (EBNA2-EBNA6) in nasal lymphoma may enable tumour cells to evade host cytotoxic T-cell surveillance.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="915" length="920"></location>
        <text>EBNAI</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="978" length="982"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="987" length="988"></location>
        <text>2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1045" length="1050"></location>
        <text>EBNA2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1257" length="1261"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1267" length="1271"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1365" length="1370"></location>
        <text>BZLF1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1375" length="1380"></location>
        <text>BHRF1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1447" length="1452"></location>
        <text>ZEBRA</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1842" length="1847"></location>
        <text>EBNA2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1848" length="1853"></location>
        <text>EBNA6</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="905" length="914"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Transcription</infon>
        <location offset="1034" length="1041"></location>
        <text>absence</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="1392" length="1400"></location>
        <text>detected</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Transcription</infon>
        <location offset="1439" length="1443"></location>
        <text>lack</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Transcription</infon>
        <location offset="1529" length="1538"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Negative_regulation</infon>
        <location offset="1801" length="1816"></location>
        <text>Down-regulation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8910577</id>
    <passage>
      <offset>0</offset>
      <text>Identification and characterization of a leukocyte-specific component of the nuclear body.</text>
    </passage>
    <passage>
      <offset>92</offset>
      <text>The nuclear body (NB) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection. The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis. In this study, serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB, a 140-kDa protein designated Sp140. The predicted amino acid sequence of the amino-terminal portion of Sp140 was similar to Sp100, a previously identified NB protein. The carboxyl portion of Sp140 contained a zinc-finger domain and a bromodomain, motifs that are present in proteins regulating gene transcription. High levels of Sp140 mRNA were detected in human spleen and peripheral blood leukocytes, but not other human tissues. The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation. Immunohistochemical techniques were used to demonstrate that SP140 localized to the NB in differentiated HL60 and NB4 cells. The location of Sp140 in the NB, and expression of this gene in cells involved in host defense, suggest that Sp140 may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="508" length="513"></location>
        <text>Sp140</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="582" length="587"></location>
        <text>Sp140</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="603" length="608"></location>
        <text>Sp100</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="670" length="675"></location>
        <text>Sp140</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="808" length="813"></location>
        <text>Sp140</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="924" length="929"></location>
        <text>SP140</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1120" length="1125"></location>
        <text>SP140</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1200" length="1205"></location>
        <text>Sp140</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1293" length="1298"></location>
        <text>Sp140</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Positive_regulation</infon>
        <location offset="793" length="804"></location>
        <text>High levels</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Transcription</infon>
        <location offset="824" length="832"></location>
        <text>detected</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Transcription</infon>
        <location offset="915" length="920"></location>
        <text>level</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="989" length="998"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Localization</infon>
        <location offset="1126" length="1135"></location>
        <text>localized</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Entity</infon>
        <location offset="1143" length="1145"></location>
        <text>NB</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Localization</infon>
        <location offset="1188" length="1196"></location>
        <text>location</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Entity</infon>
        <location offset="1213" length="1215"></location>
        <text>NB</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Gene_expression</infon>
        <location offset="1221" length="1231"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T15" role="ToLoc"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T17" role="AtLoc"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8913871</id>
    <passage>
      <offset>0</offset>
      <text>Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3.</text>
    </passage>
    <passage>
      <offset>109</offset>
      <text>The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="897" length="908"></location>
        <text>VD receptor</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="910" length="913"></location>
        <text>VDR</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Binding</infon>
        <location offset="875" length="889"></location>
        <text>lower affinity</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T1" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T3" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-8925904</id>
    <passage>
      <offset>0</offset>
      <text>Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145.</text>
    </passage>
    <passage>
      <offset>161</offset>
      <text>A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="220" length="237"></location>
        <text>androgen receptor</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="239" length="241"></location>
        <text>AR</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="377" length="379"></location>
        <text>AR</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="494" length="513"></location>
        <text>CRE binding protein</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="515" length="519"></location>
        <text>CREB</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="574" length="576"></location>
        <text>AR</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="637" length="641"></location>
        <text>CREB</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="667" length="669"></location>
        <text>AR</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Regulation</infon>
        <location offset="186" length="212"></location>
        <text>transcriptional regulation</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Positive_regulation</infon>
        <location offset="251" length="254"></location>
        <text>via</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="389" length="400"></location>
        <text>upregulated</text>
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      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="619" length="631"></location>
        <text>interactions</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Regulation</infon>
        <location offset="658" length="666"></location>
        <text>regulate</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="675" length="688"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Negative_regulation</infon>
        <location offset="725" length="729"></location>
        <text>lost</text>
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    </passage>
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      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T1" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9032466</id>
    <passage>
      <offset>0</offset>
      <text>Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid.</text>
    </passage>
    <passage>
      <offset>101</offset>
      <text>Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release.</text>
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        <infon key="type">Protein</infon>
        <location offset="709" length="712"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="713" length="716"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="889" length="902"></location>
        <text>IkappaB-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="919" length="923"></location>
        <text>p105</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1378" length="1406"></location>
        <text>tumour necrosis factor-alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1408" length="1417"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1458" length="1467"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1649" length="1658"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1933" length="1942"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Positive_regulation</infon>
        <location offset="634" length="642"></location>
        <text>promoted</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Localization</infon>
        <location offset="647" length="657"></location>
        <text>appearance</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Entity</infon>
        <location offset="725" length="732"></location>
        <text>nucleus</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein_catabolism</infon>
        <location offset="903" length="914"></location>
        <text>proteolysis</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="940" length="947"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="1343" length="1355"></location>
        <text>treated with</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Localization</infon>
        <location offset="1369" length="1377"></location>
        <text>released</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Localization</infon>
        <location offset="1468" length="1475"></location>
        <text>release</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1922" length="1932"></location>
        <text>subsequent</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Localization</infon>
        <location offset="1943" length="1950"></location>
        <text>release</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T12" role="AtLoc"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T12" role="AtLoc"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9043959</id>
    <passage>
      <offset>0</offset>
      <text>Thymocytes control the CD4 gene differently from mature T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="23" length="26"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Regulation</infon>
        <location offset="11" length="18"></location>
        <text>control</text>
      </annotation>
    </passage>
    <passage>
      <offset>72</offset>
      <text>We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="157" length="160"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="386" length="389"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="561" length="564"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="819" length="822"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="845" length="848"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="937" length="940"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="965" length="968"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1146" length="1149"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1185" length="1188"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1216" length="1219"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1308" length="1311"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1364" length="1367"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="374" length="381"></location>
        <text>control</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="569" length="584"></location>
        <text>double-negative</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="655" length="670"></location>
        <text>double-positive</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="832" length="840"></location>
        <text>required</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="854" length="864"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Negative_regulation</infon>
        <location offset="956" length="964"></location>
        <text>inhibits</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="974" length="984"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="1132" length="1145"></location>
        <text>down-regulate</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="1155" length="1165"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="1289" length="1297"></location>
        <text>required</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Negative_regulation</infon>
        <location offset="1301" length="1307"></location>
        <text>resume</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Gene_expression</infon>
        <location offset="1312" length="1322"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="1376" length="1385"></location>
        <text>regulated</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Negative_regulation</infon>
        <location offset="1376" length="1385"></location>
        <text>regulated</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E12" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9149909</id>
    <passage>
      <offset>0</offset>
      <text>Triggering of HLA-DR antigens differentially modulates tumor necrosis factor alpha release by B cells at distinct stage of maturation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="55" length="82"></location>
        <text>tumor necrosis factor alpha</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Regulation</infon>
        <location offset="45" length="54"></location>
        <text>modulates</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Localization</infon>
        <location offset="83" length="90"></location>
        <text>release</text>
      </annotation>
    </passage>
    <passage>
      <offset>136</offset>
      <text>Triggering of HLA class II antigens by the anti-HLA-DR monoclonal antibody (mAb) L243 significantly (P &lt; 0.05) and differentially enhanced the release of tumor necrosis factor alpha (TNF-alpha) by the non-Hodgkin's lymphoma cells Ri-I, Ci-I, and Sc-I, which are at a distinct stage of B-cell differentiation, and by the more mature Burkitt lymphoma cell Raji; in contrast, it did not induce TNF-alpha release by the pre-B leukemia cells Nalm-6 and BV173. TNF-alpha release peaked at 24 h and decreased thereafter, and it was dose dependent and preceded by an increase of TNF-alpha mRNA detectable after 3 h of stimulation with mAb L243. Secreted TNF-alpha mediated the enhancement of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding activity; in fact, the triggering of HLA-DR antigens in the presence of antihuman TNF-alpha-neutralizing antibodies did not upregulate NF-kappa B and AP-1. In contrast, released TNF-alpha was not responsible for the homotypic aggregation of Ri-I, Ci-I, Sc-I, and Raji cells induced by mAb L243, and it did not affect the proliferation of B cells investigated. Altogether, our data demonstrate that: (a) the ability of B cells to release TNF-alpha after triggering of HLA-DR antigens depends on their stage of differentiation; (b) levels of released TNF-alpha seem to correlate with the stage of B-cell maturation but do not correlate with the amounts of cell surface HLA-DR antigens; (c) secreted TNF-alpha regulates the levels of expression of NF-kappa B and AP-1 by an autocrine loop; and (d) intracellular signals mediating TNF-alpha release by B cells are distinct from those regulating homotypic aggregation and proliferation.</text>
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        <text>tumor necrosis factor alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="319" length="328"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="527" length="536"></location>
        <text>TNF-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="591" length="600"></location>
        <text>TNF-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="707" length="716"></location>
        <text>TNF-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="782" length="791"></location>
        <text>TNF-alpha</text>
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        <location offset="977" length="986"></location>
        <text>TNF-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1073" length="1082"></location>
        <text>TNF-alpha</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1332" length="1341"></location>
        <text>TNF-alpha</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1444" length="1453"></location>
        <text>TNF-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1592" length="1601"></location>
        <text>TNF-alpha</text>
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        <location offset="1722" length="1731"></location>
        <text>TNF-alpha</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="266" length="274"></location>
        <text>enhanced</text>
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        <infon key="type">Localization</infon>
        <location offset="279" length="286"></location>
        <text>release</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="520" length="526"></location>
        <text>induce</text>
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        <infon key="type">Localization</infon>
        <location offset="537" length="544"></location>
        <text>release</text>
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        <infon key="type">Localization</infon>
        <location offset="601" length="608"></location>
        <text>release</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="609" length="615"></location>
        <text>peaked</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="680" length="688"></location>
        <text>preceded</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="695" length="703"></location>
        <text>increase</text>
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        <text>Secreted</text>
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        <infon key="type">Localization</infon>
        <location offset="1064" length="1072"></location>
        <text>released</text>
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        <infon key="type">Localization</infon>
        <location offset="1324" length="1331"></location>
        <text>release</text>
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        <location offset="1342" length="1347"></location>
        <text>after</text>
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        <infon key="type">Localization</infon>
        <location offset="1425" length="1443"></location>
        <text>levels of released</text>
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        <infon key="type">Localization</infon>
        <location offset="1583" length="1591"></location>
        <text>secreted</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1712" length="1721"></location>
        <text>mediating</text>
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      <annotation id="T31">
        <infon key="type">Localization</infon>
        <location offset="1732" length="1739"></location>
        <text>release</text>
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      <infon key="type">Equiv</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
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    <relation id="E6">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
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    <relation id="E7">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="E10" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9154298</id>
    <passage>
      <offset>0</offset>
      <text>Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="49" length="62"></location>
        <text>interleukin-6</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="38" length="48"></location>
        <text>upregulate</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Gene_expression</infon>
        <location offset="68" length="78"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>81</offset>
      <text>BACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection.</text>
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        <infon key="type">Protein</infon>
        <location offset="243" length="256"></location>
        <text>Interleukin-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="258" length="262"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="332" length="345"></location>
        <text>Interleukin-6</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="456" length="460"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="633" length="637"></location>
        <text>IL-6</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="735" length="748"></location>
        <text>Interleukin-6</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="761" length="765"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="874" length="878"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="879" length="913"></location>
        <text>chloramphenicol acetyl transferase</text>
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        <infon key="type">Protein</infon>
        <location offset="915" length="918"></location>
        <text>CAT</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="923" length="927"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="969" length="973"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1002" length="1015"></location>
        <text>Interleukin-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1257" length="1261"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1288" length="1292"></location>
        <text>IL-6</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1293" length="1296"></location>
        <text>CAT</text>
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        <infon key="type">Protein</infon>
        <location offset="1301" length="1305"></location>
        <text>IL-6</text>
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        <infon key="type">Protein</infon>
        <location offset="1459" length="1463"></location>
        <text>IL-6</text>
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        <infon key="type">Protein</infon>
        <location offset="1620" length="1624"></location>
        <text>IL-6</text>
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        <location offset="1666" length="1670"></location>
        <text>IL-6</text>
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        <text>IL-6</text>
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        <text>TNF alpha</text>
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        <text>IL-6</text>
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        <text>regulation</text>
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        <location offset="624" length="632"></location>
        <text>modulate</text>
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        <text>production</text>
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        <location offset="1033" length="1043"></location>
        <text>production</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1063" length="1072"></location>
        <text>increased</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1229" length="1237"></location>
        <text>enhanced</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1242" length="1253"></location>
        <text>stimulation</text>
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        <infon key="type">Entity</infon>
        <location offset="1262" length="1270"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1597" length="1605"></location>
        <text>enhanced</text>
      </annotation>
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        <location offset="1606" length="1616"></location>
        <text>expression</text>
      </annotation>
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        <infon key="type">Entity</infon>
        <location offset="1767" length="1775"></location>
        <text>promoter</text>
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        <infon key="type">Regulation</infon>
        <location offset="1776" length="1786"></location>
        <text>responsive</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Positive_regulation</infon>
        <location offset="1895" length="1902"></location>
        <text>enhance</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Gene_expression</infon>
        <location offset="1908" length="1918"></location>
        <text>production</text>
      </annotation>
    </passage>
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      <infon key="type">Equiv</infon>
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    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
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    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
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      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T35" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
      <node refid="T38" role="Site"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T39" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
      <node refid="T23" role="Cause"></node>
      <node refid="T38" role="Site"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T24" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9159166</id>
    <passage>
      <offset>0</offset>
      <text>Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="30" length="64"></location>
        <text>interleukin 4 receptor alpha chain</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="136" length="170"></location>
        <text>interleukin 2 receptor gamma chain</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Binding</infon>
        <location offset="0" length="16"></location>
        <text>Homodimerization</text>
      </annotation>
    </passage>
    <passage>
      <offset>173</offset>
      <text>The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="187" length="205"></location>
        <text>interleukin (IL)-4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="210" length="215"></location>
        <text>IL-13</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="391" length="395"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="404" length="420"></location>
        <text>IL-4Ralpha chain</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="463" length="488"></location>
        <text>IL-2Rgamma (gammac) chain</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="511" length="516"></location>
        <text>IL-7R</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="518" length="523"></location>
        <text>IL-9R</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="537" length="541"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="636" length="648"></location>
        <text>gammac chain</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="707" length="711"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="737" length="749"></location>
        <text>gammac chain</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="807" length="823"></location>
        <text>IL-4Ralpha chain</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1093" length="1107"></location>
        <text>erythropoietin</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1152" length="1162"></location>
        <text>IL-4Ralpha</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1171" length="1185"></location>
        <text>Janus kinase 1</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1187" length="1191"></location>
        <text>Jak1</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1205" length="1210"></location>
        <text>STAT6</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1302" length="1306"></location>
        <text>Jak1</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1343" length="1353"></location>
        <text>IL-4Ralpha</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1441" length="1478"></location>
        <text>chimeric CD8alpha/IL-4Ralpha receptor</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1642" length="1658"></location>
        <text>IL-4Ralpha chain</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1686" length="1690"></location>
        <text>Jak1</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="396" length="403"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Negative_regulation</infon>
        <location offset="649" length="659"></location>
        <text>deficiency</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Gene_expression</infon>
        <location offset="728" length="732"></location>
        <text>lack</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="1163" length="1170"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="1193" length="1203"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="1422" length="1433"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Gene_expression</infon>
        <location offset="1422" length="1433"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Gene_expression</infon>
        <location offset="1489" length="1498"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="1519" length="1533"></location>
        <text>as a homodimer</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Binding</infon>
        <location offset="1618" length="1634"></location>
        <text>homodimerization</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T18" role="Event"></node>
      <node refid="T17" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
      <node refid="T6" role="Theme2"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T22" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T23" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9160887</id>
    <passage>
      <offset>0</offset>
      <text>Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="74" length="77"></location>
        <text>Tax</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="51" length="61"></location>
        <text>transduced</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Positive_regulation</infon>
        <location offset="51" length="61"></location>
        <text>transduced</text>
      </annotation>
    </passage>
    <passage>
      <offset>131</offset>
      <text>Tax1, a transcriptional trans-activator of the Human T-cell leukemia virus type I (HTLV-I), induces the expression of many cellular genes through interaction with at least three distinct cellular transcription factors; CREB/ATF, NF-kappaB, and SRF. This Tax1-induced activation of cellular genes is considered to be a critical event in T-cell transformation by HTLV-I. To elucidate the role of each Tax1-inducible transcriptional pathway in T-cell transformation, we introduced Tax1 mutants with different trans-activating phenotypes into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kappaB pathway is sufficient to promote the growth response to IL-2. However, for the clonal expansion of CD4+ T-cells, which is a characteristic result of HTLV-I infection, activation of the CREB/ATF and SRF pathways is also required.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="131" length="135"></location>
        <text>Tax1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="375" length="378"></location>
        <text>SRF</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="385" length="389"></location>
        <text>Tax1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="530" length="534"></location>
        <text>Tax1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="609" length="613"></location>
        <text>Tax1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="849" length="853"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="892" length="895"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="991" length="994"></location>
        <text>SRF</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="277" length="288"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="598" length="608"></location>
        <text>introduced</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="598" length="608"></location>
        <text>introduced</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T3" role="Theme2"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9164841</id>
    <passage>
      <offset>0</offset>
      <text>Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="50" length="76"></location>
        <text>mineralocorticoid receptor</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="126" length="133"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <passage>
      <offset>177</offset>
      <text>To gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Leger and Auzou (1996) J.Steroid Biochem.Mol.Biol.57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="249" length="275"></location>
        <text>mineralocorticoid receptor</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="278" length="280"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="478" length="480"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="619" length="621"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="662" length="664"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="690" length="692"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="918" length="920"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="960" length="962"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1065" length="1067"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1328" length="1330"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1356" length="1358"></location>
        <text>MR</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1433" length="1438"></location>
        <text>hsp90</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1471" length="1474"></location>
        <text>hMR</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Binding</infon>
        <location offset="228" length="235"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Binding</infon>
        <location offset="578" length="588"></location>
        <text>recognized</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Regulation</infon>
        <location offset="847" length="853"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Binding</infon>
        <location offset="902" length="909"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="951" length="959"></location>
        <text>liganded</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Negative_regulation</infon>
        <location offset="1117" length="1126"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Binding</infon>
        <location offset="1141" length="1148"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Regulation</infon>
        <location offset="1154" length="1160"></location>
        <text>effect</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Binding</infon>
        <location offset="1176" length="1183"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Binding</infon>
        <location offset="1331" length="1338"></location>
        <text>complex</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="1359" length="1366"></location>
        <text>complex</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="1449" length="1461"></location>
        <text>cross-linked</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Negative_regulation</infon>
        <location offset="1509" length="1522"></location>
        <text>still able to</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Regulation</infon>
        <location offset="1523" length="1528"></location>
        <text>react</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Binding</infon>
        <location offset="1641" length="1648"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Binding</infon>
        <location offset="2001" length="2008"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T14" role="Theme2"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
      <node refid="E14" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E15" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9164919</id>
    <passage>
      <offset>0</offset>
      <text>Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="72" length="85"></location>
        <text>IkappaB alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Positive_regulation</infon>
        <location offset="54" length="68"></location>
        <text>overexpression</text>
      </annotation>
    </passage>
    <passage>
      <offset>88</offset>
      <text>A role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="317" length="330"></location>
        <text>IkappaB alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Gene_expression</infon>
        <location offset="297" length="307"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T3" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T4" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9164948</id>
    <passage>
      <offset>0</offset>
      <text>Sequential development of structural and functional alterations in T cells from tumor-bearing mice.</text>
    </passage>
    <passage>
      <offset>101</offset>
      <text>The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 deltaepsilongamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (&gt;26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="191" length="200"></location>
        <text>CD3 delta</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="200" length="207"></location>
        <text>epsilon</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="207" length="212"></location>
        <text>gamma</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="217" length="225"></location>
        <text>TCR zeta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="653" length="667"></location>
        <text>TCR zeta chain</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="700" length="724"></location>
        <text>tyrosine kinase p56(lck)</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="868" length="871"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1047" length="1051"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1056" length="1065"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1127" length="1141"></location>
        <text>TCR zeta chain</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1146" length="1154"></location>
        <text>p56(lck)</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Negative_regulation</infon>
        <location offset="857" length="864"></location>
        <text>failure</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Localization</infon>
        <location offset="872" length="885"></location>
        <text>translocation</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Entity</infon>
        <location offset="893" length="900"></location>
        <text>nucleus</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="902" length="907"></location>
        <text>occur</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="945" length="953"></location>
        <text>decrease</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Positive_regulation</infon>
        <location offset="987" length="997"></location>
        <text>paralleled</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Negative_regulation</infon>
        <location offset="1004" length="1020"></location>
        <text>impaired ability</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="1024" length="1031"></location>
        <text>produce</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="1094" length="1102"></location>
        <text>followed</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Negative_regulation</infon>
        <location offset="1119" length="1123"></location>
        <text>loss</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T14" role="ToLoc"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
      <node refid="E1" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9166418</id>
    <passage>
      <offset>0</offset>
      <text>An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="110" length="133"></location>
        <text>class II transactivator</text>
      </annotation>
    </passage>
    <passage>
      <offset>136</offset>
      <text>Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="262" length="285"></location>
        <text>class II transactivator</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="287" length="292"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="650" length="655"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="783" length="788"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="865" length="870"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="997" length="1002"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1049" length="1054"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1383" length="1388"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="851" length="861"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="1066" length="1076"></location>
        <text>detectable</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9170401</id>
    <passage>
      <offset>0</offset>
      <text>Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets.</text>
    </passage>
    <passage>
      <offset>93</offset>
      <text>BACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="654" length="658"></location>
        <text>CD41</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="909" length="917"></location>
        <text>thrombin</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1033" length="1056"></location>
        <text>interleukin (IL),1 beta</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1058" length="1062"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1068" length="1108"></location>
        <text>monocyte chemoattractant protein (MCP)-1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1154" length="1163"></location>
        <text>IL-1 beta</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1168" length="1172"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1288" length="1293"></location>
        <text>MCP-1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1495" length="1505"></location>
        <text>P selectin</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1699" length="1708"></location>
        <text>IL-1 beta</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1710" length="1714"></location>
        <text>IL-8</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1720" length="1725"></location>
        <text>MCP-1</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="1002" length="1023"></location>
        <text>resulted in increased</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="1220" length="1229"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="1306" length="1315"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="1691" length="1698"></location>
        <text>induces</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9171108</id>
    <passage>
      <offset>0</offset>
      <text>The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="4" length="28"></location>
        <text>class II trans-activator</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="29" length="34"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="54" length="57"></location>
        <text>TBP</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="76" length="83"></location>
        <text>TAFII32</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Binding</infon>
        <location offset="35" length="44"></location>
        <text>interacts</text>
      </annotation>
    </passage>
    <passage>
      <offset>86</offset>
      <text>The class II trans- activator (CIITA) is the main transcriptional co-activator for the expression of MHC class II proteins. Its N-terminal 125 amino acids function as an independent transcriptional activation domain. Analyses of the primary amino acid sequence of the activation domain predict the presence of three alpha-helices, each with a high proportion of acidic residues. Using site-directed mutagenesis, we found that two of these predicted alpha-helices are required for full transcriptional activation by CIITA. Moreover, a CIITA protein in which both functional alpha-helices have been deleted displays a dominant negative phenotype. This activation domain of CIITA interacts with the 32 kDa subunit of the general transcription complex TFIID, TAFII32. Decreased transcriptional activation by N-terminal deletions of CIITA is correlated directly with their reduced binding to TAFII32. We conclude that interactions between TAFII32 and CIITA are responsible for activation of class II genes.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="90" length="115"></location>
        <text>class II trans- activator</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="117" length="122"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="601" length="606"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="620" length="625"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="757" length="762"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="841" length="848"></location>
        <text>TAFII32</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="914" length="919"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="973" length="980"></location>
        <text>TAFII32</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1020" length="1027"></location>
        <text>TAFII32</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1032" length="1037"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="691" length="729"></location>
        <text>displays a dominant negative phenotype</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Entity</infon>
        <location offset="736" length="753"></location>
        <text>activation domain</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="763" length="772"></location>
        <text>interacts</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Negative_regulation</infon>
        <location offset="954" length="961"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="962" length="969"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Binding</infon>
        <location offset="999" length="1011"></location>
        <text>interactions</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T1" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T6" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
      <node refid="T4" role="Theme2"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
      <node refid="T17" role="Site"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T12" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T14" role="Theme2"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9211933</id>
    <passage>
      <offset>0</offset>
      <text>Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="36" length="63"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="113" length="118"></location>
        <text>Egr-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="120" length="125"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="19" length="28"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Entity</infon>
        <location offset="64" length="72"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Regulation</infon>
        <location offset="99" length="109"></location>
        <text>Regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>165</offset>
      <text>Biosynthesis of tumor necrosis factor-alpha (TNF-alpha) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-alpha promoter localized enhancer elements in a region (-182 to -37 base pairs (bp)) that were required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contained an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contained CRE and NF-kappaB (designated kappaB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and kappaB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the kappaB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="181" length="208"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="210" length="219"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="399" length="408"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="550" length="559"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="691" length="700"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="807" length="810"></location>
        <text>Sp1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="811" length="816"></location>
        <text>Egr-1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="975" length="980"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1068" length="1073"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1179" length="1184"></location>
        <text>Egr-1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1229" length="1234"></location>
        <text>Egr-1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1239" length="1242"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1243" length="1246"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1601" length="1606"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1621" length="1624"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1625" length="1628"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1687" length="1696"></location>
        <text>TNF-alpha</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Gene_expression</infon>
        <location offset="165" length="177"></location>
        <text>Biosynthesis</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Regulation</infon>
        <location offset="297" length="301"></location>
        <text>role</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="376" length="385"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Entity</infon>
        <location offset="409" length="417"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="649" length="657"></location>
        <text>required</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Transcription</infon>
        <location offset="670" length="683"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="706" length="720"></location>
        <text>in response to</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Binding</infon>
        <location offset="1005" length="1009"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Positive_regulation</infon>
        <location offset="1043" length="1052"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Binding</infon>
        <location offset="1057" length="1064"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Binding</infon>
        <location offset="1137" length="1144"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Positive_regulation</infon>
        <location offset="1670" length="1679"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Entity</infon>
        <location offset="1697" length="1705"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Regulation</infon>
        <location offset="1709" length="1717"></location>
        <text>mediated</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T5" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T22" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T27" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T35" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
      <node refid="T36" role="Site"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9233628</id>
    <passage>
      <offset>0</offset>
      <text>Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="20" length="23"></location>
        <text>Fos</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="29" length="32"></location>
        <text>Jun</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="69" length="73"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="83" length="87"></location>
        <text>CD28</text>
      </annotation>
    </passage>
    <passage>
      <offset>138</offset>
      <text>CD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="138" length="142"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="276" length="280"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="301" length="305"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="419" length="423"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="473" length="477"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="681" length="685"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="802" length="806"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="924" length="928"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1192" length="1196"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1484" length="1489"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1518" length="1521"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1526" length="1530"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1567" length="1572"></location>
        <text>c-Fos</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1577" length="1582"></location>
        <text>c-Jun</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1748" length="1752"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1774" length="1778"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="281" length="286"></location>
        <text>leads</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="290" length="300"></location>
        <text>high level</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="306" length="316"></location>
        <text>production</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="436" length="447"></location>
        <text>contributes</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="455" length="465"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Entity</infon>
        <location offset="478" length="486"></location>
        <text>promoter</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="1140" length="1156"></location>
        <text>binding activity</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Entity</infon>
        <location offset="1164" length="1184"></location>
        <text>CD28RE/AP-1 sequence</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Binding</infon>
        <location offset="1269" length="1276"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Binding</infon>
        <location offset="1547" length="1551"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Regulation</infon>
        <location offset="1738" length="1747"></location>
        <text>influence</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Entity</infon>
        <location offset="1753" length="1761"></location>
        <text>promoter</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T26" role="Site"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T28" role="Site"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T28" role="Site"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
      <node refid="T32" role="Site"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9233802</id>
    <passage>
      <offset>0</offset>
      <text>c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="5"></location>
        <text>c-Myb</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="75" length="78"></location>
        <text>ZEB</text>
      </annotation>
    </passage>
    <passage>
      <offset>81</offset>
      <text>The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the alpha4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="266" length="269"></location>
        <text>ZEB</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="432" length="435"></location>
        <text>ZEB</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="597" length="602"></location>
        <text>c-Myb</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="771" length="774"></location>
        <text>ZEB</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="798" length="803"></location>
        <text>c-Myb</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="935" length="940"></location>
        <text>c-Myb</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1091" length="1094"></location>
        <text>ZEB</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1129" length="1134"></location>
        <text>c-Myb</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1319" length="1334"></location>
        <text>alpha4 integrin</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="302" length="307"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Negative_regulation</infon>
        <location offset="775" length="781"></location>
        <text>blocks</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Negative_regulation</infon>
        <location offset="851" length="870"></location>
        <text>synergize to resist</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Regulation</infon>
        <location offset="1275" length="1303"></location>
        <text>positive/negative regulation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T7" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9234696</id>
    <passage>
      <offset>0</offset>
      <text>GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="30" length="43"></location>
        <text>interleukin 2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="45" length="49"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="78" length="83"></location>
        <text>c-Raf</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="105" length="109"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="13" length="17"></location>
        <text>bind</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Entity</infon>
        <location offset="51" length="59"></location>
        <text>enhancer</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Regulation</infon>
        <location offset="64" length="74"></location>
        <text>contribute</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="93" length="101"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="110" length="119"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <passage>
      <offset>122</offset>
      <text>Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E.Flory, A. Hoffmeyer, U.Smola, U.R.Rapp, and J.T.Bruder, J.Virol.70:2260- 2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.</text>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="347" length="360"></location>
        <text>interleukin 2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="361" length="366"></location>
        <text>(IL-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="516" length="521"></location>
        <text>c-Raf</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="591" length="595"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="743" length="747"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="779" length="783"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="832" length="836"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1016" length="1026"></location>
        <text>GABP alpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1031" length="1036"></location>
        <text>-beta</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1296" length="1300"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1375" length="1379"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1427" length="1430"></location>
        <text>BXB</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1465" length="1470"></location>
        <text>c-Raf</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1644" length="1649"></location>
        <text>c-Raf</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1868" length="1872"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Regulation</infon>
        <location offset="312" length="322"></location>
        <text>contribute</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Positive_regulation</infon>
        <location offset="330" length="339"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="596" length="605"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="806" length="815"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Entity</infon>
        <location offset="837" length="854"></location>
        <text>promoter-enhancer</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="868" length="875"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="904" length="915"></location>
        <text>independent</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Binding</infon>
        <location offset="983" length="990"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Negative_regulation</infon>
        <location offset="1228" length="1238"></location>
        <text>interfered</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="1248" length="1257"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Entity</infon>
        <location offset="1301" length="1318"></location>
        <text>promoter-enhancer</text>
      </annotation>
      <annotation id="T36">
        <infon key="type">Positive_regulation</infon>
        <location offset="1326" length="1340"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T37">
        <infon key="type">Gene_expression</infon>
        <location offset="1326" length="1340"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T38">
        <infon key="type">Positive_regulation</infon>
        <location offset="1362" length="1370"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T39">
        <infon key="type">Entity</infon>
        <location offset="1380" length="1397"></location>
        <text>promoter-enhancer</text>
      </annotation>
      <annotation id="T40">
        <infon key="type">Positive_regulation</infon>
        <location offset="1398" length="1407"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T41">
        <infon key="type">Gene_expression</infon>
        <location offset="1409" length="1423"></location>
        <text>Overexpression</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Positive_regulation</infon>
        <location offset="1409" length="1423"></location>
        <text>Overexpression</text>
      </annotation>
      <annotation id="T43">
        <infon key="type">Regulation</infon>
        <location offset="1804" length="1816"></location>
        <text>contribution</text>
      </annotation>
      <annotation id="T44">
        <infon key="type">Positive_regulation</infon>
        <location offset="1853" length="1864"></location>
        <text>enhancement</text>
      </annotation>
      <annotation id="T45">
        <infon key="type">Positive_regulation</infon>
        <location offset="1873" length="1882"></location>
        <text>induction</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T5" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
      <node refid="T21" role="Site"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T29" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
      <node refid="T35" role="Site"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T37" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T36" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T38" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
      <node refid="E18" role="Cause"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T40" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T39" role="Site"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T41" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T42" role="Trigger"></node>
      <node refid="E21" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T43" role="Trigger"></node>
      <node refid="E24" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T44" role="Trigger"></node>
      <node refid="E25" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T45" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9299399</id>
    <passage>
      <offset>0</offset>
      <text>Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="76" length="83"></location>
        <text>eotaxin</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Regulation</infon>
        <location offset="0" length="20"></location>
        <text>Genomic organization</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Regulation</infon>
        <location offset="36" length="62"></location>
        <text>transcriptional regulation</text>
      </annotation>
    </passage>
    <passage>
      <offset>91</offset>
      <text>Eotaxin is an eosinophil specific beta-chemokine assumed to be involved in eosinophilic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma and parasitic infections. Its expression is stimulus- and cell-specific. We here describe the genomic organisation (3 exons of 132, 112 and 542 bp and 2 introns of 1211 and 378 bp) and sequence including 3 kb of DNA from the immediate 5' upstream region of the human eotaxin gene. Among the regulatory promoter elements potentially regulating eotaxin gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-1, a NF-kappa-B like consensus sequence and gamma-interferon- as well as glucocorticoid response elements.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="91" length="98"></location>
        <text>Eotaxin</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="521" length="528"></location>
        <text>eotaxin</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="597" length="604"></location>
        <text>eotaxin</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="755" length="761"></location>
        <text>NF-IL6</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="810" length="826"></location>
        <text>gamma-interferon</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Gene_expression</infon>
        <location offset="284" length="294"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Regulation</infon>
        <location offset="586" length="596"></location>
        <text>regulating</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="610" length="620"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Binding</infon>
        <location offset="720" length="728"></location>
        <text>interact</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9351352</id>
    <passage>
      <offset>0</offset>
      <text>Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein.</text>
    </passage>
    <passage>
      <offset>88</offset>
      <text>Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="850" length="865"></location>
        <text>I kappa B-alpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="1044" length="1059"></location>
        <text>I kappa B-alpha</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1142" length="1169"></location>
        <text>tumor necrosis factor-alpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1174" length="1192"></location>
        <text>interleukin-1 beta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Negative_regulation</infon>
        <location offset="837" length="846"></location>
        <text>depletion</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Positive_regulation</infon>
        <location offset="897" length="905"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Negative_regulation</infon>
        <location offset="990" length="999"></location>
        <text>prevented</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Negative_regulation</infon>
        <location offset="1031" length="1040"></location>
        <text>depletion</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Negative_regulation</infon>
        <location offset="1079" length="1089"></location>
        <text>inhibition</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="1128" length="1138"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T10" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9352360</id>
    <passage>
      <offset>0</offset>
      <text>Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain.</text>
    </passage>
    <passage>
      <offset>116</offset>
      <text>The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="120" length="144"></location>
        <text>class II trans-activator</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="146" length="151"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="572" length="577"></location>
        <text>CIITA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="655" length="664"></location>
        <text>IFN-gamma</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Positive_regulation</infon>
        <location offset="548" length="559"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Gene_expression</infon>
        <location offset="548" length="559"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Gene_expression</infon>
        <location offset="726" length="738"></location>
        <text>transfection</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Positive_regulation</infon>
        <location offset="726" length="738"></location>
        <text>transfection</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T1" role="Event"></node>
      <node refid="T2" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9353251</id>
    <passage>
      <offset>0</offset>
      <text>CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="4"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="30" length="35"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Regulation</infon>
        <location offset="5" length="14"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein_catabolism</infon>
        <location offset="15" length="26"></location>
        <text>degradation</text>
      </annotation>
    </passage>
    <passage>
      <offset>127</offset>
      <text>CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="127" length="131"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="277" length="281"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="414" length="418"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="496" length="533"></location>
        <text>type 1 tumor necrosis factor receptor</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="535" length="540"></location>
        <text>TNFR1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="612" length="616"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="686" length="691"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="749" length="754"></location>
        <text>TNFR1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="845" length="849"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="896" length="901"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="931" length="935"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="970" length="975"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1003" length="1008"></location>
        <text>TRAF1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1061" length="1065"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1160" length="1165"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1194" length="1199"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1351" length="1356"></location>
        <text>TNFR1</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1433" length="1438"></location>
        <text>TRAF2</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1463" length="1468"></location>
        <text>TNFR1</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1543" length="1547"></location>
        <text>CD30</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Entity</infon>
        <location offset="582" length="600"></location>
        <text>TRAF-binding sites</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="662" length="667"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="885" length="892"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="936" length="943"></location>
        <text>results</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Protein_catabolism</infon>
        <location offset="1012" length="1023"></location>
        <text>proteolysis</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="1304" length="1314"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T6" role="Event"></node>
      <node refid="T7" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T8" role="Theme"></node>
      <node refid="T9" role="Theme2"></node>
      <node refid="T25" role="Site"></node>
    </relation>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T13" role="Theme2"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="E4" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9356353</id>
    <passage>
      <offset>0</offset>
      <text>TNFalpha cooperates with the protein kinase A pathway to synergistically increase HIV-1 LTR transcription via downstream TRE-like cAMP response elements.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="8"></location>
        <text>TNFalpha</text>
      </annotation>
    </passage>
    <passage>
      <offset>155</offset>
      <text>Activating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFalpha. Ctx combined with TNFalpha or IL-1beta to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFalpha, whereas the synergy induced by Ctx and IL-1beta was unaffected, suggesting TNFalpha and IL-1beta cooperate differently with the cAMP/PKA activation pathway to induce HIV-1 expression in U1 cells. Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFalpha as well as IL-1beta cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data indicate that the TRE-like cAMP-responsive DSE sites within the 5'-untranslated leader can mediate the transcriptional cooperativity between TNFalpha and the cAMP/PKA pathway. Since the DSE and TRE sites cannot bind CREB/ATF homodimers, we propose a mechanism in which the HIV-1 DSE bind heterodimers composed of both AP-1 and CREB/ATF proteins. Copyright 1997 Academic Press.</text>
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        <infon key="type">Protein</infon>
        <location offset="822" length="830"></location>
        <text>TNFalpha</text>
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        <infon key="type">Protein</infon>
        <location offset="850" length="858"></location>
        <text>TNFalpha</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="862" length="870"></location>
        <text>IL-1beta</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="908" length="919"></location>
        <text>p24 antigen</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1100" length="1108"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1149" length="1157"></location>
        <text>IL-1beta</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1185" length="1193"></location>
        <text>TNFalpha</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1198" length="1206"></location>
        <text>IL-1beta</text>
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      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1426" length="1434"></location>
        <text>TNFalpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1446" length="1454"></location>
        <text>IL-1beta</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1695" length="1703"></location>
        <text>TNFalpha</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
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        <text>to produce a synergistic increase</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="920" length="930"></location>
        <text>production</text>
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      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
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      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T5" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9356494</id>
    <passage>
      <offset>0</offset>
      <text>The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB.</text>
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        <infon key="type">Protein</infon>
        <location offset="40" length="65"></location>
        <text>latent membrane protein 1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="78" length="108"></location>
        <text>tumor necrosis factor receptor</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Binding</infon>
        <location offset="66" length="73"></location>
        <text>engages</text>
      </annotation>
    </passage>
    <passage>
      <offset>212</offset>
      <text>The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth.</text>
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        <infon key="type">Protein</infon>
        <location offset="235" length="260"></location>
        <text>latent membrane protein 1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="262" length="266"></location>
        <text>LMP1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="400" length="404"></location>
        <text>LMP1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1272" length="1334"></location>
        <text>tumor necrosis factor receptor-associated death domain protein</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1336" length="1341"></location>
        <text>TRADD</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1344" length="1349"></location>
        <text>TRADD</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1445" length="1450"></location>
        <text>TRADD</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1477" length="1481"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1514" length="1518"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1543" length="1548"></location>
        <text>TRADD</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1580" length="1584"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1635" length="1640"></location>
        <text>TRADD</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1645" length="1649"></location>
        <text>TES2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1736" length="1740"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1754" length="1759"></location>
        <text>TRADD</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="425" length="434"></location>
        <text>activated</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="1236" length="1247"></location>
        <text>interacting</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Binding</infon>
        <location offset="1376" length="1386"></location>
        <text>interacted</text>
      </annotation>
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        <infon key="type">Binding</infon>
        <location offset="1451" length="1461"></location>
        <text>associated</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="1564" length="1574"></location>
        <text>associated</text>
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    </passage>
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      <infon key="type">Equiv</infon>
      <node refid="T7" role="Event"></node>
      <node refid="T6" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T10" role="Theme2"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
      <node refid="T11" role="Theme2"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
      <node refid="T13" role="Theme2"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9360945</id>
    <passage>
      <offset>0</offset>
      <text>Transcription factor NF-kappaB regulates inducible Oct-2 gene expression in precursor B lymphocytes.</text>
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        <infon key="type">Protein</infon>
        <location offset="51" length="56"></location>
        <text>Oct-2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Regulation</infon>
        <location offset="31" length="40"></location>
        <text>regulates</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="41" length="50"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="57" length="72"></location>
        <text>gene expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>102</offset>
      <text>The POU transcription factors Oct-1 and Oct-2 regulate the activity of octamer-dependent promoters, including those that direct transcription from rearranged immunoglobulin genes. Unlike Oct-1, which is constitutively expressed in many cell types, Oct-2 expression is restricted primarily to B lymphocytes and can be induced in precursor B cells by stimulation with bacterial lipopolysaccharide (LPS). However, the precise factors that mediate this induction mechanism remain unknown. In the present study, we monitored Oct-2 expression in cells arrested for the activation of NF-kappaB, an LPS-responsive member of the Rel transcription factor family. Despite stimulation with LPS, disruption of the NF-kappaB signaling pathway in precursor B cells led to the loss of inducible Oct-2 DNA binding activity in vitro and the suppression of Oct-2-directed transcription in vivo. This biochemical defect correlated with a specific block to Oct-2 gene expression at the level of transcription, whereas the expression of Oct-1 was unaffected. The finding that Oct-2 is under NF-kappaB control highlights an important cross-talk mechanism involving two distinct transcription factor families that regulate B lymphocyte function.</text>
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        <infon key="type">Protein</infon>
        <location offset="132" length="137"></location>
        <text>Oct-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="142" length="147"></location>
        <text>Oct-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="289" length="294"></location>
        <text>Oct-1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="350" length="355"></location>
        <text>Oct-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="622" length="627"></location>
        <text>Oct-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="881" length="886"></location>
        <text>Oct-2</text>
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        <infon key="type">Protein</infon>
        <location offset="940" length="945"></location>
        <text>Oct-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1038" length="1043"></location>
        <text>Oct-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1117" length="1122"></location>
        <text>Oct-1</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="1156" length="1161"></location>
        <text>Oct-2</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="320" length="329"></location>
        <text>expressed</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="356" length="366"></location>
        <text>expression</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="370" length="380"></location>
        <text>restricted</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="419" length="426"></location>
        <text>induced</text>
      </annotation>
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        <infon key="type">Positive_regulation</infon>
        <location offset="538" length="545"></location>
        <text>mediate</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="628" length="638"></location>
        <text>expression</text>
      </annotation>
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        <infon key="type">Negative_regulation</infon>
        <location offset="755" length="762"></location>
        <text>Despite</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Negative_regulation</infon>
        <location offset="863" length="867"></location>
        <text>loss</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="871" length="880"></location>
        <text>inducible</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Binding</infon>
        <location offset="891" length="898"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Negative_regulation</infon>
        <location offset="1029" length="1034"></location>
        <text>block</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Transcription</infon>
        <location offset="1049" length="1089"></location>
        <text>expression at the level of transcription</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Gene_expression</infon>
        <location offset="1103" length="1113"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Negative_regulation</infon>
        <location offset="1127" length="1137"></location>
        <text>unaffected</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Regulation</infon>
        <location offset="1181" length="1188"></location>
        <text>control</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="E14" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
    <relation id="M5">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E11" role="Event"></node>
    </relation>
    <relation id="M6">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
    <relation id="M7">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E18" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9361029</id>
    <passage>
      <offset>0</offset>
      <text>Paternal expression of WT1 in human fibroblasts and lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="23" length="26"></location>
        <text>WT1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Gene_expression</infon>
        <location offset="0" length="19"></location>
        <text>Paternal expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>66</offset>
      <text>The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier.</text>
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        <infon key="type">Protein</infon>
        <location offset="101" length="104"></location>
        <text>WT1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="270" length="273"></location>
        <text>WT1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="572" length="575"></location>
        <text>WT1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="729" length="733"></location>
        <text>IGF2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="738" length="741"></location>
        <text>H19</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="891" length="894"></location>
        <text>WT1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1078" length="1081"></location>
        <text>WT1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Gene_expression</infon>
        <location offset="167" length="186"></location>
        <text>maternal expression</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="240" length="266"></location>
        <text>allele-specific expression</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="405" length="415"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="549" length="568"></location>
        <text>paternal expression</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Gene_expression</infon>
        <location offset="667" length="693"></location>
        <text>allele-specific expression</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="867" length="887"></location>
        <text>biallelic expression</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Gene_expression</infon>
        <location offset="877" length="887"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="978" length="998"></location>
        <text>biallelic expression</text>
      </annotation>
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        <infon key="type">Gene_expression</infon>
        <location offset="988" length="998"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="1041" length="1067"></location>
        <text>allele-specific regulatory</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Regulation</infon>
        <location offset="1103" length="1113"></location>
        <text>controlled</text>
      </annotation>
    </passage>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E13" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9372447</id>
    <passage>
      <offset>0</offset>
      <text>Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner.</text>
    </passage>
    <passage>
      <offset>253</offset>
      <text>When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="1049" length="1054"></location>
        <text>Oct-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="1056" length="1061"></location>
        <text>OBF-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="1063" length="1067"></location>
        <text>PU.1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="1128" length="1131"></location>
        <text>E47</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="1313" length="1318"></location>
        <text>Oct-2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Negative_regulation</infon>
        <location offset="999" length="1004"></location>
        <text>block</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Transcription</infon>
        <location offset="1008" length="1021"></location>
        <text>transcription</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Negative_regulation</infon>
        <location offset="1086" length="1094"></location>
        <text>inactive</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9372912</id>
    <passage>
      <offset>0</offset>
      <text>Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="14" length="17"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="41" length="46"></location>
        <text>Cdk-2</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="0" length="10"></location>
        <text>Regulation</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="47" length="56"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Phosphorylation</infon>
        <location offset="57" length="72"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <passage>
      <offset>75</offset>
      <text>The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E.Hara, M.Hall, and G.Peters, EMBO J.16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="538" length="546"></location>
        <text>cyclin E</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="547" length="551"></location>
        <text>Cdk2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="557" length="565"></location>
        <text>cyclin A</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="566" length="570"></location>
        <text>Cdk2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="644" length="647"></location>
        <text>Id2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="765" length="768"></location>
        <text>Id2</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="979" length="982"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1009" length="1012"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1191" length="1194"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1199" length="1202"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1328" length="1331"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1437" length="1440"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1450" length="1454"></location>
        <text>Cdk2</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1601" length="1604"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1696" length="1699"></location>
        <text>Id3</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1894" length="1898"></location>
        <text>Cdk2</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="571" length="580"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Phosphorylation</infon>
        <location offset="581" length="596"></location>
        <text>phosphorylation</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Entity</infon>
        <location offset="635" length="639"></location>
        <text>Ser5</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Regulation</infon>
        <location offset="950" length="959"></location>
        <text>regulated</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Phosphorylation</infon>
        <location offset="960" length="975"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
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      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
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      <node refid="T1" role="Theme"></node>
      <node refid="E3" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T24" role="Site"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9488049</id>
    <passage>
      <offset>0</offset>
      <text>Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="99" length="111"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Negative_regulation</infon>
        <location offset="85" length="95"></location>
        <text>inhibition</text>
      </annotation>
    </passage>
    <passage>
      <offset>114</offset>
      <text>We previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="318" length="322"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="343" length="347"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="467" length="479"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="562" length="574"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="657" length="669"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="708" length="712"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="722" length="726"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="855" length="867"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1182" length="1194"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="458" length="463"></location>
        <text>level</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Regulation</infon>
        <location offset="488" length="497"></location>
        <text>regulated</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Transcription</infon>
        <location offset="552" length="558"></location>
        <text>levels</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="552" length="558"></location>
        <text>levels</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="670" length="679"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Negative_regulation</infon>
        <location offset="695" length="704"></location>
        <text>inhibited</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Negative_regulation</infon>
        <location offset="829" length="837"></location>
        <text>inhibits</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Positive_regulation</infon>
        <location offset="842" length="851"></location>
        <text>induction</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Regulation</infon>
        <location offset="1168" length="1178"></location>
        <text>modulation</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="1195" length="1205"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="T7" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E4" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E7" role="Event"></node>
    </relation>
    <relation id="M4">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9507734</id>
    <passage>
      <offset>0</offset>
      <text>Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B.</text>
    </passage>
    <passage>
      <offset>170</offset>
      <text>Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P &lt; 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P &lt; 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="1447" length="1450"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Gene_expression</infon>
        <location offset="1413" length="1430"></location>
        <text>positive staining</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T2" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9619918</id>
    <passage>
      <offset>0</offset>
      <text>IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-kappaB induction and IL-2 receptor alpha expression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="4"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="101" length="120"></location>
        <text>IL-2 receptor alpha</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="121" length="131"></location>
        <text>expression</text>
      </annotation>
    </passage>
    <passage>
      <offset>134</offset>
      <text>The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="149" length="162"></location>
        <text>interleukin-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="164" length="168"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="287" length="320"></location>
        <text>IL-2 receptor (IL-2R) alpha chain</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="584" length="594"></location>
        <text>IL-2Ralpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="599" length="603"></location>
        <text>beta</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="677" length="687"></location>
        <text>IL-2Ralpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="726" length="730"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="772" length="776"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="799" length="803"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="907" length="911"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1031" length="1035"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1103" length="1108"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1113" length="1117"></location>
        <text>KBF1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1134" length="1138"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1174" length="1178"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1336" length="1340"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Binding</infon>
        <location offset="138" length="145"></location>
        <text>binding</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="204" length="211"></location>
        <text>induces</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Positive_regulation</infon>
        <location offset="269" length="279"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Gene_expression</infon>
        <location offset="574" length="583"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Positive_regulation</infon>
        <location offset="665" length="673"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Gene_expression</infon>
        <location offset="688" length="698"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Transcription</infon>
        <location offset="765" length="771"></location>
        <text>detect</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="1036" length="1045"></location>
        <text>increased</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Transcription</infon>
        <location offset="1050" length="1068"></location>
        <text>abundance of mRNAs</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Binding</infon>
        <location offset="1139" length="1146"></location>
        <text>binding</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T4" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="E2" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E8" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E13" role="Theme"></node>
      <node refid="T13" role="Cause"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E12" role="Theme"></node>
      <node refid="T13" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E14" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9733846</id>
    <passage>
      <offset>0</offset>
      <text>Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="145" length="157"></location>
        <text>CD4 receptor</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="112" length="122"></location>
        <text>expressing</text>
      </annotation>
    </passage>
    <passage>
      <offset>161</offset>
      <text>We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J.Corbeil, M.Tremblay, and D.D.Richman, J.Exp.Med.183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild- type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF- kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="211" length="214"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="423" length="426"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="520" length="523"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="633" length="636"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="748" length="756"></location>
        <text>p56(lck)</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="917" length="920"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1038" length="1046"></location>
        <text>p56(lck)</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1103" length="1115"></location>
        <text>CD4 receptor</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1290" length="1293"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1456" length="1459"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1460" length="1470"></location>
        <text>Fas ligand</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="1514" length="1517"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1654" length="1657"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1729" length="1732"></location>
        <text>Fas</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1733" length="1743"></location>
        <text>Fas ligand</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1774" length="1782"></location>
        <text>p56(lck)</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1832" length="1835"></location>
        <text>CD4</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Gene_expression</infon>
        <location offset="553" length="562"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Gene_expression</infon>
        <location offset="666" length="675"></location>
        <text>expressed</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="692" length="702"></location>
        <text>expressing</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Binding</infon>
        <location offset="733" length="742"></location>
        <text>associate</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Binding</infon>
        <location offset="1281" length="1286"></location>
        <text>binds</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Binding</infon>
        <location offset="1471" length="1482"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Binding</infon>
        <location offset="1744" length="1755"></location>
        <text>interaction</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Binding</infon>
        <location offset="1836" length="1848"></location>
        <text>dimerization</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T11" role="Theme"></node>
      <node refid="T12" role="Theme2"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T15" role="Theme"></node>
      <node refid="T16" role="Theme2"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E5" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9747720</id>
    <passage>
      <offset>0</offset>
      <text>NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="53" length="78"></location>
        <text>latent membrane protein 1</text>
      </annotation>
    </passage>
    <passage>
      <offset>103</offset>
      <text>The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel- responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="107" length="132"></location>
        <text>latent membrane protein 1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="134" length="138"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="282" length="286"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="436" length="440"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="738" length="744"></location>
        <text>ICAM-1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="749" length="753"></location>
        <text>CD71</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="757" length="761"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="799" length="803"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="833" length="836"></location>
        <text>p50</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="837" length="840"></location>
        <text>p65</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="846" length="851"></location>
        <text>c-Rel</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="981" length="987"></location>
        <text>ICAM-1</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="992" length="996"></location>
        <text>CD71</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1030" length="1034"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1115" length="1119"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1236" length="1240"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1377" length="1381"></location>
        <text>LMP1</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="287" length="297"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Regulation</infon>
        <location offset="650" length="660"></location>
        <text>importance</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Positive_regulation</infon>
        <location offset="691" length="704"></location>
        <text>up-regulation</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Gene_expression</infon>
        <location offset="785" length="795"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Positive_regulation</infon>
        <location offset="1014" length="1026"></location>
        <text>up-regulated</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T2" role="Event"></node>
      <node refid="T3" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T8" role="Cause"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T14" role="Theme"></node>
      <node refid="T15" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T23" role="Trigger"></node>
      <node refid="T13" role="Theme"></node>
      <node refid="T15" role="Cause"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E2" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E3" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9794238</id>
    <passage>
      <offset>0</offset>
      <text>Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="21" length="33"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="59" length="67"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Gene_expression</infon>
        <location offset="47" length="57"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Gene_expression</infon>
        <location offset="68" length="77"></location>
        <text>synthesis</text>
      </annotation>
    </passage>
    <passage>
      <offset>132</offset>
      <text>In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="238" length="250"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="413" length="425"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="885" length="893"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="966" length="978"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1025" length="1033"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1121" length="1129"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1266" length="1274"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1416" length="1424"></location>
        <text>TNFalpha</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Gene_expression</infon>
        <location offset="219" length="237"></location>
        <text>stably transfected</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Positive_regulation</infon>
        <location offset="219" length="237"></location>
        <text>stably transfected</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Gene_expression</infon>
        <location offset="371" length="394"></location>
        <text>constitutive expression</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Negative_regulation</infon>
        <location offset="915" length="922"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Gene_expression</infon>
        <location offset="979" length="990"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="979" length="990"></location>
        <text>transfected</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Regulation</infon>
        <location offset="1300" length="1307"></location>
        <text>targets</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T11" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T12" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9794375</id>
    <passage>
      <offset>0</offset>
      <text>Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="32" length="35"></location>
        <text>HS1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="206" length="209"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="214" length="218"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="22" length="31"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T35">
        <infon key="type">Phosphorylation</infon>
        <location offset="36" length="51"></location>
        <text>phosphorylation</text>
      </annotation>
    </passage>
    <passage>
      <offset>221</offset>
      <text>CD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HSI phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HSI. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="226" length="229"></location>
        <text>CD2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="235" length="239"></location>
        <text>CD28</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="439" length="442"></location>
        <text>Vav</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="444" length="447"></location>
        <text>Cbl</text>
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      <annotation id="T8">
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  </document>
  <document>
    <id>PMID-9794389</id>
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      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="39" length="44"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="5" length="18"></location>
        <text>reconstitutes</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="54" length="63"></location>
        <text>signaling</text>
      </annotation>
    </passage>
    <passage>
      <offset>66</offset>
      <text>The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="141" length="146"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="224" length="228"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="233" length="237"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="287" length="314"></location>
        <text>Janus protein kinase (JAK)1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="316" length="320"></location>
        <text>JAK3</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="333" length="338"></location>
        <text>STAT6</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="370" length="375"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="449" length="454"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="552" length="556"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="561" length="565"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="622" length="627"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="658" length="662"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="688" length="692"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="785" length="789"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="834" length="839"></location>
        <text>c-Myc</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="841" length="846"></location>
        <text>Bcl-2</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="852" length="858"></location>
        <text>Bcl-xL</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="906" length="910"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="928" length="933"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1058" length="1062"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1108" length="1112"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1153" length="1158"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Protein</infon>
        <location offset="1201" length="1205"></location>
        <text>IL-4</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Protein</infon>
        <location offset="1210" length="1214"></location>
        <text>IL-7</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Protein</infon>
        <location offset="1257" length="1262"></location>
        <text>v-Abl</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="273" length="283"></location>
        <text>activation</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Negative_regulation</infon>
        <location offset="795" length="802"></location>
        <text>delayed</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Negative_regulation</infon>
        <location offset="807" length="816"></location>
        <text>decreases</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T1" role="Cause"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
      <node refid="T16" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T19" role="Theme"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T17" role="Theme"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9796702</id>
    <passage>
      <offset>0</offset>
      <text>p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="0" length="6"></location>
        <text>p21ras</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="17" length="22"></location>
        <text>Rac-1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="62" length="65"></location>
        <text>PKB</text>
      </annotation>
    </passage>
    <passage>
      <offset>114</offset>
      <text>p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.</text>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="114" length="120"></location>
        <text>p21ras</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="368" length="373"></location>
        <text>Raf-1</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="401" length="406"></location>
        <text>Rac-1</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="578" length="583"></location>
        <text>Rac-1</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="618" length="624"></location>
        <text>p21ras</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="685" length="688"></location>
        <text>Akt</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="689" length="692"></location>
        <text>PKB</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="854" length="859"></location>
        <text>Rac-1</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="950" length="953"></location>
        <text>Akt</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Protein</infon>
        <location offset="954" length="957"></location>
        <text>PKB</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Protein</infon>
        <location offset="1056" length="1061"></location>
        <text>Rac-1</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Protein</infon>
        <location offset="1179" length="1185"></location>
        <text>p21ras</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Protein</infon>
        <location offset="1341" length="1344"></location>
        <text>PKB</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein</infon>
        <location offset="1509" length="1515"></location>
        <text>p21ras</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Protein</infon>
        <location offset="1545" length="1548"></location>
        <text>Akt</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Protein</infon>
        <location offset="1549" length="1552"></location>
        <text>PKB</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Protein</infon>
        <location offset="1605" length="1608"></location>
        <text>Akt</text>
      </annotation>
      <annotation id="T21">
        <infon key="type">Protein</infon>
        <location offset="1609" length="1612"></location>
        <text>PKB</text>
      </annotation>
      <annotation id="T22">
        <infon key="type">Protein</infon>
        <location offset="1705" length="1708"></location>
        <text>Akt</text>
      </annotation>
      <annotation id="T23">
        <infon key="type">Protein</infon>
        <location offset="1709" length="1712"></location>
        <text>PKB</text>
      </annotation>
      <annotation id="T24">
        <infon key="type">Protein</infon>
        <location offset="1772" length="1777"></location>
        <text>Rac-1</text>
      </annotation>
      <annotation id="T25">
        <infon key="type">Positive_regulation</infon>
        <location offset="124" length="133"></location>
        <text>activated</text>
      </annotation>
      <annotation id="T26">
        <infon key="type">Regulation</infon>
        <location offset="251" length="268"></location>
        <text>Effector pathways</text>
      </annotation>
      <annotation id="T27">
        <infon key="type">Positive_regulation</infon>
        <location offset="328" length="336"></location>
        <text>mediated</text>
      </annotation>
      <annotation id="T28">
        <infon key="type">Positive_regulation</infon>
        <location offset="611" length="617"></location>
        <text>couple</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Regulation</infon>
        <location offset="961" length="970"></location>
        <text>regulated</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Positive_regulation</infon>
        <location offset="1348" length="1357"></location>
        <text>activated</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Positive_regulation</infon>
        <location offset="1449" length="1472"></location>
        <text>required and sufficient</text>
      </annotation>
      <annotation id="T32">
        <infon key="type">Positive_regulation</infon>
        <location offset="1538" length="1544"></location>
        <text>induce</text>
      </annotation>
      <annotation id="T33">
        <infon key="type">Positive_regulation</infon>
        <location offset="1592" length="1600"></location>
        <text>required</text>
      </annotation>
      <annotation id="T34">
        <infon key="type">Positive_regulation</infon>
        <location offset="1613" length="1623"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T12" role="Event"></node>
      <node refid="T13" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T22" role="Event"></node>
      <node refid="T23" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T20" role="Event"></node>
      <node refid="T21" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T9" role="Event"></node>
      <node refid="T10" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T18" role="Event"></node>
      <node refid="T19" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T6" role="Cause"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
      <node refid="T5" role="Cause"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T16" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T32" role="Trigger"></node>
      <node refid="T18" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T33" role="Trigger"></node>
      <node refid="E11" role="Theme"></node>
      <node refid="T17" role="Cause"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T34" role="Trigger"></node>
      <node refid="T20" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E9" role="Event"></node>
    </relation>
    <relation id="M3">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
      <node refid="E10" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9796963</id>
    <passage>
      <offset>0</offset>
      <text>Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy.</text>
    </passage>
    <passage>
      <offset>149</offset>
      <text>Studies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P &lt; 0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in &gt;50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="360" length="367"></location>
        <text>TCRzeta</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="372" length="380"></location>
        <text>p56(lck)</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="450" length="454"></location>
        <text>RelA</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="844" length="851"></location>
        <text>TCRzeta</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="853" length="861"></location>
        <text>p56(lck)</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="1021" length="1039"></location>
        <text>interleukin (IL)-2</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="1041" length="1050"></location>
        <text>IFN alpha</text>
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      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1135" length="1142"></location>
        <text>TCRzeta</text>
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        <infon key="type">Protein</infon>
        <location offset="1147" length="1155"></location>
        <text>p56(lck)</text>
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        <infon key="type">Protein</infon>
        <location offset="1778" length="1785"></location>
        <text>TCRzeta</text>
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        <infon key="type">Protein</infon>
        <location offset="1790" length="1798"></location>
        <text>p56(lck)</text>
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        <infon key="type">Protein</infon>
        <location offset="2057" length="2064"></location>
        <text>TCRzeta</text>
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        <infon key="type">Protein</infon>
        <location offset="2081" length="2089"></location>
        <text>p56(lck)</text>
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        <infon key="type">Protein</infon>
        <location offset="2110" length="2120"></location>
        <text>CD3epsilon</text>
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        <infon key="type">Protein</infon>
        <location offset="2201" length="2205"></location>
        <text>IL-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="2206" length="2215"></location>
        <text>IFN alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="2250" length="2257"></location>
        <text>TCRzeta</text>
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        <infon key="type">Protein</infon>
        <location offset="2361" length="2368"></location>
        <text>TCRzeta</text>
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        <infon key="type">Protein</infon>
        <location offset="2372" length="2380"></location>
        <text>p56(lck)</text>
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        <location offset="2441" length="2448"></location>
        <text>TCRzeta</text>
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        <location offset="3009" length="3016"></location>
        <text>TCRzeta</text>
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        <infon key="type">Protein</infon>
        <location offset="3062" length="3069"></location>
        <text>TCRzeta</text>
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        <location offset="3213" length="3220"></location>
        <text>TCRzeta</text>
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        <location offset="3225" length="3233"></location>
        <text>p56(lck)</text>
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        <location offset="3276" length="3283"></location>
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        <text>FN alpha</text>
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        <location offset="3588" length="3595"></location>
        <text>TCRzeta</text>
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        <text>Diminished</text>
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        <text>expression</text>
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        <text>associated</text>
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        <text>reduced</text>
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        <infon key="type">Entity</infon>
        <location offset="426" length="433"></location>
        <text>nuclear</text>
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      <annotation id="T34">
        <infon key="type">Localization</infon>
        <location offset="434" length="446"></location>
        <text>localization</text>
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      <annotation id="T35">
        <infon key="type">Negative_regulation</infon>
        <location offset="532" length="539"></location>
        <text>defects</text>
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      <annotation id="T36">
        <infon key="type">Negative_regulation</infon>
        <location offset="822" length="829"></location>
        <text>altered</text>
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        <location offset="830" length="840"></location>
        <text>expression</text>
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      <annotation id="T38">
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        <location offset="1125" length="1131"></location>
        <text>levels</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="1691" length="1698"></location>
        <text>Changes</text>
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      <annotation id="T40">
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        <location offset="1804" length="1812"></location>
        <text>detected</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="1880" length="1887"></location>
        <text>reduced</text>
      </annotation>
      <annotation id="T42">
        <infon key="type">Negative_regulation</infon>
        <location offset="2044" length="2053"></location>
        <text>decreases</text>
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      <annotation id="T43">
        <infon key="type">Positive_regulation</infon>
        <location offset="2258" length="2267"></location>
        <text>increased</text>
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      <annotation id="T44">
        <infon key="type">Negative_regulation</infon>
        <location offset="2435" length="2440"></location>
        <text>lower</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="2996" length="3005"></location>
        <text>reduction</text>
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      <annotation id="T46">
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        <location offset="3017" length="3027"></location>
        <text>expression</text>
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        <infon key="type">Gene_expression</infon>
        <location offset="3205" length="3212"></location>
        <text>express</text>
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        <infon key="type">Negative_regulation</infon>
        <location offset="3252" length="3266"></location>
        <text>reduced levels</text>
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        <infon key="type">Positive_regulation</infon>
        <location offset="3323" length="3331"></location>
        <text>increase</text>
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      <infon key="type">Event</infon>
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      <infon key="event type">Negative_regulation</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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    <relation id="E9">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E10">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E16">
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    <relation id="E18">
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      <infon key="type">Event</infon>
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    <relation id="E19">
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      <infon key="type">Event</infon>
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  </document>
  <document>
    <id>PMID-9799798</id>
    <passage>
      <offset>0</offset>
      <text>Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2.</text>
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        <location offset="30" length="51"></location>
        <text>lysosomal acid lipase</text>
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        <text>Transcriptional regulation</text>
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        <text>mediated</text>
      </annotation>
    </passage>
    <passage>
      <offset>133</offset>
      <text>Human lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between -182 bp and -107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2.</text>
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        <location offset="162" length="165"></location>
        <text>LAL</text>
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        <text>LAL</text>
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        <text>LAL</text>
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        <text>LAL</text>
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        <location offset="1221" length="1224"></location>
        <text>Sp1</text>
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        <text>LAL</text>
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        <text>lysosomal acid lipase</text>
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        <location offset="1433" length="1436"></location>
        <text>LAL</text>
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        <text>increase</text>
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        <text>expression</text>
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        <text>binding</text>
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        <text>increased</text>
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        <text>important role</text>
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        <text>enhanced</text>
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        <text>expression</text>
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      <annotation id="T28">
        <infon key="type">Regulation</infon>
        <location offset="1388" length="1397"></location>
        <text>dependent</text>
      </annotation>
      <annotation id="T29">
        <infon key="type">Positive_regulation</infon>
        <location offset="1398" length="1406"></location>
        <text>increase</text>
      </annotation>
      <annotation id="T30">
        <infon key="type">Gene_expression</infon>
        <location offset="1438" length="1448"></location>
        <text>expression</text>
      </annotation>
      <annotation id="T31">
        <infon key="type">Regulation</infon>
        <location offset="1467" length="1475"></location>
        <text>mediated</text>
      </annotation>
    </passage>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T12" role="Event"></node>
      <node refid="T11" role="Event"></node>
    </relation>
    <relation id="*">
      <infon key="type">Equiv</infon>
      <node refid="T3" role="Event"></node>
      <node refid="T4" role="Event"></node>
    </relation>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E1" role="Theme"></node>
      <node refid="T2" role="Cause"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="E7" role="Theme"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T21" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
      <node refid="T8" role="Theme2"></node>
      <node refid="T23" role="Site2"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
      <node refid="T22" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
      <node refid="T23" role="Site"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E10" role="Theme"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T24" role="Trigger"></node>
      <node refid="E9" role="Theme"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T25" role="Trigger"></node>
      <node refid="E15" role="Theme"></node>
      <node refid="T9" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T26" role="Trigger"></node>
      <node refid="E16" role="Theme"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T27" role="Trigger"></node>
      <node refid="T10" role="Theme"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T28" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T29" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T30" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T31" role="Trigger"></node>
      <node refid="E17" role="Theme"></node>
    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E6" role="Event"></node>
    </relation>
    <relation id="M2">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E8" role="Event"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9802971</id>
    <passage>
      <offset>0</offset>
      <text>A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes.</text>
    </passage>
    <passage>
      <offset>150</offset>
      <text>The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="1180" length="1185"></location>
        <text>Kv1.3</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Negative_regulation</infon>
        <location offset="1166" length="1174"></location>
        <text>reducing</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Negative_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T2" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9804806</id>
    <passage>
      <offset>0</offset>
      <text>Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="8" length="12"></location>
        <text>IKK1</text>
      </annotation>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="17" length="21"></location>
        <text>IKK2</text>
      </annotation>
    </passage>
    <passage>
      <offset>81</offset>
      <text>Mononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-kappaB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IkappaB cytoplasmic inhibitors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, by these kinases triggers proteolytic degradation and the release of NF-kappaB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IkappaBs and the nuclear translocation of NF-kappaB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced kappaB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of kappaB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of kappaB-dependent gene expression in human monocytic cells requires activation of IKK2.</text>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="421" length="425"></location>
        <text>IKK1</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Protein</infon>
        <location offset="430" length="434"></location>
        <text>IKK2</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Protein</infon>
        <location offset="491" length="503"></location>
        <text>IkappaBalpha</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Protein</infon>
        <location offset="505" length="516"></location>
        <text>IkappaBbeta</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Protein</infon>
        <location offset="522" length="536"></location>
        <text>IkappaBepsilon</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Protein</infon>
        <location offset="1094" length="1098"></location>
        <text>IKK1</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Protein</infon>
        <location offset="1127" length="1131"></location>
        <text>IKK1</text>
      </annotation>
      <annotation id="T10">
        <infon key="type">Protein</infon>
        <location offset="1153" length="1157"></location>
        <text>IKK2</text>
      </annotation>
      <annotation id="T11">
        <infon key="type">Protein</infon>
        <location offset="1279" length="1283"></location>
        <text>IKK2</text>
      </annotation>
      <annotation id="T12">
        <infon key="type">Protein</infon>
        <location offset="1496" length="1500"></location>
        <text>IKK2</text>
      </annotation>
      <annotation id="T13">
        <infon key="type">Positive_regulation</infon>
        <location offset="399" length="407"></location>
        <text>activate</text>
      </annotation>
      <annotation id="T14">
        <infon key="type">Phosphorylation</infon>
        <location offset="436" length="451"></location>
        <text>Phosphorylation</text>
      </annotation>
      <annotation id="T15">
        <infon key="type">Positive_regulation</infon>
        <location offset="538" length="540"></location>
        <text>by</text>
      </annotation>
      <annotation id="T16">
        <infon key="type">Positive_regulation</infon>
        <location offset="555" length="563"></location>
        <text>triggers</text>
      </annotation>
      <annotation id="T17">
        <infon key="type">Protein_catabolism</infon>
        <location offset="564" length="587"></location>
        <text>proteolytic degradation</text>
      </annotation>
      <annotation id="T18">
        <infon key="type">Positive_regulation</infon>
        <location offset="1066" length="1080"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T19">
        <infon key="type">Gene_expression</infon>
        <location offset="1066" length="1080"></location>
        <text>overexpression</text>
      </annotation>
      <annotation id="T20">
        <infon key="type">Positive_regulation</infon>
        <location offset="1482" length="1492"></location>
        <text>activation</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T4" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T13" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Phosphorylation</infon>
      <infon key="type">Event</infon>
      <node refid="T14" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E7">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E8">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E4" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E9">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T3" role="Cause"></node>
    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E5" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E11">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T15" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
      <node refid="T4" role="Cause"></node>
    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
      <node refid="E9" role="Cause"></node>
    </relation>
    <relation id="E13">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E20" role="Theme"></node>
      <node refid="E10" role="Cause"></node>
    </relation>
    <relation id="E14">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E8" role="Cause"></node>
    </relation>
    <relation id="E15">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
      <node refid="E6" role="Cause"></node>
    </relation>
    <relation id="E16">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E19" role="Theme"></node>
      <node refid="E11" role="Cause"></node>
    </relation>
    <relation id="E17">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T16" role="Trigger"></node>
      <node refid="E18" role="Theme"></node>
      <node refid="E7" role="Cause"></node>
    </relation>
    <relation id="E18">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T7" role="Theme"></node>
    </relation>
    <relation id="E19">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T5" role="Theme"></node>
    </relation>
    <relation id="E20">
      <infon key="event type">Protein_catabolism</infon>
      <infon key="type">Event</infon>
      <node refid="T17" role="Trigger"></node>
      <node refid="T6" role="Theme"></node>
    </relation>
    <relation id="E21">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E22" role="Theme"></node>
    </relation>
    <relation id="E22">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T9" role="Theme"></node>
    </relation>
    <relation id="E23">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T19" role="Trigger"></node>
      <node refid="T8" role="Theme"></node>
    </relation>
    <relation id="E24">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T18" role="Trigger"></node>
      <node refid="E23" role="Theme"></node>
    </relation>
    <relation id="E25">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T20" role="Trigger"></node>
      <node refid="T12" role="Theme"></node>
    </relation>
  </document>
  <document>
    <id>PMID-9808178</id>
    <passage>
      <offset>0</offset>
      <text>Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.</text>
      <annotation id="T1">
        <infon key="type">Protein</infon>
        <location offset="85" length="89"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T4">
        <infon key="type">Regulation</infon>
        <location offset="0" length="7"></location>
        <text>Effects</text>
      </annotation>
      <annotation id="T5">
        <infon key="type">Gene_expression</infon>
        <location offset="90" length="100"></location>
        <text>production</text>
      </annotation>
    </passage>
    <passage>
      <offset>170</offset>
      <text>The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.</text>
      <annotation id="T2">
        <infon key="type">Protein</infon>
        <location offset="800" length="804"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="939" length="943"></location>
        <text>IL-2</text>
      </annotation>
      <annotation id="T6">
        <infon key="type">Gene_expression</infon>
        <location offset="805" length="815"></location>
        <text>production</text>
      </annotation>
      <annotation id="T7">
        <infon key="type">Positive_regulation</infon>
        <location offset="826" length="834"></location>
        <text>enhanced</text>
      </annotation>
      <annotation id="T8">
        <infon key="type">Positive_regulation</infon>
        <location offset="931" length="938"></location>
        <text>induced</text>
      </annotation>
      <annotation id="T9">
        <infon key="type">Gene_expression</infon>
        <location offset="944" length="954"></location>
        <text>production</text>
      </annotation>
    </passage>
    <relation id="E1">
      <infon key="event type">Regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T4" role="Trigger"></node>
      <node refid="E2" role="Theme"></node>
    </relation>
    <relation id="E2">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T5" role="Trigger"></node>
      <node refid="T1" role="Theme"></node>
    </relation>
    <relation id="E3">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T6" role="Trigger"></node>
      <node refid="T2" role="Theme"></node>
    </relation>
    <relation id="E4">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T7" role="Trigger"></node>
      <node refid="E3" role="Theme"></node>
    </relation>
    <relation id="E5">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
      <node refid="T8" role="Trigger"></node>
      <node refid="E6" role="Theme"></node>
    </relation>
    <relation id="E6">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
      <node refid="T9" role="Trigger"></node>
      <node refid="T3" role="Theme"></node>
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  <document>
    <id>PMID-9808189</id>
    <passage>
      <offset>0</offset>
      <text>Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-kappaB.</text>
    </passage>
    <passage>
      <offset>112</offset>
      <text>IL-12 is a 75-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 chains. This pro-inflammatory cytokine plays a prominent role in the development of Th1 cell-mediated immune responses. Th1 cell-mediated immune responses have been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Thus, IL-12 appears to be a critical factor in the generation and maintenance of chronic inflammatory conditions. In this study, we investigated the effects of a commonly prescribed anti-inflammatory drug, acetyl salicylic acid (ASA), on IL-12 production and Th1 cell development. ASA was found to inhibit secretion of the IL-12 heterodimer as well as p40 monomer by human monocytic cells. This was associated with the down-regulation of IL-12p40 mRNA expression. Analysis of the regulation of the p40 gene promoter revealed that ASA inhibited NF-kappaB activation and binding to the p40-kappaB site in the p40 promoter, leading to transcriptional repression of the p40 gene. Addition of ASA to an in vitro T helper cell differentiation system, at concentrations compatible with plasma levels reached during anti-inflammatory therapy, resulted in reduced development of Th1 cells. These results suggest that the inhibition of NF-kappaB activation by ASA leads to down-regulation of IL-12 production and inhibition of Th1 cell development.</text>
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        <text>p35</text>
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        <infon key="type">Protein</infon>
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        <text>p40</text>
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        <text>p40</text>
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        <text>IL-12p40</text>
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        <text>p40</text>
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        <text>p40</text>
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        <text>p40</text>
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        <text>p40</text>
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        <text>inhibit</text>
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        <text>secretion</text>
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        <text>down-regulation</text>
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        <infon key="type">Transcription</infon>
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        <text>mRNA expression</text>
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        <infon key="type">Regulation</infon>
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        <text>regulation</text>
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        <infon key="type">Entity</infon>
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        <text>promoter</text>
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        <text>leading to transcriptional repression</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event modification</infon>
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  </document>
  <document>
    <id>PMID-9825820</id>
    <passage>
      <offset>0</offset>
      <text>Membrane-associated lymphotoxin on natural killer cells activates endothelial cells via an NF-kappaB-dependent pathway.</text>
    </passage>
    <passage>
      <offset>121</offset>
      <text>BACKGROUND: Inhibition of complement in small animal models of xenotransplantation has demonstrated graft infiltration with natural killer (NK) cells and monocytes associated with endothelial cell (EC) activation. We have previously demonstrated that human NK cells activate porcine EC in vitro, which results in adhesion molecule expression and cytokine secretion. In this study, we used the NK cell line NK92 to define the molecular and cellular basis of NK cell-mediated EC activation. METHODS: EC were transfected with either reporter constructs containing the luciferase gene driven either by E-selectin or interleukin (IL)-8 promoters or a synthetic NF-kappaB-dependent promoter. In addition, a dominant-negative mutant tumor necrosis factor receptor I (TNFRI) expression vector was co-transfected in inhibition studies. Forty-eight hours after transfection, EC were stimulated with NK cells or NK cell membrane extracts for 7 hr and activation was measured by a luciferase assay. RESULTS: Co-culture of NK cells with transfected EC enhanced E-selectin, IL-8, and NF-kappaB-dependent promoter activity. NK cell membrane extracts retained the capacity to activate EC and induced nuclear translocation of NF-kappaB (p50 and p65). Western blotting of NK cell and membrane extracts detected the presence of Lymphotoxin-alpha (LTalpha) but not tumor necrosis factor-alpha. Furthermore, LTalpha was secreted in NK:EC co-cultures. Co-transfection with dominant-negative mutant TNFRI inhibited EC activation by NK cell membrane extracts and by NK cells by 80% and 47%, respectively. The same pattern of inhibition was observed using anti-human LT sera. CONCLUSIONS: Human NK cell membrane-bound LT signals across species via TNFRI, leading to NF-kappaB nuclear translocation and transcription of E-selectin and IL-8, which results in EC activation. The discrepancy in the degree of inhibition by membrane extracts and NK cells with mutant TNFRI suggests that additional pathways are utilized by NK cells to activate EC.</text>
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        <text>interleukin (IL)-8</text>
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        <text>tumor necrosis factor receptor I</text>
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        <text>TNFRI</text>
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        <text>E-selectin</text>
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        <location offset="1181" length="1185"></location>
        <text>IL-8</text>
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        <location offset="1341" length="1344"></location>
        <text>p50</text>
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        <text>p65</text>
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        <infon key="type">Protein</infon>
        <location offset="1430" length="1447"></location>
        <text>Lymphotoxin-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1449" length="1456"></location>
        <text>LTalpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1466" length="1493"></location>
        <text>tumor necrosis factor-alpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1508" length="1515"></location>
        <text>LTalpha</text>
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        <infon key="type">Protein</infon>
        <location offset="1597" length="1602"></location>
        <text>TNFRI</text>
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        <infon key="type">Protein</infon>
        <location offset="1844" length="1849"></location>
        <text>TNFRI</text>
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        <location offset="1915" length="1925"></location>
        <text>E-selectin</text>
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        <location offset="1930" length="1934"></location>
        <text>IL-8</text>
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        <location offset="2058" length="2063"></location>
        <text>TNFRI</text>
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        <text>co-transfected</text>
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        <text>co-transfected</text>
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        <text>induced</text>
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        <text>nuclear</text>
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        <infon key="type">Localization</infon>
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        <text>translocation</text>
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        <text>presence</text>
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        <text>secreted</text>
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        <text>Co-transfection</text>
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        <text>Co-transfection</text>
      </annotation>
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        <text>leading</text>
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        <infon key="type">Transcription</infon>
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        <text>transcription</text>
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    <relation id="E7">
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      <infon key="type">Event</infon>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E9">
      <infon key="event type">Localization</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E10">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E11">
      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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    </relation>
    <relation id="E12">
      <infon key="event type">Positive_regulation</infon>
      <infon key="type">Event</infon>
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    <relation id="E13">
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      <infon key="type">Event</infon>
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      <infon key="event type">Transcription</infon>
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      <infon key="type">Event</infon>
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    <relation id="M1">
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      <infon key="modification type">Negation</infon>
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    </relation>
  </document>
  <document>
    <id>PMID-9852070</id>
    <passage>
      <offset>0</offset>
      <text>The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily.</text>
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        <infon key="type">Protein</infon>
        <location offset="116" length="147"></location>
        <text>receptor activator of NF-kappaB</text>
      </annotation>
    </passage>
    <passage>
      <offset>184</offset>
      <text>Receptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events.</text>
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        <text>Receptor activator of NF-kappaB</text>
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        <location offset="217" length="221"></location>
        <text>RANK</text>
      </annotation>
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        <location offset="384" length="389"></location>
        <text>RANKL</text>
      </annotation>
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        <location offset="531" length="535"></location>
        <text>RANK</text>
      </annotation>
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        <location offset="541" length="600"></location>
        <text>tumor necrosis factor receptor-associated factors (TRAFs) 1</text>
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        <location offset="602" length="603"></location>
        <text>2</text>
      </annotation>
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        <location offset="605" length="606"></location>
        <text>3</text>
      </annotation>
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        <text>5</text>
      </annotation>
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        <text>6</text>
      </annotation>
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        <location offset="693" length="697"></location>
        <text>RANK</text>
      </annotation>
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        <location offset="788" length="792"></location>
        <text>RANK</text>
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        <location offset="886" length="890"></location>
        <text>RANK</text>
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        <text>c-Jun</text>
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        <text>TRAF6</text>
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        <text>TRAFs 1</text>
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        <location offset="1197" length="1198"></location>
        <text>2</text>
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        <text>3</text>
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        <text>5</text>
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        <location offset="1238" length="1243"></location>
        <text>TRAF6</text>
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        <text>RANK</text>
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        <text>c-Jun</text>
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        <text>TRAF6</text>
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        <text>RANKL</text>
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        <text>TRAF6</text>
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        <text>expressed</text>
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        <text>interaction</text>
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        <text>interaction</text>
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        <text>interacted</text>
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        <infon key="type">Binding</infon>
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        <text>binding</text>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
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      <infon key="type">Event</infon>
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    </relation>
    <relation id="E4">
      <infon key="event type">Binding</infon>
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  <document>
    <id>PMID-9878621</id>
    <passage>
      <offset>0</offset>
      <text>Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP.</text>
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        <location offset="14" length="31"></location>
        <text>interleukin-1beta</text>
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        <text>Regulation</text>
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        <text>transcription</text>
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    <passage>
      <offset>138</offset>
      <text>Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both. Copyright 1998 Academic Press.</text>
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        <text>interleukin-1beta</text>
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        <text>IL-1beta</text>
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        <text>IL-1beta</text>
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        <text>IL-1beta</text>
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        <text>EBV nuclear proteins 2</text>
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    <relation id="E17">
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      <infon key="type">Event</infon>
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    <relation id="E19">
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    </relation>
    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Speculation</infon>
      <node refid="E5" role="Event"></node>
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  </document>
  <document>
    <id>PMID-9880555</id>
    <passage>
      <offset>0</offset>
      <text>Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes.</text>
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        <location offset="30" length="35"></location>
        <text>elk-1</text>
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        <location offset="99" length="104"></location>
        <text>elk-1</text>
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        <location offset="56" length="60"></location>
        <text>role</text>
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        <location offset="105" length="120"></location>
        <text>gene expression</text>
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    </passage>
    <passage>
      <offset>136</offset>
      <text>To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes.</text>
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        <location offset="162" length="167"></location>
        <text>elk-1</text>
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        <location offset="232" length="237"></location>
        <text>elk-1</text>
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        <location offset="646" length="651"></location>
        <text>elk-1</text>
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        <text>elk-1</text>
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        <location offset="893" length="898"></location>
        <text>Elk-1</text>
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        <location offset="1002" length="1007"></location>
        <text>Elk-1</text>
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        <text>elk-1</text>
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        <text>Egr-1</text>
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        <text>serum response factor</text>
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        <text>Egr-1</text>
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        <location offset="1359" length="1364"></location>
        <text>elk-1</text>
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        <text>Egr-1</text>
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        <text>elk-1</text>
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        <text>important</text>
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        <text>transcription</text>
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        <text>contribute</text>
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        <text>expression</text>
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        <text>inducibility</text>
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        <text>expression</text>
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        <text>interact</text>
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        <text>increase</text>
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        <text>induction</text>
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        <text>expression</text>
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        <text>transcription</text>
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    </relation>
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      <infon key="type">Event</infon>
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      <infon key="event type">Gene_expression</infon>
      <infon key="type">Event</infon>
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      <infon key="event type">Transcription</infon>
      <infon key="type">Event</infon>
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  </document>
  <document>
    <id>PMID-9989503</id>
    <passage>
      <offset>0</offset>
      <text>Functional association of Nmi with Stat5 and Stat1 in IL-2- and IFNgamma-mediated signaling.</text>
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        <infon key="type">Protein</infon>
        <location offset="26" length="29"></location>
        <text>Nmi</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Stat1</text>
      </annotation>
      <annotation id="T3">
        <infon key="type">Protein</infon>
        <location offset="54" length="58"></location>
        <text>IL-2</text>
      </annotation>
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        <text>IFNgamma</text>
      </annotation>
    </passage>
    <passage>
      <offset>94</offset>
      <text>Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, we identified the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor. We further show that Nmi interacts with all STATs except Stat2. We evaluated two cytokine systems, IL-2 and IFNgamma, and demonstrate that Nmi augments STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 can augment IL-2- and IFNgamma-dependent transcription. Therefore, our data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors.</text>
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        <text>Stat5b</text>
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        <text>Nmi</text>
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        <text>N-Myc interactor</text>
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        <infon key="type">Protein</infon>
        <location offset="308" length="311"></location>
        <text>Nmi</text>
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        <text>Stat2</text>
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        <text>IL-2</text>
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        <text>IFNgamma</text>
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        <text>Nmi</text>
      </annotation>
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        <location offset="514" length="517"></location>
        <text>Nmi</text>
      </annotation>
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        <location offset="581" length="584"></location>
        <text>Nmi</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>Stat1</text>
      </annotation>
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        <text>IL-2</text>
      </annotation>
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        <infon key="type">Protein</infon>
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        <text>IFNgamma</text>
      </annotation>
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        <infon key="type">Protein</infon>
        <location offset="789" length="792"></location>
        <text>Nmi</text>
      </annotation>
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        <text>association</text>
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        <text>interacts</text>
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        <text>enhances</text>
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        <text>association</text>
      </annotation>
    </passage>
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      <infon key="type">Event</infon>
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      <infon key="event type">Binding</infon>
      <infon key="type">Event</infon>
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    <relation id="M1">
      <infon key="type">Event modification</infon>
      <infon key="modification type">Negation</infon>
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    </relation>
  </document>
</collection>
