TI - ERK-responsive transcription factors Ets1 and Ets2 bind the endogenous DUSP6/MKP-3 gene promoter . AB - Our results thus far implicate a member or members of the Ets group of transcription factors in mediating ERK -dependent DUSP6/MKP-3 transcription in response to FGFs . To explore this further , we performed EMSAs using a labelled double-stranded oligonucleotide probe spanning the conserved Ets site (MKP-3) . These experiments revealed a specific protein complex , which was not seen when labelled mutant oligonucleotide ( MKP-3m ) is used as a probe ( Figure 7A ) . This complex is effectively competed away by unlabelled wild-type MKP-3 oligonucleotide (M) , but not by a mutant oligonucleotide with a single substitution in the Ets site (m) . This complex is also effectively competed by a wild-type (E) but not a mutant (e) oligonucleotide containing a canonical Ets -binding site . A complex that co-migrated with that seen using the wild-type MKP-3 oligonucleotide , was also observed using the wild-type canonical Ets site (E74) , but not a mutant oligonucleotide ( E74m ) as a probe ( Figure 7A ) . This complex was competed away by both wild-type Ets (E) oligonucleotide and , albeit less effectively , by the wild-type MKP-3 oligonucleotide (M) . Further experiments showed that the complex detected using the MKP-3 oligonucleotide was present when nuclear extracts from either control or FGF-treated cells was used , indicating that proteins may be constitutively bound to the DUSP6/MKP-3 promoter ( results not shown ) . With this information in hand , we decided to take a candidate approach to examine the binding of individual Ets factors to the endogenous DUSP6/MKP-3 promoter using ChIP assays . For these experiments , NIH 3T3 cells were cultured in 10% serum before treatment with 1% formaldehyde for 10 min to cross-link DNA and proteins in vivo , followed by cell lysis and the preparation of chromatin . Chromatin from approx. 2x107 cells was sonicated to an average length of 600 bp before immunoprecipitation with either a non-specific ( HA ) antibody or specific antisera against Ets factors . Precipitated DNA was amplified by PCR , separated by agarose gel electrophoresis , and visualized by ethidium bromide staining (Figure 7B) . Two sets of oligonucleotide PCR primers were used , one of which was specific for the Ets -binding site and , as a control , primers that annealed to sequences upstream of the Ets -binding site . Non-precipitated ( input ) chromatin was a positive control for these PCRs . We could detect significant association of both Ets1 and Ets2 with the Ets -binding site , whereas no detectable signal was seen using either the control antibody or an antibody against ERM81 , a member of the closely related Pea3 subfamily of Ets factors . Additional ChIP experiments using antibodies specific for Elk1 also resulted in a failure to detect binding to the DUSP6/MKP-3 promoter ( results not shown ) . Finally , we assessed the impact of expressing wild-type and mutant forms of Ets2 on the activity of the 508 bp DUSP6/MKP-3 luciferase reporter . Co-transfection of wild-type Ets2 caused a significant increase in reporter activity . In contrast , co-expression of either a deletion mutant of Ets2 ( Delta410-425 ) lacking a functional DNA -binding domain (Ets2 DBDmut) or a mutant form of Ets2 ( Ets2 T72A ) lacking a critical MAPK PHOSphorylation site [38] did not increase DUSP6/MKP-3 reporter activity (Figure 7C) .