TI - Discussion Genetic Evidence for Activator -Dependent Gene Positioning . AB - We have used a genetic model to show that a transcriptional activator regulates the subnuclear positioning of its direct endogenous targets in primary differentiated cells . We documented Hnf1alpha -dependent differential gene positioning with respect to : . . . . . . . a ) subnuclear regions enriched in H3-Lys27me3 , H3-Lys4me2 and PHOSphoserine-5 RNA polymerase II ( Figure 3 ) , b ) radial nuclear zones ( Figure 4 ) , c ) genomic regions adjacent to an Hnf1alpha -dependent gene ( Figure 5 ) , and d ) chromosomal territories ( Figure S8 ) . The analysis of four control loci in trans allowed us to conclude that the observed Hnf1alpha -dependent spatial changes are specific . Experiments comparing the position of an Hnf1alpha -dependent locus to adjacent chromosomal regions and its chromosomal territory further demonstrated specificity , and revealed that changes were locus -selective and did not reflect broad chromosomal reconfigurations . Although numerous studies have shown a relationship between gene transcription and subnuclear positioning , several variables that are only indirectly related to gene transcription , such as regional gene density or nucleotide composition , also appear to impact the subnuclear location of genomic regions , independent of their actual transcriptional activity [27] -[29] , [45] . Our new findings demonstrate that transactivator -dependent functions are dominant over such variables in the regulation of subnuclear gene positioning . Earlier reports have linked the function of sequence specific-DNA binding proteins such as Ikaros and NF-E2p18 with the repositioning of endogenous loci [21] , [46] , [47] . In such examples , repressor-mediated repositioning of silenced loci to pericentromeric compartments was observed during developmentally regulated gene -silencing processes . This clearly represents a different situation compared to the current analysis where gene inactivity results from the sheer lack of an activator and gives rise to a different pattern of subnuclear positioning that does not involve association with chromocenters . Previous evidence supporting the role of transactivators in gene positioning comes from studies of transgenes . Some of these studies took advantage of a lac repressor-VP16 acidic activation domain fusion protein , which was shown to cause repositioning of targeted multicopy loci away from the nuclear periphery [31] , [32] . Another study has analyzed transgenes with intact or mutated transactivator binding sites and showed that intact sites prevent association of transgenes with pericentromeric heterochromatin [48] . The role of transactivators in the positioning of endogenous loci , however , has not been directly assessed . One study showed that the deletion of a 24 Kb endogenous genomic region containing the beta globin locus control region results in gene silencing and increased perinuclear localization of the endogenous locus [49] . These effects were probably due to activator functions because the deleted region contained multiple binding sites for essential transcription factors . Nevertheless , it could not be excluded that structural changes due to deletion of an extended genomic segment also affected nuclear positioning by transactivator independent mechanisms . Our results provide genetic evidence in primary cells that positioning of endogenous genes can be dependent on a single transactivator . Together with previous studies , this suggests that the regulation of the subnuclear location of target gene loci might be a general function of sequence -specific DNA binding transcriptional regulators .