Cohesin Mediates Chromatin Conformation Figure 8. Biallelic IGF2 expression after cohesin depletion. (A) Sequencing of genomic DNA (gDNA) shows that the HB2 cell line is heterozygous for a SNP in IGF2. Pyrosequencing of cDNA of cohesin depleted G2 phase cells shows both IGF2 alleles indicating biallelic IGF2 expression. (B) Sequencing of genomic DNA (gDNA) shows that the HB2 cell line is heterozygous for a SNP in H19. Pyrosequencing of cDNA of cohesin depleted and control G2 cells shows only the C allele indicating that H19 expression remains monoallelic after cohesin depletion. (C) After labelling IGF2 RNA by RNA fluorescence in situ hybridisation (FISH; green) on cohesin depleted (SCC1 RNAi) and control interphase HB2 cells (RNAi control), DNA FISH (red) was performed to detect genomic IGF2 sequence. Nuclei were counterstained with DAPI (blue) and imaged by confocal microscopy. Representative images are shown; first panel: overlay, second panel: DNA signal, third panel: RNA signal. IGF2 expression increases and becomes biallelic upon cohesin depletion. doi:10.1371/journal.pgen.1000739.g008 cohesin mediates these effects. Our results suggest that cohesin contributes to gene regulation by mediating the formation of higher order chromatin conformation, at least at the imprinted IGF2-H19 locus. Recent studies of the apolipoprotein cluster [38] and of the interferon c locus [39] have revealed that cohesin also has roles in long-range chromatin interactions at these loci. It is, therefore, possible that cohesin has a widespread role in the formation of chromatin loops in mammalian genomes and regulates gene expression through this mechanism at numerous sites. In our experiments 3C interactions were reduced but not abolished after cohesin depletion. It is possible that the remaining chromatin interactions were caused by residual amounts of cohesin, which is difficult to deplete completely by RNAi. However, we can not exclude the possibility that other proteins maintain the chromatin loops in the absence of cohesin. Our first systematic q3C analysis of the human IGF2-H19 locus has brought to light an unexpected complexity of chromatin interactions. We found evidence for IGF2 promoter-enhancer interactions on the putative paternal allele [17,18] as well as ICR interactions with CTCF sites at the 59 end of the IGF2 gene (CTCF AD sites). Previous studies in mice have identified allele specific interactions of the ICR and the enhancers, but CTCF sites other than the ICR have not yet been analysed at the mouse locus. By extending our analysis to a wider number of CTCF sites we found a previously unknown association of the ICR with a CTCF DS site on the presumed maternal allele in the human cell line, as well as biallelic CTCF mediated interactions involving the CCD site. The CCD region in mice has tissue specific silencer or enhancer activities which are independent of imprinting [26,27,30]. Genome-wide CTCF ChIP- sequencing data in adult mouse livers indicate that this region also binds CTCF (D. Odom and D. Schmidt, pers. communication), suggesting that the CCD may function as a boundary or insulator with regard to its silencer function. It is thus possible that cohesin is also required for the insulator activity of the CCD. Our data are consistent with the possibility that multiple CTCF- cohesin mediated loops come together in a chromatin ‘‘hub’’ as PLoS Genetics | www.plosgenetics.org 10 November 2009 | Volume 5 | Issue 11 | e1000739