phase. Importantly, these data also suggest that cohesin’s role in chromatin conformation would have to be independent of its function in sister chromatid cohesion, which exists in G2 but not G1 phase. Cohesin is associated with loops that are formed by interactions between CTCF sites Another prediction made by the hypothesis that cohesin mediates 3C interactions between CTCF sites at the IGF2-H19 locus is that cohesin should be present in the corresponding chromatin loops. Since our cohesin ChIP experiments (Figure 2A and 2B) did not distinguish between binding of cohesin to DNA molecules which were folded into loops and those that were not, we enriched the 3C digested templates for CTCF or cohesin bound chromatin before ligating for 3C analysis (ChIP-loop).We found that both SMC3 and CTCF antibodies can immunopre- cipitate 3C products representing associations between the ICR (j restriction site) and the CTCF AD/DMR0 region (b1 restriction site) (Figure 5). These results indicate that cohesin is present in the corresponding chromatin loops. Importantly, 3C products repre- senting enhancer-promoter associations could not be detected. These observations indicate that enhancer-promoter interactions cannot directly be mediated by cohesin, whereas the data are consistent with the possibility that cohesin mediates chromatin association between CTCF sites. Figure 5. ChIP–loop experiments indicate that CTCF and cohesin are present at the interactions between CTCF AD/ DMR0 and the ICR. Agarose gel showing a PCR product of 272 bp amplified with primers for the ICR and CTCFAD/DMR0 (primers at j and b1 restriction sites in Figure 1A) and a PCR product of 303 bp amplified with primers for the enhancer and CTCFAD/DMR0 (primers at m and b1 restriction sites in Figure 1A). Templates in each panel include 3C standard (template from PCR standard curve), 3C (cross-linked chromatin BamHI digested and ligated), a-CTCF-3C, and a-SMC3-3C (ChIP–loop templates that were immuno-precipitated with CTCF or cohesin antibodies prior to ligation). ICR interactions are found in 3C and ChIP-loop material, likely reflecting a specific CTCF/cohesin mediated association between CTCF AD and the ICR, with both proteins present in the loop. In contrast, enhancer interactions are not found in ChIP–loop material confirming that this interaction is not CTCF/cohesin dependent and reflecting an association between the enhancer and the P2 promoter rather than with DMR0. doi:10.1371/journal.pgen.1000739.g005 Cohesin Mediates Chromatin Conformation Depletion of cohesin leads to destabilisation of chromatin conformation To test directly if cohesin is functionally required for the formation or maintenance of chromatin loops at the IGF2-H19 locus, we depleted the cohesin subunit SCC1 by RNAi and thus rendered the cohesin complex non-functional. Since cohesin depleted cells delay progression through mitosis, and because it is unknown if chromatin loops are maintained during mitosis, we synchronised SCC1 depleted cells by double thymidine treatment and harvested cells in G1 and G2 phases (Figure 6A and 6B). We confirmed that CTCF was still bound to the ICR, and the CCD after cohesin depletion in HB2 cells (results not shown), as was previously demonstrated in another cell line [6]. The effect of depletion of SCC1 on IGF2 and H19 expression was an activation of IGF2 transcription, but no significant difference in H19 expression (Figure 6D). Average DNA methylation levels at the IGF2 and H19 DMRs did not change significantly (Figure 6E and 6F). In SCC1 depleted cells that were enriched in G2 phase we found a significant reduction in the association frequency between all chromatin interactions between CTCF binding sites, with the exception of the interaction between the CTCF AD/DMR0 region and the very distant CTCF DS sites (Figure 7B, 7C, 7G, and 7H and Table S1). Similar results were obtained using an ICR anchor primer in another restriction site within the ICR (Primer j, Figure S3). Using BglII as a restriction enzyme we were also able to confirm that there is a 30% reduction between the CTCF AD and CCD interactions during G2 phase (Figure S2D). Importantly, however, associations with the enhancer anchor (primer m) and the IGF2 promoters (restriction sites b2, c1) were not significantly reduced (Figure 7D and 7E). These observations indicate that 3C interactions between CTCF sites are dependent on cohesin, whereas the ability of the enhancer to associate with the IGF2 promoters is not dependent on cohesin. Importantly, when we sequenced the PCR products from the enhancer anchor, we found that the monoallelic associations found in the controls became biallelic after cohesin depletion (Figure 7F). SCC1 depleted cells harvested in G1 phase yielded less chromatin for analysis than that in G2 phase and locus wide comparisons could not be done. However we were able to show that in G1, associations between the ICR (j primer) and the CTCF AD/DMR0 regions (restriction sites a- c1) were also reduced (Figure S4). Together, these results indicate that depletion of cohesin predominantly affects CTCF mediated looping interac- tions. Importantly, our observation that cohesin depletion also affects chromatin structure in G1 phase, where no sister chromatid cohesion exists, further supports the notion that cohesin’s role in chromatin looping is independent of its cohesion function. The effect of decreased CTCF and cohesin mediated 3C associations had little or no impact on imprinted expression of H19 which remained mono-allelic (Figure 8B), presumably due to the methylation being maintained at the ICR (Figure 6E and 6F). Expression of IGF2 was biallelic after cohesin depletion as determined by SNP analysis and RNA FISH (Figure 8A and 8C). However, IGF2 expression levels were so low prior to cohesin depletion that was impossible to tell whether the basal transcrip- tion was monoallelic or biallelic. Discussion It is now well established that cohesin complexes do not only function in sister chromatid cohesion but also have important roles in gene regulation, both in proliferating and post-mitotic cells (reviewed in [3,37]). However, it remains largely unknown how PLoS Genetics | www.plosgenetics.org 7 November 2009 | Volume 5 | Issue 11 | e1000739