These data indicate that the strongest associations that can be detected at the human IGF2-H19 locus in HB2 cells exist between the CTCF AD/DMR0 region and the CCD and CTCF DS sites, whereas a weak interaction may also exist between the ICR and CTCF AD/DMR0. The interaction between the CTCF AD/ DMR0 and the ICR could be weaker than the CTCF AD/DMR0 and the CCD because the ICR is a monoallelic CTCF/cohesin binding site, and/or because our ChIP experiments indicated that less CTCF and cohesin are bound to the ICR than to the other sites (Figure 2A and 2B). Alternatively, we cannot exclude the possibility that the 3C interactions that we detected between the ICR and CTCF AD/DMR0 were caused indirectly by the strong associations between CTCF AD/DMR0 and the CCD and the CTCF DS sites, which are located to the left and the right of the ICR. Allele specificity of chromatin associations To examine which of the detected chromatin associations is allele specific we combined the 3C assays with SNP analysis of ligated products. Using polymorphisms in two separate restriction fragments, we detected associations between the enhancer and the IGF2 promoter region predominantly on one allele (Figure 4A and 4B), which presumably represents the paternally derived allele. These promoter-enhancer interactions suggest that, despite the low levels of IGF2 transcription, the chromatin conformation in HB2 cells is favourable for monoallelic expression. Associations between the CTCF AD/DMR0 region and the CCD could be detected on both alleles, consistent with our finding that CTCF and cohesin bind to CTCF AD sites and the CCD biallelically (Figure 4C). Monoallelic interactions were detected on the CTCF binding allele (presumably the maternal one) between the ICR and the CTCF DS (Figure 4D). Unexpectedly, we also detected biallelic associations between the ICR and the CTCF AD/DMR0 region (Figure 4E), despite the fact that CTCF binds the ICR predominantly on one allele. This result suggests that ICR- CTCFAD/DMR0 interactions are indirect and mediated through interactions between the CTCF AD/DMR0 with other CTCF sites near the ICR such as the CTCF DS. It is also conceivable that elements within the DMR0 and the adjacent CTCF sites interact separately with the methylated and the unmethylated ICR. However, most regulatory elements are quite close to the ICR and it is, therefore, also possible that the paternal promoter- enhancer interactions distort the conformation of the loop in a manner such that the paternal ICR may also be in close proximity to IGF2. Higher-order chromatin conformation is maintained in G1 and G2 phases of the cell cycle Because our ChIP experiments revealed the strongest CTCF and cohesin signals at the CTCF AD, CCD and CTCF DS sites (Figure 2A and 2B), and our q3C assays had identified the strongest interactions between these sites, we hypothesised that these interactions could be mediated by cohesin. First, we tested two predictions that are made by this hypothesis. Cohesin associates with chromatin throughout interphase and regulates transcription at the IGF2-H19 locus both in G1 and G2 phase [6]. If cohesin affects gene regulation by enabling the formation of chromatin loops one would, therefore, predict that these loops are also present in G1 and G2 phase. To test this we used 3C assays to compare chromatin conformation between cells that were synchronised by double thymidine arrest-release either in G1 or G2 phase. Figure 3 shows that synchronisation of cells in G1 and G2 phase did not change the overall 3C profiles using the CTCF AD/DMR0, ICR or enhancers as anchors. An exception was Cohesin Mediates Chromatin Conformation Figure 4. 3C-SNP analysis. (A–E) show SNP sequencing results with electropherograms of the sequence and allele ratios by pyrosequencing (PSQ) analysis. A diagram indicating the 3C ligation product with the orientation of primers and SNPs is given below each sequence result. (A) 3C ligation product of restriction sites a and m which represents DMR0 and the enhancer showing preferentially monoallelic interaction at a SNP within fragment a-b1. The a-b1 fragment is close to the P0 and P2 promoters in the flanking BamHI fragments. (Promoters can extend up to 5 Kb upstream of the transcription start site and the resolution of the 3C technique does not enable distinction between different promoters when they are in close proximity). (B) SNP analysis of 3C ligation product of restriction sites c2 and m representing an interaction between the enhancer and IGF2 promoters near restriction site c2. Both SNPs in this region show monoallelic interactions. (C) Allelic analysis of 3C ligation product of restriction sites a and h, using a SNP near restriction site h in the CCD shows that associations between CTCF AD/ DMR0 and CCD are biallelic. This SNP also showed that CTCF bound to both alleles at the CCD in Figure 2. (D) SNP analysis of 3C ligation product of restriction sites k and q indicative of an interaction between the ICR and the downstream CTCF DS showing a monoallelic interaction. (E) SNP analysis of 3C ligation products between ICR and the CTCF AD/DMR0 (restriction sites a–k). Both sides of the hybrid product show biallelic associations. doi:10.1371/journal.pgen.1000739.g004 observed for the association between the ICR anchor and the CTCF DS which did not stand out as a ‘‘peak’’ in G1 cells, but could be detected as a ‘‘shoulder’’ in the 3C profile of the G2 cells. We presently do not know if this difference is of physiological relevance. Since most interactions did not change between G1 and G2 phase, our results are consistent with the possibility that cohesin mediates chromatin loop formation throughout inter- PLoS Genetics | www.plosgenetics.org 6 November 2009 | Volume 5 | Issue 11 | e1000739