Author Summary Recent work has shown that cohesin, a protein best known for its role in holding sister chromatids together, and CTCF, a protein implicated in the formation of chromatin loops, localize to the same regions of DNA in mammalian genomes. This observation raised the intrigu- ing possibility that cohesin might facilitate the role of CTCF in structuring chromatin. CTCF is well known for its role in regulating genomic imprinting at the IGF2-H19 gene locus. Imprinted genes are widely studied due to their roles in fetal growth and cancer and have the unusual property of expressing only one parental copy of the gene. CTCF is thought to regulate imprinting of IGF2 and H19 by enabling DNA to form loops that separate the genes into silent or active domains. In this paper we describe, for the first time, the looping structure of the human IGF2-H19 locus and show that cohesin stabilises CTCF–mediated DNA loops. Depletion of cohesin leads to disruption of long-range chromatin interactions and changes expression levels of the IGF2 gene. This work adds a new level of understanding of how cohesin can play a role in gene expression. separation is thought to maintain the silence of the maternal IGF2 allele. On the methylated paternal allele CTCF is excluded from binding and the IGF2 promoters can interact with the enhancers [14,15]. In mice it has been demonstrated that higher order chromatin conformation at this locus differs between the maternal and paternal alleles and that CTCF binding is essential for the formation of chromatin loops on the maternal allele [17–22]. In addition to the ICR and the downstream enhancers, several additional regulatory regions have been described surrounding the locus. At the 59end of IGF2 there is a differentially methylated region, DMR0, which in human has variable methylation in somatic tissues and is hypomethylated in cancers [23–25]. In the intervening sequence between IGF2 and H19 there is a Centrally Conserved DNase I hypersensitive domain (CCD), which in mice has tissue specific enhancer functions [26–30]. In humans the functions of the CCD and DMR0 are unknown. Because CTCF is needed for the recruitment of cohesin to insulator sequences, we speculated that cohesin, with its unique capability of holding DNA strands together, is required for the formation and stabilisation of CTCF-dependent chromatin loops. To test this hypothesis we undertook quantitative Chromatin Conformation Capture (q3C) analysis [31] of the human IGF2- H19 locus (Figure 1) and examined CTCF-dependent chromatin loops after cohesin depletion by RNAi. Our results indicate that cohesin has an important role in long-range interactions between CTCF sites, whereas CTCF independent chromatin associations do not require the presence of cohesin. Cohesin may, therefore, contribute to gene regulation at CTCF sites by mediating the formation of chromatin loops. Results We chose to study the role of cohesin at the IGF2-H19 locus in human cells because we are interested in the effects of chromatin looping in human cells. We have previously developed protocols for depletion of cohesin subunits by RNAi for human cells [6]. We used a diploid human breast epithelial cell line, HB2, from which cohesin could be depleted, which could be synchronised in the cell cycle, and which contained informative Single Nucleotide Polymorphisms (SNPs), enabling us to distinguish between alleles Cohesin Mediates Chromatin Conformation at the IGF2-H19 locus. H19 expression in these cells is monoallelic and as with most normal adult tissues IGF2 is expressed only at basal levels [32,33]. Bisulphite sequencing confirmed that this cell line has monoallellic methylation at the H19 ICR (Figure 2E), indicating correct imprinting at this locus. The ICR acquires its methylation in the male germ line and maintains it in all normal tissues even if IGF2 is tissue specifically or developmentally down- regulated [34]. Co-localisation of CTCF and cohesin within the IGF2-H19 locus To predict higher order interactions that may be directly mediated by CTCF and cohesin at this locus in HB2 cells, we performed locus-wide ChIP experiments with antibodies specific for CTCF and SMC3 (this subunit of cohesin was analysed due to the availability of validated ChIP grade antibodies [6]). We found that CTCF and cohesin co-localise at the ICR, at CTCF sites immediately adjacent to the DMR0, at CTCF sites in the CCD region [26,27,30] and also with CTCF sites downstream of the enhancers (Figure 2A and 2B), consistent with previous data from other cell lines [6,35]. We refer to the CTCF sites adjacent to the DMR0 as ‘‘CTCF AD’’ and to those downstream of the enhancers as ‘‘CTCF DS’’ (Figure 1). In contrast to the above mentioned sites, CTCF and cohesin were not enriched at the DMR0, IGF2 promoters or the enhancers downstream of H19 (Figure 2A and 2B). SNP sequencing of ChIP-PCR samples indicated that CTCF and cohesin bind at all sites to both alleles, with exception of the ICR, where monoallelic binding was observed on the unmethy- lated allele (Figure 2C and 2E). The CTCF sites adjacent to the DMR0 and the CCD are not CpG rich and were unmethylated (data not shown). Locus-wide chromatin conformation studies at IGF2-H19 We determined the chromatin conformation at the human IGF2-H19 locus through extensive q3C analyses with a BamHI restriction enzyme, using primers within the ICR, enhancer, and two other CTCF sites (CTCF AD/DMR0 and CTCF DS) flanking the locus as anchors. BamHI cuts the locus frequently, but there are no restriction sites within the CTCF AD. The nearest site to the CTCF AD is on the edge of the DMR0 region (restriction site a). For this reason we have regarded CTCF AD/DMR0 as a single 3C element in our analysis. The resulting data represent an average of association frequencies on both parental alleles across the whole locus. Random ligations are expected to decrease exponentially the further a restriction site is away from the anchor, while specific associations occur as ‘‘spikes’’ above the random ligation curve [36]. However, because the resolution of 3C is limited, multiple adjacent restriction sites within a 5 Kb stretch of DNA will associate with similar frequencies with a distant restriction site used as an anchor. The 3C signals therefore indicate proximities to interactions, rather than pinpoint the exact sequences involved in the interactions. When a primer within the ICR was used as an anchor (primer k), weak but specific associations were detected with the CTCF AD/DMR0 region (restriction site a, Figure 3B and 3C). When the enhancer was used as an anchor (primer m) we were able to detect associations with a restriction site within the CTCF AD/ DMR0 (restriction site a) as well as at restriction sites near the P2 and P3 promoters (sites b2, c1 and d, Figure 3D and 3E), despite low levels of IGF2 expression in these cells. However, when an anchor primer was placed within the CTCF AD/DMR0 region (primer b1), we found strong association frequencies with the CCD (restriction site h) and the CTCF DS (restriction site q), but not with the ICR or the enhancer (Figure 3F). Because the DMR0 PLoS Genetics | www.plosgenetics.org 2 November 2009 | Volume 5 | Issue 11 | e1000739