depicted in Figure 9. At this hub CTCF and cohesin might bring various regulatory elements into close proximity to enable interactions between distant elements, either simultaneously as is drawn in our model, or possibly in a sequential order. Cohesin may stabilise the CTCF mediated interactions. Cross-linked chromatin enables us to study a snapshot of interactions at any given time, but it is likely that these interactions are dynamic with some occurring more rapidly than others. Transcription of IGF2 and H19 is developmentally down regulated in most adult tissues but reactivated in various cancers (reviewed in [13]). We chose a normal epithelial breast cell line to study the conformation of the adult IGF2-H19 locus. After the disruption of CTCF mediated chromatin conformation by cohesin depletion IGF2 expression was reactivated in these cells. Moreover, substantial biallelic expression of IGF2 was observed and enhancer-promoter associations changed from mono- to biallelic. Interestingly, biallelic IGF2 expression was not accom- panied by hypermethylation at the ICR, suggesting that depletion of cohesin can uncouple the relationship between IGF2 expression and methylation at the H19 ICR. Methylation profiles of the IGF2-H19 locus in many cancers indicate that loss of IGF2 imprinting and methylation are often disconnected during neoplasia [40–46]. The roles of higher order chromatin structure Cohesin Mediates Chromatin Conformation and loss of imprinting in cancer are still largely unexplored. Defects in proper positioning of cohesin on DNA could therefore contribute to abnormal gene regulation in neoplastic cells. The finding that cohesin is required to stabilise higher order chromatin conformation raises the intriguing possibility that cohesin physically connects two DNA sequences on the same DNA molecule in cis, to form loop structures similar to how cohesin interacts with two DNA molecules in trans, to mediate sister chromatid cohesion. It is conceivable that cohesin forms chromatin loops by embracing two DNA strands at the base of a loop, similar to how cohesin has been proposed to mediate cohesion as a ring [2]. Alternatively, it is possible that cohesin complexes bound to two DNA sites can interact with each other, as has been suggested for CTCF molecules [47]. Our finding that cohesin depletion interferes with chromatin loop formation, although cohesin depletion does not abrogate CTCF binding [6], supports our hypothesis that one of CTCF’s main roles as a transcriptional insulator may be to recruit cohesin to insulator sequences. Hypomorphic mutations in the cohesin subunits SMC1 and SMC3 and in the cohesin loading factor NIPBL have been identified as the molecular cause of Cornelia de Lange syndrome, a rare human developmental disorder [48–50]. The results in this study show a modest reduction in looping interactions after Figure 9. Simplified model of the cohesin and CTCF–mediated interactions in the human IGF2-H19 locus. DNA elements are indicated as follows: CTCF AD (red bar); CCD (green bar), ICR (purple bar), and CTCF DS (Cerise bar), Enhancer is yellow oval. Pink and pale blue ovals represent the CTCF/cohesin complexes. CpG methylation is depicted as filled lollipops. (A) Linear representation of the IGF2-H19 locus. Elements above the bar represent the maternal allele with CTCF and cohesin binding the ICR and an active H19 gene. Elements below the bar represent the paternal allele with active IGF2 gene and methylated ICR. ChIP data indicate that cohesin and CTCF co-localise at the CCD, CTCF AD and CTCF DS on both alleles. 3C data indicate that these CTCF/cohesin sites interact strongly with each other; while the ICR and enhancer have limited allele specific interactions (long curved arrows indicate 3C interactions between 3C elements). Based on these data we propose the following model: (B) On the paternal allele, co- localisation of CTCF and cohesin at CTCF AD, CCD, and CTCF DS brings these regions together. The methylated ICR does not bind CTCF and is thus excluded from CTCF/cohesin interacting regions. The exclusion of the ICR may enable the IGF2 gene promoters and H19 enhancer region to interact, (shown by yellow oval close to IGF2 arrow) even though they are on different looping domains. (The H19 domain is shaded.) (C) On the maternal allele, CTCF/cohesin can bind to the unmethylated ICR which can then interact with other CTCF/cohesin sites. An interaction between CTCF AD/ DMR0 and the ICR may be indirectly mediated through the interaction between CTCF AD/DMR0 and CTCF DS. A monoallelic interaction between the ICR and CTCF DS could redefine the H19 domain and constrain the enhancer to prevent interaction with IGF2 promoters on the maternal allele. Without cohesin, CTCF does not maintain stable loops and IGF2 promoters can access the enhancers and perhaps even other regulatory elements from neighbouring genes. Interactions between the various CTCF sites are likely to be dynamic and may occur sequentially. doi:10.1371/journal.pgen.1000739.g009 PLoS Genetics | www.plosgenetics.org 11 November 2009 | Volume 5 | Issue 11 | e1000739