Cohesin Mediates Chromatin Conformation Figure 1. Schematic representation of the IGF2-H19 locus. (A) The Insulin (INS), Insulin-like Growth Factor 2 (IGF2), and H19 genes (blue boxes) are displayed together with the downstream enhancer (Enh; green oval). CTCF/cohesin binding regions are indicated by red rectangles, BamHI restriction sites by vertical blue lines, and 3C restriction sites (3C RS) by letters. The 3C elements (yellow triangles) represent regions analysed by 3C; the IGF2 upstream region (CTCF AD/DMR0), the Centrally Conserved DNase I hypersensitive domain (CCD), the Imprinting Control Region (ICR), the H19 downstream enhancers, and a CTCF binding Downstream Site (CTCF DS). (B–F) Enlargements of the 3C elements and surrounding genomic areas indicating primer positions relative to restriction sites. Anchor primers are indicated with dark arrows, reciprocal 3C primers with grey arrowheads, and primers used for SNP analysis with open arrowheads. (B) Enlarged region of the IGF2 gene. The IGF2 upstream region is composed of the CTCF region adjacent to DMR0 (CTCF AD; red box) and the DMR0 (yellow bar). Promoters (P1 to P4), exons (empty boxes for non-coding and blue boxes for coding), and single nucleotide polymorphisms (SNP; rs numbers) are indicated. Red type and underlying bars represent ChIP amplicons. The closest BamHI restriction site to the CTCF AD is restriction site a. Restriction site b1 is within DMR0, but is also close to the P2 promoter. Restriction sites b2, c1, c2, and d are between the P2 and P3 promoters. (C) Detail of the DMR0 region to indicate SNP positions in the a-b1 restriction fragment. (D) The intervening region between IGF2 and H19 showing the position of the CCD relative to restriction sites e to h. (E) Enlargement of the ICR (yellow bar) and H19 gene (open boxes). Restriction sites i to k lie in the CTCF binding regions (red box) and ICR rs115647398* is a SNP in HB2 that is 2 bp away from the annotated rs115647398. (F) Detail of the enhancers region and CTCF DS. Primer information is available in Table S2 and Table S3. doi:10.1371/journal.pgen.1000739.g001 does not bind CTCF, it is possible that the associations between the CTCF AD/DMR0 and the CCD are caused by CTCF binding at the CTCF AD. To confirm that the CTCF AD/DMR0 region interacts with the distant CTCF DS sites we also used a primer within the CTCF DS (primer q) as an anchor. As expected, this revealed associations between CTCF DS and the CTCF AD/ DMR0 region (restriction sites a and b1), and in addition showed associations with the CCD (restriction site h) (Figure 3G). Locus- wide q3C analysis was also performed using BglII as a restriction enzyme which cuts in the CTCF AD site. These experiments revealed interactions between the CTCF AD anchor, the CCD and CTCF DS as well as ICR anchor and the CTCF AD (Figure S1 and Figure S2), similar to what we had obtained for BamHI experiments. PLoS Genetics | www.plosgenetics.org 3 November 2009 | Volume 5 | Issue 11 | e1000739