Cohesin Mediates Chromatin Conformation Figure 7. Depletion of cohesin by siRNA results in ablation of the CTCF-mediated loops. (A) Schematic representation of a 350 Kb genomic region including INS and the IGF2-H19 locus as shown in Figure 3. (B) Associations detected with an anchor site in the ICR (primer k) in G2 phase cells treated with control RNAi (G2 control) or RNAi against the cohesin subunit SCC1 (SCC1 RNAi). Association between the ICR and CTCF/ cohesin sites, i.e. CTCF AD, CCD, and CTCF DS, are reduced in SCC1 depleted cells indicating a general ‘‘loosening’’ of the chromatin structure. (C) Histogram of association frequencies between ICR anchor and restriction sites at the CTCF AD/DMR0 peak. (D) Enhancer associations (m primer) after cohesin RNAi are not generally reduced. Reduction at the intervening CCD and ICR sites may be due to the influence of disrupted associations at CTCF/cohesin bound sites. (E) Histogram of association frequencies between Enh anchor and restriction sites at the CTCF AD/DMR0 peak. (F) Sequencing of b1-m interaction shows a change from mono-allelic interactions in controls to biallelic interactions after cohesin depletion. (G) Association frequencies of CTCF AD/DMR0 (restriction site b1 as anchor) show a significant reduction in the association with CCD. (H) Histogram representing reduction in CTCF AD/DMR0 -CCD (h-b1) association after cohesin depletion. Table S1 has statistical analysis of all interactions between anchor primers and BamHI sites. # Denotes significant differences (P,0.05) between control and SCC1 RNAi. doi:10.1371/journal.pgen.1000739.g007 PLoS Genetics | www.plosgenetics.org 9 November 2009 | Volume 5 | Issue 11 | e1000739