Cohesin participates in enhancer-promoter interactions. (A) SMC1A ChIA-PET interaction between Pitx1 and a distal site 133 kb upstream, bound by CTCF and SMC1A. Although E11.5 forelimb and hindlimb tissue were combined for ChIP, it has previously been shown that both Pitx1 and the distal site are marked only by H3K27ac in hindlimb and only by H3K27me3 in forelimb (Cotney et al. 2012). CTCF binding sites in E14.5 cortex are also shown (Shen et al. 2012). (B) 3C analysis of interactions at the Pitx1 locus in E11.5 forelimb, E11.5 hindlimb, and E14.5 cortex, using an anchor primer located in the gene (dashed line) and primers tiled across the locus. Interactions are normalized to a crosslinking control at Ercc3. Each data point represents average interaction frequency and error bars represent standard error from three independent qPCR reactions (see Supplemental Fig. S4A for biological replicate). (C) Promoters within 2 kb of an interaction anchor that loop to a distal site show significantly higher expression than promoters contained within the loop ([*] Wilcoxon P-value < 2 × 10−16). (D) E11.5 limb H3K27ac (green) and H3K4me2 (blue) normalized ChIP-seq signal aggregated at all distal sites looping to a promoter.
