Figure 5. ChIP–loop experiments indicate that CTCF and cohesin are present at the interactions between CTCF AD/DMR0 and the ICR.

Agarose gel showing a PCR product of 272 bp amplified with primers for the ICR and CTCFAD/DMR0 (primers at j and b1 restriction sites in Figure 1A) and a PCR product of 303 bp amplified with primers for the enhancer and CTCFAD/DMR0 (primers at m and b1 restriction sites in Figure 1A). Templates in each panel include 3C standard (template from PCR standard curve), 3C (cross-linked chromatin BamHI digested and ligated), α-CTCF-3C, and α-SMC3-3C (ChIP–loop templates that were immuno-precipitated with CTCF or cohesin antibodies prior to ligation). ICR interactions are found in 3C and ChIP-loop material, likely reflecting a specific CTCF/cohesin mediated association between CTCF AD and the ICR, with both proteins present in the loop. In contrast, enhancer interactions are not found in ChIP–loop material confirming that this interaction is not CTCF/cohesin dependent and reflecting an association between the enhancer and the P2 promoter rather than with DMR0.