Figure 2.

Chromatin immunoprecipitation analysis for CTCF at the KvDMR. (A) Schematic of the wild-type (WT) and Kcnq1ot1^PO alleles. Primers a^wt and b amplify CTCF binding site 1 (CTCF-1) in the WT allele only, since a^wt anneals to a sequence in the promoter, which is absent in the mutant allele; primers a^m and b amplify CTCF binding site 1 in the mutant allele (PO). Primers c and d amplify CTCF binding site 2 (CTCF-2) of both the WT and Kcnq1ot^PO allele. Asterisk denotes SNP analyzed with the primer depicted with the hatched arrowhead. (B) Quantitative PCR of immunoprecipitated substrates from neonatal hearts of WT mice and Kcnq1ot1^+/PO littermates. Assays were performed on mice from three litters each, in triplicate, and results expressed as percentage input normalized to H19 R1 (repeat 1 of the differentially methylated domain). Error bars indicate SEM. CTCF-1 shows occupancy of CTCF in both parental alleles of in both the WT mouse and the Kcnq1ot1^+/PO mouse. For the Kcnq1ot1^+/PO mouse (MUT), the white bar represents the data for the WT maternal (primers a^wt and b) and the hatched bar represents the mutant paternal allele (primers a^m and b) CTCF-2 shows occupancy of CTCF for both WT and mutant alleles. (C) Allele-specific restriction digests of ChIP-PCR products for primer pairs for CTCF-1 (left panel) and CTCF-2 (right panel). ND, non-digested; H, Hpy1881 digest. For CTCF-1, Hpy1881 cuts only the castaneus allele; for CTCF-2, MfeI cuts only the castaneus allele.