Figure 6.
Binding of p68 to the maternal allele of IGF2/H19 ICR is both CTCF- and SRA-dependent. (A) Cross-linked DNA–protein complexes from HeLa cells were immunoprecipitated with anti-CTCF or normal rabbit serum, followed by qPCR amplification with primers specific to the IGF2/H19 ICR CTCF-binding region and a negative control region (chr11:1983833–1983994) (Wendt et al. 2008). Values are presented as percent of input. (B) ChIP analysis of p68 occupancy at the IGF2/H19 ICR CTCF-binding site was performed using chromatin immunoprecipitated from HeLa cells with mouse anti-p68, rabbit anti-p68, and goat anti-p68 antibodies or IgG alone. Precipitated DNA was analyzed by qPCR. (C) Allele-specific ChIP assays were carried out with antibodies against CTCF, p68, or IgG in MEF cells. Input and precipitated samples were subjected to semi-qPCR for the paternal and maternal H19 ICR alleles and then digested with a restriction enzyme, Tsp45I, a polymorphic restriction site. (D) Venn diagram showing overlap of p68- and CTCF-binding sites. (E) Western blot analysis of CTCF and p68 levels in NEs from CTCF shRNA or empty vector stably expressed HeLa cells. β-Actin was used as a loading control. (F,G) ChIP-qPCR analysis of isolated DNA associated with CTCF (F) and p68 (G) at IGF2/H19 ICR in CTCF-depleted HeLa cells (mean ± SD of n = 3). (H) RT–qPCR analysis to SRA knockdown efficiency in HeLa cells (mean ± SD of n = 3). (I,J) ChIP-qPCR analysis of isolated DNA associated with CTCF (I) and p68 (J) on the CTCF-binding site at IGF2/H19 ICR in SRA-depleted HeLa cells (mean ± SD of n = 3).