Methylation patterns of the CRH promoter bound by RNA-polymerase 2 (Pol-II) and phospho-cAMP response element-binding protein (pCREB).
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(A) Pol-II binding and (C) pCREB binding to the CRH promoter were determined by ChIP. PCR-amplified promoter regions are shown in Fig 1A. Bars represent means ± SEM (N = 6 and 3 placentae for Pol-II and pCREB, respectively). Asterisks indicate significant pairwise differences between treatments at each region (*, P < 0.05, ANOVA with repeated measures). Binding to the CRH1 region was undetectable (n.d.). (B) and (D), methylation patterns of CRH promoter copies bound by Pol-II and pCREB, respectively, were determined by clonal bisulfite sequencing of DNA isolated by ChIP. Ten randomly selected clones generated from 8-Br-cAMP treated cultures from each placenta were sequenced. Placenta numbers are on the left; filled and open circles indicate methylated and unmethylated CpGs, respectively. CpG Sites numbers are as shown in Fig 1A.