Figure 4.

CTCF occupancy at HSIV is specific to primary human placental syncytiotrophoblasts. (A) CTCF is selectively recruited to HSIV in primary human STBs. CTCF recruitment within the HSIII–V LCR was assessed in the human STB chromatin by ChIP analysis in parallel with analysis of primary human skin fibroblasts. A serial dilution of input DNA was used to confirm that the semi-quantitative PCR was in a linear range in each assay. A pre-immune IgG antibody established nonspecific background levels generated by the IP; IgG antibody (I); CTCF antibody (C). DNAs isolated from each IP pellet was assessed with primer sets corresponding to the predicted CTCF binding sites at HSIII, HSIV and HSV. The PCR analysis showed that CTCF proteins were recruited to the HSIII and HSV in both tissues while CTCF enrichment at HSIV was STB-specific. (B) Constitutive recruitment of CTCF to the hGH/hCS promoters. The ChIP assay demonstrates CTCF occupancy at hGH/hCS promoter regions. This occupancy, in combination with similar results in multiple other tissues (ENCODE), confirms that promoter occupancy by CTCF in the cluster is a constitutive property of this region. Additional sequence analysis of PCR amplicons demonstrated CTCFs are enriched at all five promoters within the cluster (Supplementary Figure S2).