FIGURE 6.
CTCF acts upstream of the pioneer factor FOXA1. A, FOXA1 binding pattern in the TFF locus. Soluble chromatins were prepared from MCF-7 cells, and qChIP assays were performed with primers covering the indicated boundary elements and gene promoters and antibodies against FOXA1. Each bar represents the mean ± S.D. for triplicate experiments. B, the binding of FOXA1 at the TFF1 locus is dependent on CTCF. MCF-7 cells were transfected with control siRNA (siNS) or CTCF-specific siRNA (siCTCF). Soluble chromatins were prepared from the cells and qChIP assays were performed with primers covering the 5′1 boundary, 3′1 boundary, and the TFF1 promoter (TFF1 Pro) and with antibodies against FOXA1 (left) or CTCF (right). Each bar represents the mean ± S.D. for triplicate experiments. C, the dependence of FOXA1 recruitment on CTCF in estrogen responsive gene loci. MCF-7 cells were transfected with control siRNA or CTCF-specific siRNA. Soluble chromatins were prepared from the cells, and qChIP assays were performed with primers covering the FOXA1-binding sites and CTCF-binding sites of the indicated genes and antibodies against FOXA1 (left) or CTCF (right). The L or R following gene symbols represents the primer set used to amplify FOXA1- or CTCF-binding sites located to the left or right (from the 5′ to 3′ direction) within the CTCF-confined regions in these genes. Each bar represents the mean ± S.D. for triplicate experiments. D, the binding of CTCF does not rely on FOXA1. MCF-7 cells were transfected with control siRNA (siNS) or FOXA1-specific siRNA (siFOXA1). Soluble chromatins were prepared from the cells, and qChIP assays were performed with antibodies against CTCF (left) or FOXA1 (right) and with primers described in C. Each bar represents the mean ± S.D. for triplicate experiments.