FIGURE 4.
CTCF-dependent and transcription-independent spatial interaction of the 5′1 and 3′1 boundary elements. A, estrogen-independent spatial interaction of the 5′1 and 3′1 boundaries. MCF-7 cells were deprived of estrogen for 3 days and treated with E2 for 15, 45, or 90 min. DNAs were extracted from the cells for 3C assays with 3′1 primer as the anchoring primer and the 5′1 and TFF1/2 promoter primers (pro) as the interacting primers. Fragments covering two adjacent DpnII sites were amplified as a loading control. Arrowheads indicate the expected PCR products. B, transcription-independent spatial interaction of the 5′1 and 3′1 boundaries. MCF-7 cells were treated with tamoxifen (TAM) for 12 h or α-amanitin for 2 h. Genomic DNAs were extracted from the cells for 3C analysis with the 3′1-5′1 PCR primers. C, CTCF-dependent spatial interaction of the 5′1 and 3′1 boundaries. MCF-7 cells were transfected with control siRNA (siNS), or specific siRNA molecules for ERα, FOXA1, or CTCF, deprived of estrogen for 3 days, and then subjected to the 3C assay with the 3′1-5′1 primers (upper panel). The expression of ERα, FOXA1, and CTCF in these cells was examined by Western blotting. Each bar represents the mean ± S.D. for triplicate experiments. D, cell type-specific spatial interaction of the 5′1 and 3′1 boundaries. The spatial interaction of the 5′1 and 3′1 boundaries was examined in MCF-7 and MDA-MB-231 cells by 3C assays. The DNA looping of the GAPDH locus was analyzed as a control. siNS, control siRNA; siCTCF, CTCF-specific siRNA.