FIGURE 4.

Detection of ERβ or p53 complex on the BRCA2 promoter. A, ERβ binding to the BRCA2 promoter in MCF-7 cells transfected with ERβ expression vector. MCF-7 cells were precultured as in Fig. 3A, then ERβ expression vector was transiently transfected into MCF-7 cells, and cells were cultured for 24 h. ChIP assays were performed using antibody against ERβ as described under “Experimental Procedures.” Con, pcDNA3. B, detection of ERβ complex on the BRCA2 promoter. A ChIP-reChIP assay was performed in MCF-7 cells transfected with ERβ vector. Chromatin was incubated with ERβ antibody and then immunoprecipitated sequentially with ERα, CBP, p300, and MyoD antibodies. The BRCA2 promoter DNA bound to ERα/ERβ, ERβ/CBP, ERβ/p300, or ERβ/MyoD was amplified by PCR. Con, pcDNA3. C, binding of p53, HDAC1, and CtBP1 to the BRCA2 promoter in MCF-7 cells transfected with p53 vector. MCF-7 cells were precultured as in Fig. 3A, then p53 expression vector was transiently transfected into MCF-7 cells, and cells were cultured for 24 h. ChIP assays were performed using antibody against p53, HDAC1, and CtBP1 as described under “Experimental Procedures.” Con, pcDNA3. D, detection of p53 complex on the BRCA2 promoter. A ChIP-reChIP assay was performed in MCF-7 cells transfected with p53 vector. Chromatin was incubated with p53 antibody and then immunoprecipitated sequentially with HDAC1 and CtBP1 antibodies. The BRCA2 promoter DNA bound to p53/HDAC1 and p53/CtBP1 was amplified by PCR. Con, pcDNA3. E, p53 and CtBP1 synergistically represses BRCA2 promoter activity. MCF-7 cells were precultured as in Fig. 3A, then BRCA2 promoter construct (pGL3-BRCA2) was transiently co-transfected with p53 or CtBP1 expression vector or pcDNA3 control vector into MCF-7 cells, and cells were cultured for 24 h. Luciferase activity in the cells transfected with p53 or CtBP1 expression vector was compared with that in the cells transfected with pcDNA3 control vector. *, p < 0.05; **, p < 0.01.