FIGURE 2.

ERα, CBP/p300, and MyoD increase BRCA2 promoter activity in MCF-7 cells during E₂ treatment. A, binding of ERα, CBP/p300, MyoD, and Sp1 to the BRCA2 promoter in MCF-7 cells with E₂ treatment. MCF-7 cells were precultured for 4 days in phenol red-free RPMI 1640 medium containing 5% charcoal dextran-stripped FBS, and then MCF-7 cells were cultured for 24 h in RPMI 1640 medium plus 10 nm E₂. Nucleic extracts were prepared from MCF-7 cells or MCF-7 cells treated with 10 nm E₂. ChIP assays were performed using antibody against ERα, CBP, p300, MyoD, and Sp1, respectively, as described under “Experimental Procedures.” The primers corresponding to the BRCA2 promoter region –191 and +30 upstream of the transcriptional start site were used for PCR to detect the presence of the BRCA2 promoter DNA. Con, control. B, ERα, CBP/p300, and MyoD cannot bind to exon 7 in the BRCA2 gene in MCF-7 cells with E₂ treatment. Con, control. ChIP assays were performed using antibody against ERα, CBP, p300, and MyoD, respectively, as described above. The primers correspond to the BRCA2 exon 7 region. C, detection of ERα complex on the BRCA2 promoter. A ChIP-reChIP assay was performed in MCF-7 cells with E₂ treatment. Chromatin was incubated with ERα antibody and then immunoprecipitated sequentially with CBP, p300, MyoD, or Sp1 antibody. The BRCA2 promoter DNA bound to ERα/CBP, ERα/p300, ERα/MyoD, or ERα/Sp1 was amplified by PCR. Con, control. D, histone modifications on the BRCA2 promoter. Nucleic extracts were prepared from MCF-7 cells or MCF-7 cells treated with E₂. ChIP assays were performed using antibody against acetyl-H2A, acetyl-H2B, acetyl-H3, and acetyl-H4, respectively, as described under “Experimental Procedures.” Con, control. E, activation of the BRCA2 promoter by ERα, CBP, p300, and MyoD. MCF-7 cells were plated in 6-well tissue culture plates and then co-transfected with 0.5 μg of pGL3-BRCA2 with 0.5 μg of ERα, CBP, p300, or MyoD expression vector or pcDNA3 control vector. Renilla luciferase reporter construct pRL-TK (20 ng) was used as an internal control for transfection efficiency. Forty hours after transfection, luciferase activity was measured with equivalent amounts of protein extracts. Luciferase activity of the BRCA2 promoter was normalized to the activity of a co-transfected Renilla luciferase expression vector and protein content. Luciferase activity in the cells transfected with ERα, CBP, p300, or MyoD expression vector was compared with that in the cells transfected with pcDNA3 control vector. **, p < 0.01. F, effect of siRNA on MyoD expression in MCF-7 cells. MCF-7 cells were transfected with control siRNA or MyoD siRNA for 48 h, and then Western blot was performed. G, reduction of MyoD expression by siRNA attenuates the E₂-induced BRCA2 promoter activity in MCF-7 cells. BRCA2 promoter construct (pGL3-BRCA2) was transiently co-transfected with MyoD siRNA or non-targeting siRNA (Control siRNA) into MCF-7 cells, cells were cultured for 24 h and then exposed to 10 nm E₂ for 24 h, and luciferase activity was detected. Luciferase activity in the cells treated with MyoD siRNA was compared with that in the cells treated with non-targeting siRNA. *, p < 0.05.