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. 2008 Oct 31;283(44):29671–29680. doi: 10.1074/jbc.M802785200

FIGURE 3.

FIGURE 3.

Overexpression of ERβ or p53 attenuates BRCA2 expression and the recruitment of ERα, CBP/p300, MyoD, and histone acetylations on the BRCA2 promoter region induced by E2. A, overexpression of ERβ or p53 in MCF-7 cells attenuates BRCA2 expression induced by E2. MCF-7 cells were precultured for 4 days in phenol red-free RPMI 1640 medium containing 5% charcoal dextran-stripped FBS, then ERβ or p53 expression vector was transiently transfected into MCF-7 cells, and cells were cultured for 6 h. Then the medium was replaced with RPMI 1640 medium plus 10 nm E2 for 24 h, and Western blot analysis was performed. B, ERα increases BRCA2 promoter activity induced by E2, and ERβ or p53 attenuates BRCA2 promoter activity induced by E2. MCF-7 cells were precultured as in A. Then BRCA2 promoter construct (pGL3-BRCA2) was transiently co-transfected with ERα, ERβ, or p53 expression vector or pcDNA3 control vector, respectively, into MCF-7 cells, and cells were cultured for 24 h in RPMI 1640 medium or RPMI 1640 medium plus 10 nm E2. Luciferase activity in the cells transfected with ERα, ERβ, or p53 expression vector was compared with that in the cells transfected with pcDNA3 control vector. *, p < 0.05; **, p < 0.01. C, ERβ or p53 attenuates the recruitment of ERα, p300, CBP, and MyoD to the BRCA2 promoter region stimulated by E2. MCF-7 cells were precultured as in A, and then ERβ or p53 expression vector was transiently transfected with into MCF-7 cells. Cells were cultured for 24 h in RPMI 1640 medium or RPMI 1640 medium plus 10 nm E2. ChIP assays were performed using antibody against ERα, p300, CBP, and MyoD as described under “Experimental Procedures.” Con, pcDNA3. D, ERβ or p53 attenuates histone acetylations on the BRCA2 promoter region stimulated by E2. MCF-7 cells were treated as in C. ChIP assays were performed using antibody against acetylated histones H2A, H2B, H3, and H4 as described under “Experimental Procedures.” Con, pcDNA3. E, ERα or ERβ increases BRCA2 promoter activity, and p53 represses BRCA2 promoter activity. MCF-7 cells were precultured as in A, and then BRCA2 promoter construct (pGL3-BRCA2) was transiently co-transfected with ERα, ERβ, or p53 expression vector or pcDNA3 control vector, respectively, into MCF-7 cells. Cells were cultured for 24 h. Luciferase activity in the cells transfected with ERα, ERβ, or p53 expression vector was compared with that in the cells transfected with pcDNA3 control vector. **, p < 0.01.