Fig. 1.

ERα recruits transcriptional corepressors to repress p53-mediated transcriptional activation. (A) ChIP and sequential ChIP assays were performed on MCF-7 cells with primers specific to the p53-binding site of the p21 promoter. The primary ChIP was performed with anti-p53 antibody, and the immunoprecipitate was subjected to a second ChIP with anti-ERα antibody. The immunoprecipitate from the ERα ChIP was then subjected to the third ChIP with antibodies against HDAC1, NCoR, SMRT, and RIP140. ChIP with IgG was the negative control. (B) ChIP assay as in A with antibodies against NCoR, SMRT, HDAC1, and IgG in MCF-7 cells transfected with NS or ERα siRNAs. (C) Western analysis of protein expression of NCoR, SMRT, RIP140, HDAC1, and ERα in MCF-7 cells transfected with NS siRNA or ERα siRNA. (D) ChIP assay performed on ERα knockdown MCF-7 cells using antibodies against p53, ERα, or IgG. (E) Recruitment of NCoR, SMRT, and HDAC1 to the p53-binding site of the p21 promoter in MCF-7 cells treated with E2 (10 nM) or vehicle for 3 h, as analyzed by ChIP assay. (F) MCF-7 cells were transfected with −2326 p21-luc reporter and NCoR shRNA plasmid or a nonspecific (NS) shRNA plasmid. Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Data are averages from three samples with SD. (G) Transcription of the endogenous p21 gene in MCF-7 cells treated as in F was assayed by qPCR. Data are averages from three samples with SD. (H) Western analysis of protein expression of NCoR, ERα, and p53 in MCF-7 cells transfected with NS or NCoR siRNAs.