Figure 4

MPA induces in vivo binding of c-Jun, c-Fos and PR to the cyclin D1 promoter. (A) Protein recruitment to the cyclin D1 promoter was analyzed by ChIP in cells treated with MPA or pretreated with U0126 when indicated. Immunoprecipitated DNA was amplified by qPCR using primers flanking the TRE site. The arbitrary qPCR number obtained for each sample was normalized to the input, setting the value of the untreated sample as 1. Data are expressed as n-fold chromatin enrichment over untreated cells. For b vs. a and c vs. b: P <0.001. (B) Sequential ChIP chromatins from cells treated with MPA were first immunoprecipitated with c-Jun or c-Fos antibodies and were then re-immunoprecipitated using a PR antibody. qPCR and data analysis were performed as detailed in A. For b vs. a: P <0.001. Results in A and B are the mean ± SEM from three independent experiments. IgG was used as a negative control. MPA effects in T47D-Y-C587A-PR cells. (C) Protein recruitment to the cyclin D1 promoter was studied as described in A. For b vs. a: P <0.001. (D) Cyclin D1 promoter activation was detected as in Figure 3A and data shown represent the mean of three independent experiments ± SEM. (E) Cyclin D1 expression was studied by WB. This experiment was repeated three times with similar results. ChIP, chromatin immunoprecipitation; MPA, Medroxyprogesterone acetate; PR, Progesterone receptor; WB, Western blot.