MPA induces a cooperative transcriptional interaction at the cyclin D1 promoter among AP-1, Stat3, PR and ErbB-2. (A) Cells were transfected with the indicated siRNAs or expression vectors and were then treated with MPA for 30 minutes. Recruitment of proteins to the cyclin D1 promoter was analyzed by ChIP. Immunoprecipitated DNA was amplified by qPCR using primers (red arrows) flanking the GAS and TRE sites indicated in the top panels. Amounts of immunoprecipitated DNA were normalized to inputs and reported relative to the amount obtained by IgG immunoprecipitation, which was set to one. (B) Sequential ChIP. Chromatins from cells treated with MPA as described in A were first immunoprecipitated with c-Jun or c-Fos antibodies and were then re-immunoprecipitated using an ErbB-2 antibody. 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 fold chromatin enrichment over untreated cells. For b vs. a: P <0.001. (C) Recruitment of CBP and p300, and H3 and H4 acetylation levels (AcH3 and AcH4) at the sites described in A were studied by ChIP and data were also analyzed as in A. Results in A to C are the mean ± SEM from three independent experiments. For b vs. a: P <0.001. ChIP, chromatin immunoprecipitation; MPA, Medroxyprogesterone acetate.