Transcriptional repression of GATA3 gene by co-recruitment of PR and EZH2. A) Protein recruitment to the GATA3 promoter was analyzed by ChIP in cells treated as indicated. Immunoprecipitated DNA was amplified by q-PCR using primers flanking the potential PRE located at position −1,504 bp. Each sample was normalized to the input. Data are expressed as n-fold chromatin enrichment over isotype control. B) T47D or T47D-Y-C587A cells were treated and ChIP was performed as described in A. C) Chromatins from T47D cells treated or not with MPA were first immunoprecipitated with PR antibody and then re-immunoprecipitated using EZH2 antibody. The inverse immunoprecipitation order is also shown. q-PCR analysis of immunoprecipitated DNA was performed as detailed in A. D) Nuclear extracts from T47D cells treated as indicated were immunoprecipitated (IP) with PR antibody and analyzed by WB with EZH2 antibody . As control of specificity, lysates were immunoprecipitated with an isotype control. Input cell lysates were blotted in parallel. E) A DNAse sensitivity assay was performed as described in the Methods section, using the indicated primers detailed in Additional file 1, Table S1. F) T47D or T47D-Y-C587A cells were treated as indicated and GATA3 mRNA expression levels were determined by RT-qPCR. The fold change of mRNA expression levels upon MPA treatment for the indicated times was calculated by normalizing the absolute levels of GATA3 mRNA to GAPDH levels, which were used as an internal control, and setting the value of untreated cells as 1.0. For A, B, C and F, (*P <0.05, **P <0.01 and ***P <0.001, one-way ANOVA). G) Schematic representation of the mechanism proposed for progestin-induced GATA3 transcriptional repression. ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; EZH2, enhancer of zeste homolog 2; MPA, medroxyprogesterone acetate; PR, progesterone receptor; PRE, progesterone response element; WB, Western blot.