Binding of estrogen receptor alpha/beta heterodimers to chromatin in MCF-7 cells

Estrogen receptors (ERs), ERalpha and ERbeta, belong to a group of transcription factors that, upon ligand binding, regulate gene expression by binding to specific DNA regions in chromatin as dimers. In this article, we applied the sequential chromatin immunoprecipitation assay (Re-ChIP) to study the simultaneous presence of ERalpha and ERbeta on various DNA-binding regions in intact chromatin. ERalpha/beta heterodimers were isolated by precipitation with anti-ERbeta antibody followed by anti-ERalpha antibody from a stable MCF-7-derived cell line that expresses endogenous ERalpha and an inducible version of ERbeta. The Re-ChIP method was first validated based on the detection of ERalpha/beta heterodimers bound to a promoter region of the pS2 gene known to bind both ERalpha and ERbeta. We next examined 12 ER-binding sites using Re-ChIP assays for ERalpha/beta heterodimer recruitment. Our results confirmed the recruitment of ERalpha/beta heterodimers to all these regions. This study represents the first demonstration of binding of ERalpha/beta heterodimers to various DNA-binding regions in intact chromatin.