Estrogen receptors (ERs), ERα and ERβ, 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 ERα and ERβ on various DNA-binding regions in intact chromatin. ERα/β heterodimers were isolated by precipitation with anti-ERβ antibody followed by anti-ERα antibody from a stable MCF-7-derived cell line that expresses endogenous ERα and an inducible version of ERβ. The Re-ChIP method was first validated based on the detection of ERα/β heterodimers bound to a promoter region of the pS2 gene known to bind both ERα and ERβ. We next examined 12 ER-binding sites using Re-ChIP assays for ERα/β heterodimer recruitment. Our results confirmed the recruitment of ERα/β heterodimers to all these regions. This study represents the first demonstration of binding of ERα/β heterodimers to various DNA-binding regions in intact chromatin.
ASJC Scopus subject areas
- Molecular Biology