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Department of Biosciences and Nutrition, Karolinska Institutet, NOVUM, SE 14157 Huddinge, Sweden

(Correspondence should be addressed to Z Papoutsi; Email: zoi.papoutsi{at}ki.se)

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

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