Functional genomics identifies a mechanism for estrogen activation of the retinoic acid receptor 1 gene in breast cancer cells

Abstract

The identification of estrogen receptor (ERα) target genes is crucial to our understanding of its predominant role in breast cancer. In this study, we used a chromatin immunoprecipitation (ChIP)-cloning strategy to identify ERα-regulatory modules and associated target genes in the human breast cancer cell line MCF-7. We isolated 12 transcriptionally active genomic modules that recruit ERα and the coactivator steroid receptor coactivator (SRC)-3 to different intensities in vivo. One of the ERα-regulatory modules identified is located 3.7 kb downstream of the first transcriptional start site of the RARA locus, which encodes retinoic acid receptor α1 (RARα1). This module, which includes an estrogen response element (ERE), is conserved between the human and mouse genomes. Direct binding of ERα to the ERE was shown using EMSAs, and transient transfections in MCF-7 cells demonstrated that endogenous ERα can induce estrogen-dependent transcriptional activation from the module or the ERE linked to a heterologous promoter. Furthermore, ChIP assays showed that the coregulators SRC-1, SRC-3, and receptor-interacting protein 140 are recruited to this intronic module in an estrogen-dependent manner. As expected from previous studies, the transcription factor Sp1 can be detected at the RARA α1 promoter by ChIP. However, treatment with estradiol did not influence Sp1 recruitment nor help recruit ERα to the promoter. Finally, ablation of the intronic ERE was sufficient to abrogate the up-regulation of RARA α1 promoter activity by estradiol. Thus, this study uncovered a mechanism by which ERα significantly activates RARα1 expression in breast cancer cells and exemplifies the utility of functional genomics strategies in identifying long-distance regulatory modules for nuclear receptors. Copyright © 2005 by The Endocrine Society.