Genome-wide analysis of unliganded estrogen receptor binding sites in breast cancer cells

Estrogen Receptor alpha (ER) is a ligand-dependent transcription factor central to the growth and differentiation of epithelial mammary cells among others. Genomic actions of ER in response to ligands have been widely described. However, recent studies suggest that unliganded ERα is necessary and sufficient to maintain basal expression of epithelial genes (Cardamone et al., 2009).Therefore, we set out to examine the binding of unliganded ERα to chromatin and possible epigenetic and transcriptional effects, in human breast cancer cells. First, we have analyzed available ER ChIP-seq (chromatin immunoprecipitation followed by mass-sequencing) datasets from experiments of MCF7 and T47D cells cultured in absence of estrogen (Cicatiello et al., 2010; JS Carroll, unpublished). Data obtained from MCF7 and T47D experiments were crossed: common peaks were mapped on genome and validated on individual ERChIP experiments, by comparing MCF7 cells transfected with control and ER siRNA in hormone-deprived medium. These preliminary experiments demonstrated that a number of bona-fide ER binding sites are indeed present in absence of ligand. Next, we have performed ER-ChIP-sequencing using MCF7 cells transfected with control and ER siRNA, as above. 10,778 ER-binding peaks (p-value0.005) were found, confirming the constitutive presence of ER at gene promoters as well at intergenic regions. Intersections with different published ChIP-Seq datasets showed large overlaps demonstrating the presence of ERa binding in hormone-deprived conditions. Furthemore, we have studied gene expression by microarray experiments in the same conditions, obtaining a list of genes that are regulated by ER siRNA, suggesting that unliganded ERmay indeed regulate basal expression of a number of genes.