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

Estrogen Receptor alpha (ERalpha) 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 ERalpha 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datasets from experiments of MCF7 and T47D cells cultured in absence of estrogen (Cicatiello et al., 2010; JS Carroll, unpublished).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 ERalpha 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 ERalpha binding sites are indeed present in absence of ligand. Next, we have performed ERalpha-ChIP-sequencing using MCF7 cells transfected with control and ERalpha siRNA, as above. 10,778 ER-binding peaks (p-value < 0.005) were found, confirming the constitutive presence of ERalpha in intronic and intergenic regions (45.90% and 43.93%, respectively) as well as in gene promoters and exonic regions (4.62% and 2.51%, respectively). The searching for the transcription factor binding sites shows a significant enrichment for EREs motifs (identified in 47% of ER-binding peaks), as well as a number of other putative binding motifs (SP1, AP1, AP2, RXR, CTCF). Furthemore, we have studied gene expression by microarray experiments in the same conditions, obtaining a list of genes that are regulated by ERalpha siRNA, suggesting that unliganded ER may indeed regulate basal expression of a number of genes.