Estrogen Receptor alpha (ERa) is a ligand-dependent transcription factor central to the growth and differentiation of epithelialmammary cells among others. Genomic actions of ERa in response to ligands have been widely described. However, recent studies suggest that unliganded ERa 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 ERa to chromatin and possible epigenetic and transcriptional effects, in human breast cancer cells. First, we have analyzed available ERa 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datasets from experiments of MCF7 and T47D cells cultured in absence of estrogen (Cicatiello et al. 2010; Carroll, JS unpublished). Data obtained from MCF7 and T47D experiments were crossed: common peaks were mapped on genome and validated on individual ERa ChIP experiments, by comparing MCF7 cells transfected with control and ERa siRNA in hormone-deprived medium. These preliminary experiments demonstrated that a number of bonafide ERa binding sites are indeed present in absence of ligand. Next, we have performed ERa-ChIP-sequencing using MCF7 cells transfected with control and ERa siRNA, as above. 10,778 ER-binding peaks (p-value 0.005) were found, confirming the constitutive presence of ERa 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 search for transcription factor binding sites showed 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). Furthermore, we have studied gene expression by microarray experiments in the same conditions, obtaining a list of genes that are regulated by ERa siRNA, suggesting that unliganded ERa may indeed regulate basal expression of a number of genes.
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