Novel Estrogen Receptor-α Binding Sites and Estradiol Target Genes Identified by Chromatin Immunoprecipitation Cloning in Breast Cancer

Abstract

Estrogen receptor-A (ERA) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ERA was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ERA target loci in breast cancer cells. The ERA-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ERA-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/ transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ERA responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ERA that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer. [Cancer Res 2007;67(10):5017-24]

Methylation of a Novel CpG Island of Intron I Is Associated With Steroidogenic Factor I Expression in Endometriotic Stromal Cells

“…Moreover, although some of the TDMRs are located in promoter regions, many functionally relevant binding sites for transcription factors probably exist in regions outside of gene promoters, particularly in introns. [21][22][23][24] We found evidence for this in our previous study, where we reported a positive association between increased methylation of the CpG island at the exon 2/intron 3 region and SF-1 expression in endometriotic tissue. 12 Intriguingly, hypermethylation of this exon/ intron region activates SF-1 mRNA expression in endometriotic cells; however, this region is distinct from the 5 0 promoter sequence, hypermethylation of which classically silences gene expression.…”

The role of long noncoding RNAs in cancer metastasis

“…Specifically, ERRLR01 is highly expressed in triple-negative breast cancers, yet not in samples derived from patients with ERα+ tumors. Follow up experiments indicated 17β-estradiol also altered the levels of ERRLR01 in ERα+ cells lines (i.e., MCF-7 and T47D) [25,153] . Given 17β-estradiol is a crucial regulator of EMT [28] , we surmise ERRLR01 and other lncRNAs are crucial mediators of the metastatic cascade.…”

In vitro and in vivo testing methods of epigenomic endpoints for evaluating endocrine disruptors

“…Recently, several hundreds of potential transcriptional coregulators that interact directly and indirectly with nuclear receptors have been identified (O'Malley, Qin et al, 2008;Kato and Fujiki, 2011), including fat-soluble ligands like vitamin A/D, steroid hormone receptors, and peroxisome proliferator-activated receptor gamma (PPARγ), which plays critical roles in metabolism and adipogenesis (Kato and Fujiki, 2011;Sugii and Evans, 2011). Genes that represent specific targets of estrogen receptor alpha have been identified using the chromatin immunoprecipitation approach (Jin et al, 2004;Lin et al, 2007), allowing insight into the downstream effectors of hormonal signaling.…”