Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation

Abstract

BackgroundEstrogen receptors alpha (ERα) and beta (ERβ) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology.ResultsExpression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions.ConclusionsResults indicate that the vast majority of the genomic targets of ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer

“…Gene-expression profiling of asynchronously growing cells showed no major differences between N-and C-TAP-ERb cells, whereas their transcriptomes were significantly different from that of C-TAP-ERa cells (Supplementary Figure S1), confirming previous results obtained in E2-stimulated cells (Grober et al, 2011). Based on these results, expression of the TAP-ERb fusion proteins appears to significantly affect ERa-mediated estrogen signal transduction to target genes and the cell cycle, confirming previous observations indicating that they are fully functional in vivo (Grober et al, 2011;Nassa et al, 2011).…”

“…Global analysis of miRNome expression was peformed with microarrays detecting the vast majority of known and characterized miRNAs (Illumina MicroRNA Expression Beadchip, Illumina Italia, Milano, Italy) as described in Material and methods. Results indicates that expression of ERb has a deep impact on BC cell miRNome, as 84 miRNAs were found differentially expressed in three ERb þ vs two ERbÀ cell lines, whereas no significant differences could be detected among cells expressing the different tagged forms of ERb, or between C-TAP-ERa, wt and MCF7-TAP cells (not shown), that express only the TAP peptide and show no differences in ERa signaling with respect to wt cells (Ambrosino et al, 2010;Grober et al, 2011). To validate this result, we performed miRNA expression profiling with a different microarray platform (Agilent Human microRNA Microarrays 18 Â 15 K v3, Agilent Technologies Italia, Milano, Italy) and compared the results obtained in the two experimental settings.…”

“…We analyzed by chromatin immunoprecipitation sequencing (ChIPSeq) the entire ERa and ERb cistromes in the ERb þ (Grober et al, 2011) and ERbÀ cells (Cicatiello et al, 2010) upon E2 stimulation. Aligning ER-binding sites and miRNA gene positioning in the genome we observed that several miRNA-encoding genes differentially expressed in ERb þ vs ERbÀ cell lines (Supplementary Table S3A) and/or mammary tumors Table S3B) display ER-binding sites within 10 kb of the transcription unit, including sites where both ERs can be found together, likely associated in heterodimers.…”

“…ERs are members of the nuclear receptors superfamily of ligand-dependent transcription factors that both regulate gene expression controlling the estrogen signal transduction cascade with distinct and even antagonistic roles. In hormoneresponsive, ERa-positive BC cells ERb inhibits estrogen-mediated cell proliferation by increasing the expression of growth-inhibitory genes and by interfering with activation of cell cycle and anti-apoptotic genes by ERa in response to 17b-estradiol (E2: Chang et al, 2006;Grober et al, 2011). ERb is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease (Sugiura et al, 2007), is a biomarker of a less aggressive clinical phenotype (Novelli et al, 2008;Shaaban et al, 2008) and its downregulation has been postulated to represent a critical stage in estrogen-dependent tumor progression (Roger et al, 2001;Bardin et al, 2004).…”

“…More recently, global mapping of ERb binding to ERa-positive, hormone-responsive BC cells chromatin in vivo showed ERb interaction with several miRNA genes, suggesting the possible involvement of this receptor in hormonal control of small noncoding RNA biogenesis in this cell type (Grober et al, 2011). Starting from this observation, we investigated here miRNA expression pattern in estrogen-responsive BC cell lines engineered to express full-length ERb and in primary-tumor samples selected according to the presence or absence of this nuclear receptor.…”