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. 2011 Jan 14:12:36.
doi: 10.1186/1471-2164-12-36.

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

Oli M V Grober  1 , Margherita Mutarelli, Giorgio Giurato, Maria Ravo, Luigi Cicatiello, Maria Rosaria De Filippo, Lorenzo Ferraro, Giovanni Nassa, Maria Francesca Papa, Ornella Paris, Roberta Tarallo, Shujun Luo, Gary P Schroth, Vladimir Benes, Alessandro Weisz
Affiliations

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

Oli M V Grober et al. BMC Genomics. .

Abstract

Background: Estrogen 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.

Results: Expression 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.

Conclusions: Results 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.

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Figures

Figure 1
Figure 1
Functional characterization of ERβ-expressing MCF-7 cells. (A) Nuclear translocation of ERα and ERβ shown by western blot analysis on cytosolic (c) and nuclear (n) protein extracts, prepared from wt MCF-7, Nt-ERβ and Ct-ERβ cells after treatment with either 17β-estradiol (10-8M, +E2) or vehicle alone (-E2) for 45 minutes. The amount of α-tubulin was also analyzed to verify the absence of cytosolic contaminants in the nuclear fractions. (B) The transcriptional activity of ERα, Nt-ERβ and Ct-ERβ was measured by transient transfection in SKBR3 cells (left) and the ability of tagged ERβ to interfere with ERα activity was assessed by comparing estrogen effects in wt, in Nt-ERβ and Ct-ERβ MCF-7 cells (right); in all cases transiently transfected ERE-tk-luc was used as reporter gene. (C) Proliferation of wt MCF-7, Nt-ERβ and Ct-ERβ cells was measured by stimulating hormone-starved cells with 10-8M E2, followed by cell counting with a colorimetric assay at the indicated times.
Figure 2
Figure 2
Gene expression differences in absence or presence of ERβ. Top: Heatmap summarizing the effects of ERβ expression of the estrogen responsive transcriptome of MCF-7 cells, showing changes in expression (log2 of the fold-change) of 921 transcripts after cell exposure to 10-8M E2 for the indicated times. Transcripts are grouped as follows: regulated only in wt MCF-7 cells (1), in both cell lines (2) and only in TAP-ERβ cells (3). Bottom: Venn diagram showing the numbers of differentially regulated by E2 in wt MCF-7 only (1), both cell lines (2) or ERβ expressing cells only (3).
Figure 3
Figure 3
Sequence analysis of ERα, ERβ and ERα+ERβ binding sites. (A) Venn diagram showing a summary of ERα and ERβ binding sites identified in TAP-ERβ cells by ChIP-Seq. (B) Classification of ERα and β binding sites based on the presence of a perfectly or imperfectly palindromic Estrogen Response Element (ERE, green), an ERE hemipalindrome (hERE, blue) or no ERE (none, red). (C) ERE motif matrices identified in each of the three ER binding regions indicated (left), classification of the binding sites belonging to each region according to the presence of ERE (center) and grid summarizing the results of TFBS matrix enrichment (overrepresentation) analyses performed on the binding sites groups indicated (right). Z-Score cut-off was 3.0 and only TFBSs showing an over-representation score ≥4.0 in at least one of the ERE- (none) binding site groups. Light grey cells indicate a Z-Score <3.0 while dark grey cells indicate absence of the matrix.
Figure 4
Figure 4
Putative ERβ primary targets. Left: Heatmap summarizing the effects of estrogen stimulation on 424 mRNAs encoded by genes showing one (left) or more (right) ERβ binding sites within 10 kb of the TU (primary response genes) transcriptome of MCF-7 cells, as changes in expression (log2 of the fold-change) after cell exposure to 10-8M E2 for the indicated times. Transcripts are grouped as follows: regulated only in wt MCF-7 cells (1), in both cell lines (2) and only in TAP-ERβ cells (3). Vertical bars to the right of each heatmap indicate the class of ERβ binding site present, as indicated in the legend. When a regulated gene showed multiple ERβ binding sites belonging to different classes it was included in a separate group, classified as 'combination of ERβ sites' (grey bar). Right: Genome Browser view of genomic loci representative of the different ERα and ERβ binding site categories identified. ChIP-Seq ERα and ChIP-Seq ERβ indicate sites identified in this study, ChIP-Seq ERα1, ChIP-Seq ERα2 and ChIP-on-chip indicates sites identified in MCF-7 cells by Cicatiello et al. [26], Fullwood et al. [27], and Hurtado et al. [28], respectively.
Figure 5
Figure 5
Mitochondrial ER-beta binding sites. (A) Genome Browser view of the ERβ binding site identified in mitochondrial genome. (B) Validation of ERβ binding to mitochondrial DNA by ChIP. Results shown are representative of duplicate analyses. E2: 10-8M 17β-estradiol; PPT: 10-8M 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (selective ERα agonist); DPN: 10-8M 2,3-bis(4-hydroxyphenyl) propionitrile (ERβ agonist). (C) Western blot analysis of ERβ and/or ERα in purified mitochondria from Ct-ERβ and Ct-ERα [33] cells. Cyt: cytosol depleted of mitochondria, H: whole cell homogenate; Mito: purified mitochondrial fraction. ATPase is a mitochondrial marker and α-tubulin was included to determine the level cytosolic contaminants in 'Mito' samples.
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