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

Grober OM, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR, Ferraro L, Nassa G, Papa MF, Paris O, Tarallo R, Luo S, Schroth GP, Benes V, Weisz A - BMC Genomics (2011)

Bottom Line: 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.Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of General Pathology, Second University of Naples, vico L, De Crecchio 7, 80138 Napoli, Italy.

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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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.
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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.

Mentions: To identify among the genes differentially regulated by estrogen in ERβ+ vs ERβ- cells those representing direct targets for transcriptional regulation by DNA-bound ERβ in our cell model, we extracted from the list in Additional Table S1 [Additional file 3] the genes bearing one or more ERβ binding sites inside or within 10 kb of the TU, and termed them 'primary', to indicate that they are most likely to respond directly to the signal conveyed by the E2-activated receptor [26]. Of these 424 genes - listed in Additional Table S3 [Additional file 3], whose kinetics of response to E2 in wt and TAP-ERβ cells is shown in Figure 4, 52 show one ERβ site of Class 1 (ERβ vs ERα competition), 90 a site of Class 2 (ERβ+ERα), 71 a site of Class 3 (ERβ specific) and only 9 a Class 4 site (ERβ+ERα specific), while 202 showed multiple ERβ sites belonging to different classes and were thus classified accordingly (grey in Figure 4). In the right panels of Figure 4 are reported examples for each of the primary gene classes described above, showing the location of the receptor binding sites respect to the promoter and structural gene. It is worth mentioning that when the gene expression data from wt MCF-7 cells stimulated with E2 for 8hrs (Figure 2) were combined with information concerning ERα binding regions identified in wt MCF-7 cells under comparable conditions (Set 3 described above), 228 putative primary ERα target genes were identified -listed in Additional Table S4 [Additional file 3], 71% of which (163 genes) showed ERβ binding in hormone-stimulated ERβ+ cells. This result supports the view that the two ER subtypes tend to interact with the same targets in BC cells genome.


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

Grober OM, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR, Ferraro L, Nassa G, Papa MF, Paris O, Tarallo R, Luo S, Schroth GP, Benes V, Weisz A - BMC Genomics (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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.
Mentions: To identify among the genes differentially regulated by estrogen in ERβ+ vs ERβ- cells those representing direct targets for transcriptional regulation by DNA-bound ERβ in our cell model, we extracted from the list in Additional Table S1 [Additional file 3] the genes bearing one or more ERβ binding sites inside or within 10 kb of the TU, and termed them 'primary', to indicate that they are most likely to respond directly to the signal conveyed by the E2-activated receptor [26]. Of these 424 genes - listed in Additional Table S3 [Additional file 3], whose kinetics of response to E2 in wt and TAP-ERβ cells is shown in Figure 4, 52 show one ERβ site of Class 1 (ERβ vs ERα competition), 90 a site of Class 2 (ERβ+ERα), 71 a site of Class 3 (ERβ specific) and only 9 a Class 4 site (ERβ+ERα specific), while 202 showed multiple ERβ sites belonging to different classes and were thus classified accordingly (grey in Figure 4). In the right panels of Figure 4 are reported examples for each of the primary gene classes described above, showing the location of the receptor binding sites respect to the promoter and structural gene. It is worth mentioning that when the gene expression data from wt MCF-7 cells stimulated with E2 for 8hrs (Figure 2) were combined with information concerning ERα binding regions identified in wt MCF-7 cells under comparable conditions (Set 3 described above), 228 putative primary ERα target genes were identified -listed in Additional Table S4 [Additional file 3], 71% of which (163 genes) showed ERβ binding in hormone-stimulated ERβ+ cells. This result supports the view that the two ER subtypes tend to interact with the same targets in BC cells genome.

Bottom Line: 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.Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of General Pathology, Second University of Naples, vico L, De Crecchio 7, 80138 Napoli, Italy.

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.

Show MeSH
Related in: MedlinePlus