ERβ Suppresses Estradiol-Stimulated Proliferation and Anti-Apoptotic Activity in ERα-Positive Breast Cancer Cells
It is well documented that estrogens stimulate cell proliferation in ERα-positive breast cancer cells . To understand the role of ERβ in regulating breast cancer cell growth, we used adenovirus-mediated gene delivery of ERβ into MCF-7 cells, and we validated ERβ expression at the protein level by western blot (Fig. 1a). As shown in Fig. 1b, the copresence of ERβ in ERα breast cancer cells greatly reduced cell proliferation stimulated by E2 (Fig. 1b). We also assessed the ability of cells to form colonies in soft agar. ERα-containing cells generated a large number of colonies with E2 treatment, and the copresence of ERβ markedly reduced the number of colonies formed (Fig. 1c).
Prior studies have suggested that ERα suppresses p53-dependent apoptosis in breast cancer . To assess the effect of ERβ on cell apoptosis, we performed flow cytometry analyses. As shown in Fig. 1d, E2 reduced apoptosis in cells containing ERα, as expected, but most notable was that expression of ERβ greatly increased the percent of cells undergoing apoptosis (from 4 to 16%) and E2 no longer affected this high level of cell apoptosis (Fig. 1d).
ERβ Antagonizes ERα-Mediated Transcriptional Repression
To investigate the molecular mechanisms involved in the ERβ-mediated anti-proliferative and pro-apoptotic effects in ERα-positive breast cancer cells, we performed RNA-seq to profile the alterations of gene expression in ERα cells and ERα+ERβ cells in response to E2 treatment . We analyzed the transcriptome between the E2-treated and control vehicle samples in ERα cells (ERα cells with E2 treatment vs ERα cells with Veh treatment, fold change (FC) ≥ 2) and identified the genes significantly up- or down-regulated by E2: 926 genes were up-regulated and 1288 genes were down-regulated (Fig. 2a). Within the set of estrogen-regulated genes, we also compared the expression of genes that were significantly up- or down-modulated by ERβ (ERα+ERβ cells with E2 treatment vs ERα cells with E2 treatment, FC ≥ 2) and found that 125 (13%) of these ERα up-regulated genes were down-regulated by ERβ and 674 (52%) of these ERα down-regulated genes were up-regulated by ERβ (Fig. 2a). In essence, the suppression of more than half of the ERα-repressed genes was reversed in the presence of ERβ.
To determine whether the 674 genes repressed by ERα but activated by ERβ were directly influenced by ERα and ERβ, we compared ERα and ERβ genomic occupancy with the 674 genes in MCF-7 cells. By chromatin immunoprecipitation followed by sequencing (ChIP-Seq) data analysis , we found that there were 289 genes with ERα binding sites in ERα cells and 221 genes with ERβ binding sites in ERα+ERβ cells within 50 kb of the transcription start site (TSS). Of these genes, 172 had both ERα and ERβ binding sites (Fig. 2b). Gene ontology (GO) analysis revealed that these 172 genes were mainly associated with secretion, cell adhesion, and cell signaling (Fig. 2c).
To explore the clinical relevance of these 172 genes in patients with breast cancer, we used the Oncomine database to analyze the expression of these genes in three large clinical datasets and observed that high expression of these genes was significantly associated with better prognosis in breast cancer patients. In these clinical studies [32,33,34], patients with tumors having a high level of expression of these genes had better overall survival than those with tumors expressing low levels of these genes (Fig. 2d, e).
ERβ Physically Interacts with p53 and Regulates a Set of Common Genes
The transcription factor p53 is crucial in regulating the cell cycle, apoptosis, senescence, and genome stability. Because we found that many of the ERβ-regulated genes were also classical p53 target genes, we wondered whether ERβ might be interacting with p53. By coimmunoprecipitation analyses of extracts from MCF-7 cells coexpressing ERβ, we found that ERβ physically interacted with p53 (Fig. 3a). Interactions between ERβ and endogenous p53 in these extracts could also be detected in reciprocal coimmunoprecipitations.
Nutlin-3a is a small molecule that activates the p53 pathway by disrupting the p53-MDM2 interaction. Activation of p53 by nutlin-3a induces apoptosis, cell cycle arrest, and growth suppression in cancer cells . We next compared the expression of genes regulated by ERβ  and compared them with the genes stimulated by Nutlin-3a treatment of MCF-7 cells . By analyzing RNA-Seq data, we found that 5212 genes were significantly up- or down- regulated by ERβ (ERα+ERβ cells with E2 treatment vs ERα cells with E2 treatment, FC ≥ 2), and 1702 genes were significantly up- or down-regulated after Nutlin-3a treatment (Nutlin-3a vs Veh, FC ≥ 2). Among these genes, 782 were commonly regulated by both ERβ and p53 (Fig. 3b). Hierarchical clustering of the set of genes commonly regulated by both ERβ and p53 revealed two major clusters: genes that were commonly up-regulated by ERβ and p53 (318 genes) and genes that were commonly down-regulated by ERβ and p53 (377 genes) (Fig. 3c). Within the common set of 782 genes regulated by both ERβ and p53, about 84% of the ERβ up-regulated genes were also up-regulated by p53, and almost 93% of the ERβ down-regulated genes were also down-regulated by p53, so there is nearly a complete overlap of ERβ- and p53-regulated genes in these cells.
GO analysis revealed that the genes commonly up-regulated by ERβ and p53 were mainly associated with cell death and apoptosis, and the genes that were commonly down-regulated by ERβ and p53 were primarily associated with cell cycle processes (Fig. 3d). Overall, this analysis revealed an activating role for ERβ and p53 in the control of genes regulating cell death and apoptosis, and a repressive role in the processes associated with cell cycle and mitosis. In essence, ERβ can replicate many of the gene regulatory effects of activated p53, and like p53 can reverse many of the gene actions of ERα that drive proliferation of breast cancer cells.
To determine whether the genes commonly regulated by both ERβ and p53 were directly influenced by ERβ and p53, we analyzed ChIP-Seq data (10,36) and compared the ERβ and p53 genomic occupancy with the co-up and co-down regulated genes in MCF-7 cells. Among the 318 genes commonly up-regulated by both ERβ and p53, there were 157 genes with ERβ binding sites and 172 genes with p53 binding sites within 50 kb of the TSS (Fig. 3e), and of these, 104 had both ERβ and p53 binding sites. Among the 377 down-regulated genes, there were 172 genes with ERβ binding sites, 188 genes with p53 binding sites, and 108 with both ERβ and p53 binding sites (Fig. 3e).
ERβ Activates ERα-Repressed Genes in a p53-Dependent Manner
The fact that ERβ physically interacts with p53 and regulates a set of common genes with p53 raised the possibility that the activation by ERβ of ERα-repressed genes might be dependent on p53. To determine whether ERβ-mediated transcriptional activation of ERα-repressed genes is p53-dependent, we examined the expression of these genes in the ERα+ERβ cells depleted of p53 by transfection with p53 siRNA. Endogenous p53 knockdown was confirmed at the messenger RNA (mRNA) and protein levels by RT-PCR and Western blot immunoassay (Fig. S1, panels A and B). As shown in Fig. 4a, p53 depletion severely impaired the ERβ activation of ERα-repressed genes. The mRNA levels of p21, BTG2, MDM2, and DR5 were significantly decreased in ERα+ERβ cells transfected with p53 siRNA. We also examined the ERβ-mediated transcriptional activation in the p53-null breast cancer cell line, MDA-MB-157 (MDA-MB-157 p53
−/− line) (Fig. S1, panels C and D). In these MDA-MB-157 cells in which we expressed ERβ using adenovirus-mediated gene delivery, little or no increase of these repressed genes was observed (Fig. 4b). These results suggest that p53 is required for ERβ-induced transcriptional activation of ERα-repressed genes.
To assess whether ERβ affects the expression of p53 and then regulates the p53 pathway, we examined the mRNA and protein levels of p53 in ERα cells and ERα+ERβ cells. As shown in Fig. 4c, the presence of ERβ did not change the level of p53 mRNA or protein. However, treatment of ERα+ERβ cells with the p53 pathway activator Nutlin-3a enhanced the activation of ERα-p53-repressed genes (Fig. 4d). The mRNA levels of p21, BTG2, MDM2, and DR5 were all significantly elevated in ERα+ERβ cells treated with Nutlin-3a (Fig. 4d). Thus, the effect that ERβ has on ERα-repressed genes could be enhanced by active p53.
ERβ Competes with ERα for p53-Mediated Transcriptional Activation
Prior studies have shown that ERα suppresses p53-mediated transcriptional activation and p53-dependent apoptosis in breast cancer [17, 18]. Given that we found that ERβ antagonizes ERα-mediated transcriptional repression and ERβ also can form a complex with p53, we investigated whether ERβ has an effect on p53-mediated transcriptional activation. To test this, we examined the effect of ERβ on the transcription of p53 direct target genes, i.e., the proliferation associated genes p21, MDM2, BTG2, ATF3, and GDF15, and the apoptosis associated genes DR5, Bax1, and TRAF4. Consistent with previous studies, ERα inhibited p53 transcriptional activities, and we observed the mRNA level of p53 target genes to be decreased in MCF-7 cells upon E2 treatment (Fig. 5a). Moreover, this E2-mediated repression was ERα dependent, as siRNA knockdown of ERα resulted in an increase in the RNA level of p53 target genes (Fig. S2). By contrast, ERβ significantly increased the mRNA levels of p53 target genes in the MCF-7 cells (Fig. 5a). These observations suggest that ERα represses p53-mediated transcriptional activities, whereas ERβ activates p53-mediated transcriptional activities.
To assess the interaction of p53 with ERα and ERβ, we performed coimmunoprecipitation experiments using p53 antibody followed by immunoblotting with ERα and ERβ antibodies. Coimmunoprecipitation experiments revealed an interaction of p53-ERα and p53-ERβ (Fig. 5b). In ERα cells, p53 coimmunoprecipitated with ERα, and the presence of ERβ reduced the interaction between p53 and ERα (anti-p53 IP ERα immunoblot lanes 3 and 4 are less intense than lanes 1 and 2). Furthermore, the interaction between p53 and ERβ was observed in cells containing both ERα and ERβ (ERβ immunoblot lanes 3 and 4 are apparent) (Fig. 5b).
To confirm that ERβ competes preferentially with ERα for p53-mediated gene transcriptional regulation, we mapped the genomic localization of ERα and ERβ using ChIP-Seq  and identified the occupancy of ERα and ERβ on the p53 target gene, p21. In ERα cells, ERα was recruited to the p21 gene locus, and with the copresence of ERβ, ERβ abrogated the recruitment of ERα to the p21 gene locus. Instead, ERβ was recruited to the same binding sites on the p21 gene (Fig. 5c). We also performed chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR), and in agreement with the ChIP-Seq analysis, the ChIP-qPCR results also showed that ERα was recruited to the p21 gene promoter locus in ERα cells and that the presence of ERβ suppressed the recruitment of ERα and increased recruitment of ERβ to the p21 gene locus (Fig. 5d). These results further confirm that ERβ competes with ERα for occupancy of this estrogen-repressed gene and that these receptors have opposite effects on p53-mediated transcriptional activity.
ERβ Abrogates H3K9me3-Mediated Heterochromatin Silencing of ERα-Repressed Genes
Chromatin conformation plays a critical role in regulating gene transcription and gene silencing. Previous studies have shown that the H3K9me3 repressive histone conformation is enriched on the promoters of both p53 anti-proliferation and pro-apoptotic targets . To investigate whether ERβ affects chromatin-modifying enzymes that could alter chromatin accessibility and allow ERα-repressed genes to become activated in the presence of ERβ, we examined the expression of histone modification enzymes and identified two histone methyltransferases, SUV39H1 and SUV39H2, which specifically tri-methylate histone H3 at lys 9 (H3K9me3). Notably, RNA-Seq data analysis and qPCR results both showed that estradiol activated the transcription of SUV39H1 and SUV39H2 in MCF-7 cells, whereas ERβ repressed the expression of these two genes in control vehicle and in E2-treated cells (Fig. 6a).
SUV39H1 and SUV39H2 are histone code writers responsible for establishing and maintaining the H3K9me3 heterochromatin mark . Previous studies have shown that downregulating SUV39H1 alone was sufficient to overcome the H3K9me3 repressive chromatin conformation barrier . As we observed that the presence of ERβ down-regulates the expression of SUV39H1/H2 and abrogates estradiol-induced transcriptional repression, we investigated the effect of ERβ on the H3K9me3 repressive heterochromatin mark. We examined how alterations in H3K9me3 marks affect the expression of estrogen-repressed genes by analyzing the relative enrichment of H3K9me3 on these genes. Consistent with increased expression of SUV39H1 and SUV39H2 in ERα cells treated with E2, ChIP analysis with anti-H3K9me3 antibody showed that H3K9me3 was significantly increased in ERα cells treated with E2, and that ERβ abrogated the H3K9me3 increase on this ERα-repressed p21 gene (Fig. 6b). These results demonstrate that E2-ERα activates the transcription of SUV39H1 and SUV39H2 and maintains higher levels of the H3K9me3 heterochromatin mark on estrogen-repressed gene loci. However, when ERβ was present, it represses the transcription of SUV39H1 and SUV39H2, thus removing the H3K9me3-repressive chromatin conformation barrier for this ERα-repressed gene.
N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) are nuclear receptor co-repressors, both of which are recruited to chromatin by nuclear hormone receptors to regulate gene transcription [18, 39, 40]. Given that ERα was reported to recruit N-CoR and SMRT to repress p53-mediated transcriptional activation , we were interested in exploring whether the copresence of ERβ affected the recruitment of nuclear receptor transcriptional repressive complexes on estrogen-repressed genes. We analyzed the occupancy of N-CoR and SMRT on the p21 gene in ERα cells and ERα+ERβ cells. ChIP experiments documented that ERβ markedly decreased occupancy of N-CoR and SMRT on the p21 gene in the presence and absence of E2 (Fig. 6 b). ERβ also eliminated the increased recruitment of H3K9me3 to p21 by E2 and ERα (Fig. 6b).
ERβ Potentiates H3K4me3-Mediated Epigenetic Activation of Estrogen-Repressed Genes
Chromatin remodeling could modulate transcription by blocking or facilitating transcription factor access to DNA. H3K4me3 is a well-known marker that is correlated with gene activation, and previous studies showed that H3K4me3 enhances p53-dependent transcription . Given that we observed that ERβ could remove the H3K9me3 repressive heterochromatin barrier for estrogen-repressed genes and increase the levels of histone active H3K4me3 marks, we analyzed the enrichment of H3K4me3 on the ERα-repressed genes in ERα cells and in ERα+ERβ cells. ChIP analysis with anti-H3K4me3 antibody showed that the occupancy of H3K4me3 on the ERα-repressed gene p21 was markedly increased in MCF-7 cells in the copresence of ERβ (Fig. 6c). We also investigated the recruitment of RNA Pol II on ERα-repressed genes in ERα and in ERα+ERβ cells. ChIP analysis using anti-RNA Pol II showed that the accumulation of RNA Pol II at the p21 gene was greatly increased (by fivefold) in the ERα+ERβ cells (Fig. 6c), supporting that increased RNA Pol II recruitment likely contributes to the activation of ERα-repressed genes in cells containing ERβ.