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. 2008 May;22(5):1032-43.
doi: 10.1210/me.2007-0356. Epub 2008 Feb 7.

Estrogen Receptors alpha and beta as determinants of gene expression: influence of ligand, dose, and chromatin binding

Edmund C Chang  1 , Tze Howe Charn, Sung-Hee Park, William G Helferich, Barry Komm, John A Katzenellenbogen, Benita S Katzenellenbogen
Affiliations

Estrogen Receptors alpha and beta as determinants of gene expression: influence of ligand, dose, and chromatin binding

Edmund C Chang et al. Mol Endocrinol. 2008 May.

Abstract

Estrogen receptors alpha and beta (ERalpha and ERbeta) mediate the actions of estrogens in a variety of normal and cancer target cells. Estrogens differ in their preference for these ERs, and many phytoestrogens bind preferentially to ERbeta. To investigate how phytoestrogens such as genistein impact ER-regulated gene expression, we used adenoviral gene delivery of ERbeta coupled with ERalpha depletion with small interfering RNA to generate human breast cancer (MCF-7) cells expressing four complements of ERalpha and ERbeta. We examined the dose-dependent effects of genistein on genome-wide gene expression by DNA microarrays and monitored the recruitment of ERs and coregulators to responsive regions of estrogen-regulated genes. At a low (6 nm) concentration, genistein regulated gene expression much more effectively in cells coexpressing ERalpha and ERbeta than in cells expressing ERalpha alone, whereas at high concentration (300 nm), genistein induced transcriptome changes very similar to that of 17beta-estradiol. We demonstrate that ERbeta is preferentially activated by genistein and is recruited to estrogen-responsive genomic sites and that differential occupancy of ERalpha and ERbeta by genistein and 17beta-estradiol in turn influences the recruitment patterns of coregulators such as steroid receptor coactivator 3 (SRC3) and receptor-interacting protein 140 (RIP140). Our observations indicate that genistein is a potency-selective ligand for gene expression regulation by ERalpha and ERbeta and that the ability of ERalpha and ERbeta to serve as determinants of gene expression is greatly influenced by the nature of the ligand, by ligand dose, and by the differential abilities of ligand-ER complexes to recruit different coregulators at ER binding sites of hormone-regulated genes.

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Figures

Figure 1
Figure 1
Gene Clustering Analysis with TightCluster Program Reveals Time-Dependent Regulation and ERβ-Mediated Modulation of Estrogen-Responsive Genes A, Heat map of average profiles (log base 2) of 32 gene clusters is shown, with n denoting the total gene membership within each cluster. V, 0.1% ethanol vehicle; E2, 6 nm E2; LG, 6 nm genistein; HG, 300 nm genistein; IG, HG plus 1 μm antiestrogen ICI 182,780. B, Average gene expression profiles (in log base 2) of select clusters are shown, with n denoting the total gene membership within each cluster. Samples are grouped by treatment time (4 and 24 h) and ordered by ligand treatment: ERα (♦) and ERα+ERβ (○). Clusters enriched for genes involved in cell cycle progression (cluster A1), regulation of transcription (cluster A4), negative regulation of cell proliferation (cluster B4), and GPCR signaling pathway (cluster B9) are shown. C, Proliferation of MCF-7 cells expressing either ERα alone or coexpressing ERα and ERβ in response to treatment with E2, LG, or HG for 6 d. Cells were seeded at a density of 1000 cells per well and monitored for cell number in quadruplicate samples at d 6.
Figure 2
Figure 2
Statistical Analysis of Biological Processes Enriched within Gene Clusters Gene ontology (GO) biological functional categories significantly enriched (EASE score < 0.05) for 32 gene clusters are mapped and arranged by EASE score. Darker shading denotes more highly significant EASE scores (ranging from 10−12 to 0.05), whereas blank entries denote that the GO term did not reach significance for the gene cluster. Major biological themes present in MCF-7 transcriptomes include transcriptional regulation, mitosis and cell cycle genes, RNA and protein processing, ion homeostasis, and cell adhesion/cytoskeletal rearrangement.
Figure 3
Figure 3
At High Concentration, Genistein Elicits Gene Regulation Very Similar to that by E2 Venn diagrams comparing probe sets regulated by E2 (6 nm) or HG (300 nm) in MCF-7 cells expressing ERα alone or ERα+ERβ. A, Genes regulated at 4 h; B, genes regulated at 24 h; C, volcano plots (log odds vs. fold change) of HG-treated vs. E2-treated samples at 4 h in ERα-only cells (panel 1) or ERα+ERβ cells (panel 2) or at 24 h in ERα-only cells (panel 3) or ERα+ERβ cells (panel 4).
Figure 4
Figure 4
ERβ Coexpression Enhances Gene Regulation by LG (6 nm) A, Venn diagrams comparing probe sets regulated by E2 (6 nm), LG (6 nm) or HG (300 nm) in MCF-7 cells expressing ERα alone or ERα+ERβ; B, Heat map depicting 24-h gene expression of 1062 probe sets regulated by control vehicle (V), E2, or LG in MCF-7 cells expressing ERα only or ERα+ERβ.
Figure 5
Figure 5
Regulation of Gene Expression by E2 and Genistein in Cells Differentially Expressing ERα and ERβ A, Transcript levels of estrogen-regulated genes (FOS, TFF1, and CASP7) were assessed by quantitative PCR after 4-h treatment of MCF-7 cells differentially expressing ERα and ERβ. To generate cells with ERα+ERβ or ERβ only vs. ERα only (parental MCF-7 cells), MCF-7 cells were infected with AdGal (control) or AdERβ for 24 h and subsequently transfected with either siCNTRL or siERα for an additional 48 h. Cells were then exposed to control vehicle (V), 6 nm E2, or 6 nm genistein (LG) for 4 h. Data represent average fold change ± sd of triplicate samples and is representative of three separate experiments.
Figure 6
Figure 6
Recruitment of ERα and ERβ to ER Binding Sites Near Estrogen-Responsive Genes in Response to E2 and Genistein Changes in chromatin binding by estrogen receptor-containing complexes were measured by quantitative PCR after 45 min ligand treatment with vehicle (V), E2, LG, or HG in MCF-7 cells differentially expressing ERα and/or ERβ. ChIP experiments were performed with ERα- and ERβ-specific antibodies. Genomic locations of estrogen receptor binding sites were from work by Carroll et al. (32) (UCSC Genome Browser). Data are expressed as percentage of input and is representative of three separate experiments.
Figure 7
Figure 7
Assessment of the Recruitment of SRC3 and RIP140 by ERα- and ERβ-Containing Complexes in Response to Treatment with E2 or Genistein Chromatin from cells differentially expressing ERα and/or ERβ was immunoprecipitated using SRC3 antibody or RIP140 antibody and quantified using real-time PCR at ER binding regions: A, The −20-kb enhancer of FOS; B, proximal promoter (−300 bp) of TFF1; C, enhancer region (+190 kb) of CASP7. Data are expressed as percentage of input and is representative of three separate experiments.
Figure 8
Figure 8
Regulation of Gene Expression by E2 and Genistein in Cells Differentially Expressing ERα and ERβ without and with SRC3 and RIP140 Targeted Knockdown siRNA to SRC3 or RIP140 was transfected into hormone-depleted MCF-7 cells differentially expressing ERα and ERβ, and the transcript level of estrogen-regulated genes (FOS, TFF1, and CASP7) was assessed after 4 h of vehicle or ligand treatment. MCF-7 cells were infected with AdGal (control) or AdERβ for 24 h and subsequently transfected with either siSRC3 or siRIP140 for an additional 48 h. Cells were then exposed to control vehicle (V), 6 nm E2 (E), or 6 nm genistein (LG) for 4 h. Data are the average of two determinations.
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