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Comparative Study
. 2010 Jan;24(1):47-59.
doi: 10.1210/me.2009-0252. Epub 2009 Nov 6.

Genome-wide dynamics of chromatin binding of estrogen receptors alpha and beta: mutual restriction and competitive site selection

Tze Howe Charn  1 , Edison Tak-Bun Liu, Edmund C Chang, Yew Kok Lee, John A Katzenellenbogen, Benita S Katzenellenbogen
Affiliations
Comparative Study

Genome-wide dynamics of chromatin binding of estrogen receptors alpha and beta: mutual restriction and competitive site selection

Tze Howe Charn et al. Mol Endocrinol. 2010 Jan.

Abstract

Estrogen receptors ERalpha and ERbeta, members of the nuclear receptor superfamily, exert profound effects on the gene expression and biological response programs of their target cells. Herein, we explore the dynamic interplay between these two receptors in their selection of chromatin binding sites when present separately or together in MCF-7 breast cancer cells. Treatment of cells (containing ERalpha only, ERbeta only, or ERalpha and ERbeta) with estradiol or ER subtype-selective ligands was followed by chromatin immunoprecipitation analysis with a custom-designed tiling array for ER binding sites across the genome to examine the effects of ligand-occupied and unoccupied ERalpha and ERbeta on chromatin binding. There was substantial overlap in binding sites for these estradiol-liganded nuclear receptors when present alone, but many fewer sites were shared when both ERs were present. Each ER restricted the binding site occupancy of the other, with ERalpha generally being dominant. Binding sites of both receptors were highly enriched in estrogen response element motifs, but when both ERs were present, ERalpha displaced ERbeta, shifting it into new sites less enriched in estrogen response elements. Binding regions of the two ERs also showed differences in their enrichments for other transcription factor binding motifs. Studies with ER subtype-specific ligands revealed that it was the liganded subtype that principally determined the spectrum of chromatin binding. These findings highlight the dynamic interplay between the two ERs in their selection of chromatin binding sites, with competition, restriction, and site shifting having important implications for the regulation of gene expression by these two nuclear receptors.

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Figures

Figure 1
Figure 1
Generation of MCF-7 cells containing different complements of ERα and ERβ for ChIP-chip studies. A, MCF-7 cells were infected with control β-galactosidase-expressing adenovirus or ERβ-expressing adenovirus to generate cells containing ERα-only and ERα plus ERβ, respectively. Cells containing ERβ only were generated by knockdown of ERα by siRNA transfection of cells containing ERα plus ERβ. B, Schematic diagram showing location of tiled probes in the custom-designed tiling arrays. Each probe is 60 bp in length, and probes are tiled approximately 100 bp from each other. RNAi, RNA interference.
Figure 2
Figure 2
Effect of ER subtype partner on ER binding site distribution with E2 treatment. A, The introduction of ERβ into the cells has a relatively minor effect on the distribution of ERα binding sites. B, ERα has a more pronounced effect on the distribution of ERβ binding sites.
Figure 3
Figure 3
Venn diagrams comparing the occupancy of ER binding sites by ERα and ERβ when they are present either separately or together in cells treated with E2. A, ERα or ERβ can each occupy many of the same sites when the other ER subtype is not present in the cells. B, When both receptors are present, ERα and ERβ share a more limited number of sites. C, Diagram showing the intersection of the intersections from A (sites in common) and B (shared sites) and how sites in common that are not shared are allocated predominantly to ERα.
Figure 4
Figure 4
Presence of ERE sequences in ERα or ERβ binding sites with E2 treatment. Binding sites were probed for the presence of full ERE, half ERE, and no ERE motifs. A, ERα binding sites in ERα-only cells; B, ERβ binding sites in ERβ-only cells; C, ERα binding sites in cells containing both ERα and ERβ; D, ERβ binding sites in cells containing both ERα and ERβ.
Figure 5
Figure 5
Analysis of enrichment of TFBSs. A, Transcription factor binding motifs that are enriched in ERα (α-cell) binding sites; B, transcription factor binding motifs that are enriched in ERα (αβ-cell) binding sites; C, transcription factor binding motifs that are enriched in ERβ (β-cell) binding sites; D, transcription factor binding motifs that are enriched in ERβ (αβ-cell) binding sites.
Figure 6
Figure 6
Correlation between ER binding and transcriptional output in response to E2. A, Correlation between E2-regulated genes and binding of ERα-unique (only ERα binds), ERβ-unique (only ERβ binds), or ERα/ERβ sites (sites shared by both ERs) within ±50 kb of the transcription start site of the genes. B, FOS mRNA levels were assessed by quantitative PCR after 4 h treatment of MCF-7 cells differentially expressing ERα and/or ERβ. Data represent average fold change ± sd for three independent experiments. C, ERα and ERβ chromatin binding (by conventional ChIP assays) were measured by quantitative PCR after 45 min E2 treatment of MCF-7 cells expressing ERα and/or ERβ. ERα and ERβ occupancy of three different ER binding sites (FOS enhancer 1, FOS enhancer 2, and FOS 3′ region) that are closest to the FOS gene are presented graphically. Enh, Enhancer.
Figure 7
Figure 7
Venn diagrams comparing ER binding site occupancy after cell treatment with the ERα-selective ligand (PPT) or ERβ-selective ligand (ERB-041) vs. E2. A, ERα binding sites in ERα-only cells (E2 vs. PPT treatment); B, ERα binding sites in cells containing ERα only or both ERα and ERβ (PPT treatment); C, ERβ binding sites in ERβ-only cells (E2 vs. ERB-041 treatment); D, ERβ binding sites in cells containing ERβ only or both ERα and ERβ (ERB-041 treatment).
Figure 8
Figure 8
Venn diagrams showing the overlap of ERα binding sites (PPT treatment) and ERβ binding sites (ERB-041 treatment). A, ERβ binding sites in ERβ-only cells (ERB-041 treatment) and ERα binding sites in ERα-only cells (PPT treatment); B, ERβ and ERα binding sites in cells containing both ERα and ERβ (ERB-041 vs. PPT treatment).
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