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. 2018 Jun 20;46(11):5487-5503.
doi: 10.1093/nar/gky260.

Widespread enhancer activation via ERα mediates estrogen response in vivo during uterine development

Wendy N Jefferson  1 , H Karimi Kinyamu  2 , Tianyuan Wang  3 , Adam X Miranda  1 , Elizabeth Padilla-Banks  1 , Alisa A Suen  1 , Carmen J Williams  1
Affiliations

Widespread enhancer activation via ERα mediates estrogen response in vivo during uterine development

Wendy N Jefferson et al. Nucleic Acids Res. .

Abstract

Little is known regarding how steroid hormone exposures impact the epigenetic landscape in a living organism. Here, we took a global approach to understanding how exposure to the estrogenic chemical, diethylstilbestrol (DES), affects the neonatal mouse uterine epigenome. Integration of RNA- and ChIP-sequencing data demonstrated that ∼80% of DES-altered genes had higher H3K4me1/H3K27ac signal in close proximity. Active enhancers, of which ∼3% were super-enhancers, had a high density of estrogen receptor alpha (ERα) binding sites and were correlated with alterations in nearby gene expression. Conditional uterine deletion of ERα, but not the pioneer transcription factors FOXA2 or FOXO1, prevented the majority of DES-mediated changes in gene expression and H3K27ac signal at target enhancers. An ERα dependent super-enhancer was located at the Padi gene locus and a topological connection to the Padi1 TSS was documented using 3C-PCR. Chromosome looping at this site was independent of ERα and DES exposure, indicating that the interaction is established prior to ligand signaling. However, enrichment of H3K27ac and transcriptional activation at this locus was both DES and ERα-dependent. These data suggest that DES alters uterine development and consequently adult reproductive function by modifying the enhancer landscape at ERα binding sites near estrogen-regulated genes.

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Figures

Figure 1.
Figure 1.
Genomic distribution of histone H3K27me3, H3K4me3 and H3K27ac following neonatal DES treatment.) (A) Venn diagrams showing overlapping peaks between Co and DES-exposed samples for each histone mark. (B) Pie charts illustrating percentage genomic distribution of each histone mark in Co and DES-exposed uterine samples. The legend on the right indicates the genomic features. (C) Metaplots depicting the enrichment patterns (reads per million uniquely mapped) of the histone marks for all TSS ± 5 kb of annotated genes. Closed triangle indicates TSS.
Figure 2.
Figure 2.
H3K27ac is enriched at TSS of differentially expressed genes following neonatal DES treatment. (A) Heatmaps displaying H3K27me3, H3K4me3 and H3K27ac ChIP-seq signal mapping to a 5 kb window around TSS of 4498 differentially expressed genes, up- and down-regulated (top and bottom panel), respectively. Closed triangle indicates TSS. Left panel: Heat maps depicting normalized expression (FPKM) for both the up- and down-regulated genes ranked by the highest to lowest ChIP signal of the respective histone mark in DES-exposed sample. Right panel: Differential change in ChIP signal DES versus Co [fold change (FC)] of each histone mark. (B) Representative UCSC browser shots showing ChIP profiles of H3K27me3 (green), H3K4me3 (red), H3K27ac (blue); differentially expressed genes are indicated by the RNA-seq track. Arrows denote the direction of transcription and gold boxes demarcate the TSS region. (C) Metaplots depicting the enrichment patterns (reads per million uniquely mapped) of H3K27ac at TSS ± 5 kb of differentially expressed genes. Closed triangle indicates TSS. (D) Immunoblot showing H3K27ac expression (top panel). Duplicate gel of same histone samples stained with Simply Blue (bottom panel) as a loading control; histone H3, H2A/B and H4 indicated. Samples were from total histones isolated from Co and DES-exposed uteri (n = 4 uteri/group).
Figure 3.
Figure 3.
DES treatment induces active and super-enhancers at ERα binding sites to modulate gene expression. (A) Heat maps displaying H3K27ac and H3K4me1 ChIP signal enriched at differential peaks ± 100 kb (excluding TSS) of altered genes. ChIP signal from up- or down-regulated genes in Co and DES-exposed samples is sorted by highest to lowest signal in DES-exposed samples. Green boxes highlight positively correlated differential ChIP signal and gene expression, whereas red boxes denote an inverse correlation. The number of altered genes in each category is shown on the left. ChIP signal is plotted against the center of the peak ± 5 kb. Open triangle indicates center of peak. (B) Metaplots of average H3K27ac ChIP signal at typical enhancers (TE; N = 6249) and super-enhancers (SE; N = 380). Graphs use same axis scales; scale bar = 10 kb. (C) Bar graph showing gene expression (FPKM of log2 DES/Co) associated with TE or SE; P< 0.05. (D) Correlation between gene expression changes (FPKM of log2 DES/Co) and density of H3K27ac signal (RPM log2 DES/Co) at active enhancers. For each gene, data plotted is the largest fold change in H3K27ac density (DES versus Co). Gene expression is positively correlated r = 0.47 with H3K27ac density at active enhancers. (E) Average PhastCons score at the enhancers’ center-of-the-peak ± 1 kb is plotted. (F) Known motifs enriched at DES-mediated active ehancers with an enrichment P-value <0.01 are plotted (��log10, P-value). (G) Metaplots of H3K27ac signal at enhancers associated with up-regulated (left) and down-regulated (right) genes. Open triangle indicates center of peak. H3K27ac peak density is sorted based on presence or absence of ERα binding site in Co or DES sample (43). (H) Genome browser view of RNA-seq paired with ChIP-seq signal for H3K27ac (blue) and H3K4me1 (purple) and ChIP-seq tracks for ERα binding sites (43) from ovariectomized adult mice (top track, vehicle; bottom track, E2-1hr) at Spsb1 locus. Gold box highlights a region with a high density of ERα binding sites and differential H3K27ac/H3K4me1 enrichment.
Figure 4.
Figure 4.
ERα is required for DES-induced gene expression and epigenetic modification. (A) Gene expression patterns (EPIG) of microarray data from Co-WT (red), DES-WT (green) and DES-Esr1 cKO (blue) samples (n = 3–4/group). Profiles of six gene expression patterns are shown; number of genes in each pattern is indicated in the box. The two gene expression patterns of interest are highlighted in gold (up-regulated genes) and green (down-regulated genes). (B) Venn diagram showing overlap between expression pattern-1 and up-regulated genes in microarray analysis comparison Co-WT versus DES-WT (2389). Venn diagram showing overlap between expression pattern-6 and down-regulated genes in microarray analysis comparison Co-WT versus DES-WT (2228). Heatmap representing the expression of up- and down-regulated genes that are protected by conditional uterine deletion of ERα. (C) Heat maps displaying differential H3K27ac signal at enhancers ±100 kb of 2801 genes protected from DES in Esr1 cKO (1422 up-regulated; 1,339 down-regulated). H3K27ac signal (±5 kb from center of the peak) shown in descending order based on the signal in the WT-DES sample and matched to gene expression changes between Co versus DES-exposed WT versus Esr1 cKO samples. Open triangle indicates center of peak. Top two panels, up-regulated genes; bottom two panels, down-regulated genes. The change in H3K27ac signal relative to gene expression is shown on the right; increased H3K27ac signal in WT DES compared to WT Co (red arrows) and decreased H3K27ac signal in WT DES compared to WT Co (green arrows). Expression (Exp): 1, Co-WT; 2, Co-Esr1 cKO; 3, DES-WT; 4, DES-Esr1 cKO. (D) Genome browser view of RNA-seq paired with ChIP-seq signal for H3K27ac and ChIP-seq tracks for ERα binding sites (43) from ovariectomized adult mice (top track, vehicle; bottom track, E2-1hr) at Spsb1 locus. Gold boxes highlight regions that display a high density of ERα binding sites and differential H3K27ac enrichment following DES exposure that is not observed in the DES-Esr1 cKO sample.
Figure 5.
Figure 5.
Altered Padi family gene expression depends on chromatin looping and ERα-mediated H3K27ac association. (A) Real time RT-PCR analysis of Padi1-4 in Co and DES-exposed WT and Esr1 cKO uterine samples collected on PND5 (n = 4–6/group). Bar graphs show mean ± S.E.M. (B) Immunoblots of modified citrulline in 20 μg total protein lysate from uteri of Co and DES-exposed mice (n = 3/group). Molecular weight (kDa) indicated. (C) Browser tracks of RNA-seq, ChIP-seq (H3K27ac, ERα, and CTCF) and ChIA-PET at the Padi locus. ERα ChIP-seq tracks (43) from ovariectomized adult mice (top track, vehicle; bottom track, E2-1hr) shown in green. CTCF ChIP-seq tracks (49) from mouse liver (upper track) and ES cells (lower track) shown in orange. ChIA-PET tracks (51) are from mouse ES cells; dashed lines represent chromatin interactions. Gold box outlines a differentially H3K27ac associated region following DES exposure and protection from H3K27ac acquisition in the DES-Esr1 cKO. (D) Enhancer RNA (eRNA). Upper panel, RNA-seq tracks for Co and DES samples near the two ERα binding sites (green bars) denoted by the asterisk on panel C. Four locations near these two ERα binding sites (1–4) were selected to confirm the presence of eRNA by real time RT-PCR. Middle panel, graph of eRNA expression at locations 1–4 in Co and DES. Lower panel, graph of eRNA expression at location 3 in WT and Esr1 cKO mice, as indicated. Graphs show mean ± S.E.M. (n = 3-4/group). (E) 3C-PCR at the Padi1 enhancer/TSS loop. Schematic diagram of the Padi locus with regions of interest indicated. Asterisk indicates the two ERα binding sites marked in panels C and D. Schematic diagram of the Padi locus following HindIII excision and ligation of bound fragments. Gel electrophoresis of 3C-PCR products from uteri of Co and DES-exposed mice. Sequence chromatogram of excised band with regions of interest denoted. Results shown are representative of two independent biological replicates.
Figure 6.
Figure 6.
Working model of the toxicoepigenetics of neonatal DES exposure in mouse uteri. DES-induced changes in gene expression result from ERα-mediated H3K27ac association at topologically associating chromatin domains at the TSS and active enhancer regions (H3K4me1 and H3K27ac) of estrogen target genes. Pioneer transcription factors and/or other co-factors likely promote ERα actions. We propose that a decrease in HDAC protein expression following neonatal DES exposure (5) inhibits histone deacetylation, leading to hyperacetylation of H3K27ac (and possibly other histone lysine residues) at these regions and allowing transcription to persist even in the absence of estrogen.
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References

    1. Ho S.-M., Cheong A., Adgent M.A., Veevers J., Suen A.A., Tam N.N.C., Leung Y.-K., Jefferson W.N., Williams C.J.. Environmental factors, epigenetics, and developmental origin of reproductive disorders. Reprod. Toxicol. 2017; 68:85–104. - PMC - PubMed
    1. Newbold R.R., Jefferson W.N., Grissom S.F., Padilla-Banks E., Snyder R.J., Lobenhofer E.K.. Developmental exposure to diethylstilbestrol alters uterine gene expression that may be associated with uterine neoplasia later in life. Mol. Carcinog. 2007; 46:783–796. - PMC - PubMed
    1. Suen A.A., Jefferson W.N., Wood C.E., Padilla-Banks E., Bae-Jump V.L., Williams C.J.. SIX1 Oncoprotein as a biomarker in a model of hormonal carcinogenesis and in human endometrial cancer HHS public access. Mol. Cancer Res. 2016; 14:849–858. - PMC - PubMed
    1. Yin Y., Lin C., Veith G.M., Chen H., Dhandha M., Ma L.. Neonatal diethylstilbestrol exposure alters the metabolic profile of uterine epithelial cells. Dis. Model. Mech. 2012; 5:870–880. - PMC - PubMed
    1. Jefferson W.N., Chevalier D.M., Phelps J.Y., Cantor A.M., Padilla-Banks E., Newbold R.R., Archer T.K., Kinyamu H.K., Williams C.J.. Persistently altered epigenetic marks in the mouse uterus after neonatal estrogen exposure. Mol. Endocrinol. 2013; 27:1666–1677. - PMC - PubMed

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