The Estrogen Receptor α Cistrome in Human Endometrium and Epithelial Organoids

Abstract

Endometrial health is affected by molecular processes that underlie estrogen responses. We assessed estrogen regulation of endometrial function by integrating the estrogen receptor α (ESR1) cistromes and transcriptomes of endometrial biopsies taken from the proliferative and mid-secretory phases of the menstrual cycle together with hormonally stimulated endometrial epithelial organoids. The cycle stage-specific ESR1 binding sites were determined by chromatin immunoprecipitation and next-generation sequencing and then integrated with changes in gene expression from RNA sequencing data to infer candidate ESR1 targets in normal endometrium. Genes with ESR1 binding in whole endometrium were enriched for chromatin modification and regulation of cell proliferation. The distribution of ESR1 binding sites in organoids was more distal from gene promoters when compared to primary endometrium and was more similar to the proliferative than the mid-secretory phase ESR1 cistrome. Inferred organoid estrogen/ESR1 candidate target genes affected formation of cellular protrusions and chromatin modification. Comparison of signaling effected by candidate ESR1 target genes in endometrium vs organoids reveals enrichment of both overlapping and distinct responses. Our analysis of the ESR1 cistromes and transcriptomes from endometrium and organoids provides important resources for understanding how estrogen affects endometrial health and function.

Keywords: chromatin; endometrium; estrogen receptor.

Figure 1.

ESR1 ChIPseq of proliferative and mid-secretory endometrial biopsies. A, Venn diagram comparing number of ESR1 peaks in one proliferative (Prolif) endometrial biopsy to ESR1 peaks in one mid-secretory (MS) endometrial biopsy. B, Locations of ESR1 peaks from proliferative or mid-secretory endometrium or from organoids, relative to annotated genes (RefSeq). C, Summaries of the top 15 HOMER known motifs enriched in ESR1 peaks from proliferative or mid-secretory endometrial biopsies. The ranks of each motif, as determined by P value, along with the range of P values covered by the ranks are indicated. Motif logos are shown.

Figure 2.

Estrogen-responsive genes are induced in organoid cultures. A, RT-PCR of RNA isolated from donor 1 derived organoid cultures treated as described in methods and in Supplementary Fig. S1 (41) with 0.1% ethanol vehicle (V) or with 10 nM estradiol (E2) 6, 9, or 12 days after initial plating (d6, d9, d12). Progesterone receptor (PGR), Indian hedgehog (IHH), and growth-regulating estrogen receptor binding 1 (GREB1). Bar indicates mean, error bars indicate SD; * indicates P < .05 vs V based on 2-way ANOVA with Fisher LSD multiple comparisons test. N = 3 for all but d6 E2 (n = 2). B, RT-PCR of RNA isolated from donor 2 derived organoids 9 days after plating treated with 0.1% ethanol V, 10 nM E2, or with 1 µM ICI 182780 + 10 nM E2 (ICI + E2). Bars indicates mean, error bars indicate SD; * indicates P < .05 vs V; +indicates P < .05 vs E2 based on 2-way ANOVA with Fisher LSD multiple comparisons test. N = 3 for all.

Figure 3.

Human epithelial organoid ESR1 transcriptome and cistrome. A, Hierarchical cluster comparing E2 vs V fold-changes of significantly regulated genes (2-fold, FDR P < .05) in either donor. B, ESR1 ChIPseq of chromatin isolated from organoids. The number of peaks of each donor sample is indicated, as well as those shared by both donors. A total of 2543 of the 2944 overlapping ESR1 peaks are within 100 kb of a gene. C, Summaries of the top 14 HOMER known motifs enriched in the 2944 shared ESR1 peaks in organoids. The motif’s ranks, as determined by P value, along with the range of P values covered by the motif, are indicated.

Figure 4.

Organoid ESR1 cistrome resembles proliferative endometrium. ESR1 ChIPseq signal centered on locations of ESR1 peaks (± 1000 bp) in organoid samples. MS, mid-secretory whole endometrium; Pro, proliferative whole endometrium.

Figure 5.

ESR1 peaks near human and mouse estrogen-responsive transcripts IHH, and GREB1. For the human genes, ESR1 ChIPseq from mid-secretory (MS) and proliferative (Pro) whole endometrium and from organoids is shown. For the mouse genes, ESR1 ChIPseq from V or E2 treated whole uterus is shown. Interacting loops from mouse uterus HiC are also shown as black arcs. A, Human IHH. The transcript is highlighted, regions with ESR1 peaks at 20 kb 5′ of the IHH transcription start site (TSS), at 70 kb 5′ of IHH, and at 100 kb 5′ of IHH are also highlighted. B, Mouse Ihh. Regions comparable to the human gene are highlighted as described in A. C, Human GREB1. The ESR1-binding super-enhancer region is highlighted. D, Mouse Greb1. ESR1-binding super-enhancer is highlighted. Orientation on the genome browser view is the opposite of the human GREB1.