The Estrogen Receptor α Cistrome in Human Endometrium and Epithelial Organoids

Abstract

Endometrial health is impacted by molecular processes that underlie estrogen responses. We assessed estrogen regulation of endometrial function by integrating the estrogen receptor alpha (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 ChIPseq and then integrated with changes in gene expression from RNAseq 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 impacted formation of cellular protrusions, and chromatin modification. Comparison of signaling impacted 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 impacts endometrial health and function.

From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid

“…Often, initially, the organoid cultures will need to be replated/passaged despite the formation of only a few organoids due to the disintegration of Matrigel or to remove remaining non-epithelial cells (tissue debris, blood cells, and small presumed immune cells). One refinement to our method that would further enrich endometrial cells and decrease immune cells would be to employ the magnetic bead sorting described by Filby et al (12) We have observed estrogen induction of IHH (Figure 3A), in organoid cultures (13), and indications of decidual response to estrogen, progesterone, and cAMP (EPC) treatment of stromal cells in terms of cell morphology (not shown) and induction of the decidual cell gene PRL (Figure 3B). Both of these responses are comparable to those observed in endometrial biopsy-derived cells.…”

“…One refinement to our method that would further enrich endometrial cells and decrease immune cells would be to employ the magnetic bead sorting described by Filby et al. ( 12 ) We have observed estrogen induction of IHH ( Figure 3A ), in organoid cultures ( 13 ), and indications of decidual response to estrogen, progesterone, and cAMP (EPC) treatment of stromal cells in terms of cell morphology (not shown) and induction of the decidual cell gene PRL ( Figure 3B ). Both of these responses are comparable to those observed in endometrial biopsy-derived cells.…”

“…Both of these responses are comparable to those observed in endometrial biopsy-derived cells. Other genes of interest that have been evaluated for estrogen response in organoids derived from endometrial biopsies include GREB1 and PGR ( 13 ). Similarly, IGFBP1 is another gene induced during decidualization ( 14 ).…”

“… Relative responses of epithelial organoid and stromal cell cultures from three different individuals. (A) RT-PCR for IHH of RNA isolated from organoid cultures treated with vehicle (Veh) or estrogen (E2) for 6 days + 6 hours as described previously ( 13 ). Organoids were isolated from three different individuals (participants A, B, and C).…”

The pathophysiological role of estrogens in the initial stages of pregnancy: molecular mechanisms and clinical implications for pregnancy outcome from the periconceptional period to end of the first trimester

“…organoids, have been employed to study the hormonal regulation of epithelial cells ( Turco et al , 2017 ; Nikolakopoulou and Turco, 2021 ). By combining advanced genomic analysis of the endometrium and organoid studies, recent work has led to the identification of potential binding sites for ERα across the genome ( Hewitt et al , 2022 ), and to the partial characterization of molecular determinants of the proliferation and differentiation responses in epithelial cells to E2 and progesterone stimulation ( Garcia-Alonso et al , 2021 ). Although organoids provide significant advantages over traditional cell lines and in vitro 2D cell systems, they are still far from recapitulating the full complexity of the endometrium, and therefore their potential translational value relies on the inclusion of stromal as well as immune components, in addition to the above-mentioned microbial partners, in a relevant spatial and functional interface ( Murphy et al , 2022 ).…”