Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape [ESR1_HiChIP]

Estrogen (E2) acting through the estrogen receptor alpha (ESR1) is a critical regulator of endometrial growth, differentiation and disease. The endometrial stromal cell (ESC) is a key target of ESR1 action and plays a crucial role in regulating paracrine communication to control uterine function. A mechanistic understanding of the role of ESR1 in ESCs is limited by low levels of expression of ESC lines. Here we engineered telomerase-immortalized hESC (THESC) to express the CRISPR activation system to activate ESR1. Among six tested guide RNAs, the ESR1-3 gRNA induced robust ESR1 activation, restoring E2 responsiveness in THESC. Bulk RNA-seq revealed ESR1-mediated E2-dependent and E2-independent transcriptional programs, regulating pathways involved in inflammation, proliferation, extracellular matrix organization, and cancer. Notably, 72% of differentially expressed genes (DEGs) overlapped with genes active in human endometrial tissue during the proliferative E2 dominant phase, supporting their physiological relevance. Cut&Run-seq identified genome-wide ESR1 binding sites, with most located at distal regulatory elements. To associate distal ESR1 binding sites with genes, we integrated H3K27ac HiChIP chromatin loops in hESC to identify distal ESR1 binding sites that loop to gene promoters. We identified genes regulated by ESR1/E2 through long-range chromatin looping that are involved in stromal cell decidualization, including FOXO1 and IL6R. Additionally, we identified genes implicated in endometrial cancer, including ERRFI1, NRIP1, and EPAS1, suggesting a role for stromal ESR1 driven endometrial pathologies. Functional assays confirmed that ESR1 promotes cell viability and, in the presence of E2, enhances migration. In this study, we reveal novel insights into ESR1-dependent transcriptional regulation using a newly developed cell culture model that restores ESR1 expression in hESCs to overcome their limited E2 responsiveness. Additionally, we demonstrate the utility of integrating HiChIP chromatin looping data with cistromic data to associate distal ESR1 binding sites to gene promoters. Primary endometrial stroma cells (hESCs) were isolated from endometrial biopsies collected under the human subject protocol number H-13062 approved by the institutional review board of Baylor College of Medicine. The biopsies were obtained from two healthy, reproductive-aged volunteers with regular menstrual cycles and no history of gynecological malignancies. hESC were treated with 0.2% ethanol (vehicle) or a mixture of 10 nM 17β-estradiol (Sigma-Aldrich, St. Louis, MO, USA), 1 µM medroxyprogesterone acetate (Sigma-Aldrich, St. Louis, MO, USA) and 100 µM dibutyral cyclic AMP (Sigma-Aldrich, St. Louis, MO, USA), termed EPC, for 3 days. H3K27ac HiChIP libraries were prepared by Arima Genomics.