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. Overall design: 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 17ß-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.

雌激素（Estrogen, E2）通过雌激素受体α（estrogen receptor alpha, ESR1）发挥生理功能，是子宫内膜生长、分化及疾病发生的关键调控因子。子宫内膜基质细胞（endometrial stromal cell, ESC）是ESR1发挥作用的核心靶标，其在调控旁分泌信号交流以维持子宫功能方面扮演关键角色。目前对ESR1在ESC中作用机制的理解，受限于ESC细胞系的低表达水平。
本研究通过基因工程手段构建了端粒酶永生化人子宫内膜基质细胞（telomerase-immortalized hESC, THESC），使其表达CRISPR激活系统以实现ESR1的激活。在6条测试的向导RNA（guide RNA, gRNA）中，ESR1-3 gRNA可高效诱导ESR1激活，恢复THESC对E2的应答能力。批量RNA测序（bulk RNA-seq）结果显示，ESR1介导了E2依赖型与E2非依赖型两类转录程序，调控炎症、细胞增殖、细胞外基质重塑及肿瘤相关通路。值得注意的是，72%的差异表达基因（differentially expressed genes, DEGs）与增殖期E2主导阶段人子宫内膜组织中的活跃表达基因存在重叠，证实了该转录程序的生理相关性。Cut&Run测序（Cut&Run-seq）在全基因组范围内鉴定出ESR1结合位点，其中多数位于远端调控元件区域。为将远端ESR1结合位点与靶基因关联起来，本研究整合了人ESC中H3K27ac HiChIP的染色质环数据，以鉴定可与基因启动子形成染色质环的远端ESR1结合位点。本研究鉴定出通过长距离染色质环受ESR1/E2调控的基质细胞蜕膜化相关基因，包括FOXO1与IL6R。此外，本研究还鉴定出与子宫内膜癌相关的基因，包括ERRFI1、NRIP1及EPAS1，提示基质ESR1驱动子宫内膜病理发生的潜在作用。功能实验证实，ESR1可促进细胞存活，且在E2存在时可增强细胞迁移能力。
本研究通过构建可恢复人ESC中ESR1表达、从而克服其E2应答能力受限问题的新型细胞培养模型，为ESR1依赖的转录调控机制提供了全新见解。此外，本研究还证实了将HiChIP染色质环数据与顺组学数据（cistromic data）整合，可有效实现远端ESR1结合位点与基因启动子的关联分析。
整体实验设计：原代人子宫内膜基质细胞（primary endometrial stroma cells, hESCs）分离自子宫内膜活检样本，该样本采集流程经贝勒医学院伦理审查委员会批准，受试者编号为H-13062。活检样本来自2名健康育龄女性志愿者，其月经周期规律，无妇科恶性肿瘤病史。将hESC用0.2%乙醇（溶剂对照）或由10 nM 17β-雌二醇（17β-estradiol, Sigma-Aldrich，美国密苏里州圣路易斯市）、1 μM醋酸甲羟孕酮（medroxyprogesterone acetate, Sigma-Aldrich，美国密苏里州圣路易斯市）及100 μM双丁酰环腺苷酸（dibutyryl cyclic AMP, Sigma-Aldrich，美国密苏里州圣路易斯市）组成的混合液（命名为EPC）处理3天。H3K27ac HiChIP文库由Arima Genomics公司构建。