PGR Isoform Modulation via Enhancer Activation Regulates Progesterone Signaling in Human Endometrial Stromal Cells [RNA-Seq]

Progesterone, acting through its isoform receptors PGR-A and PGR-B, is essential for regulating uterus function. The ratio between these isoforms is dynamic and context-dependent, driving distinct transcriptional programs. The upstream mechanisms controlling the PGR isoform balance and their functional impact in the uterus remain poorly understood. In this study, we identified two distal PGR enhancers in endometrial stromal cells located 60 kb and 220 kb upstream of the PGR transcription start site. We found that in comparison to PGR promoter-targeting which favors a PGR-B dominant profile, activation of these enhancers shifts the isoform ratio by increasing PGR-A and decreasing PGR-B expression. Bulk RNA-sequencing revealed that the isoform balance alters the progesterone-regulated transcriptome. PGR-A/B-equivalent cells displayed pro-inflammatory gene signatures, whereas PGR-B-dominant cells exhibited suppression of inflammatory signaling and cell cycle activity. Cut&Run assays identified differential genomic binding patterns associated with each isoform profile, and integration with chromatin interaction maps suggest direct regulation of distinct gene subsets. Upstream, ESR1 indirectly activated the PGR-A isoform dominantly, potentially through cooperation with FOXO1 at the U2 enhancer. This study identifies a novel enhancer-driven mechanism regulating PGR isoform dynamics and sheds light on how their imbalance in the endometrial stroma may reshape progesterone signaling in health and disease. Overall design: RNA-seq profiling of control THESC cells or two THESC cell-lines with activated PGR isoforms at different ratios, treated with vehicle or 1uM Medroxyprogesterone Acetate (MPA) for 72 hours.

孕酮通过其同工型受体——孕激素受体（Progesterone Receptor, PGR）-A与PGR-B，对子宫功能的调控至关重要。上述同工型的表达比例呈动态变化且具有情境依赖性，可驱动截然不同的转录程序。目前，调控PGR同工型平衡的上游机制，及其在子宫中的功能影响仍不甚明晰。本研究在子宫内膜基质细胞中，于PGR转录起始位点上游60 kb与220 kb处鉴定出两个远端PGR增强子。研究发现，相较于靶向PGR启动子（该方式偏好PGR-B主导的表达谱），激活上述增强子可改变同工型比例：上调PGR-A的表达并下调PGR-B的表达。批量RNA测序结果显示，PGR同工型的平衡状态会改变孕酮调控的转录组：PGR-A与PGR-B表达量相当的细胞呈现促炎基因特征，而PGR-B主导的细胞则表现出炎症信号通路与细胞周期活性的抑制。Cut&Run实验鉴定出与每种同工型表达谱相关的差异基因组结合模式，结合染色质相互作用图谱分析提示，这些增强子可直接调控不同的基因子集。上游调控层面，雌激素受体1（Estrogen Receptor 1, ESR1）可间接主导性激活PGR-A同工型，该过程可能通过与叉头框O1（Forkhead Box O1, FOXO1）在U2增强子处的协同作用实现。本研究揭示了一种全新的、由增强子驱动的PGR同工型动态平衡调控机制，并阐明了子宫内膜基质中PGR同工型失衡如何重塑健康与疾病状态下的孕酮信号通路。整体实验设计：以对照组THESC细胞，以及两种可激活不同比例PGR同工型的THESC细胞系为研究对象，分别经溶剂对照或1μM醋酸甲羟孕酮（Medroxyprogesterone Acetate, MPA）处理72小时后，进行RNA-seq测序分析。