PGR Isoform modulation via enhancer activation regulates progesterone signaling in endometrial stromal cells

To investigate enhancer-mediated regulation of progesterone receptor (PGR) isoforms, PGR-A and PGR-B, in human endometrial stromal cells, and to determine how isoform modulation shapes the progesterone-responsive transcriptome and cistrome relevant to endometrial function. A CRISPR-based functional genomic screen was used to identify distal enhancers in telomerase-immortalized human endometrial stromal cells. Subsequent CRISPR targeting of identified enhancers and the PGR promoter was used to modulate PGR isoform balance and assess functional consequences. None. Engineered endometrial stromal cells were treated with medroxyprogesterone acetate (MPA) or vehicle. PGR isoform expression was assessed by western blot, the progesterone-responsive transcriptome was characterized by bulk RNA sequencing, and the PGR cistrome was characterized by Cut&Run. Two distal PGR enhancers were identified in endometrial stromal cells located approximately 60 and 220 kb upstream of the PGR transcription start site. CRISPR-based activation of these enhancers upregulated both PGR-A and PGR-B, while promoter activation primarily upregulated PGR-B. Bulk RNA sequencing revealed that shifting the PGR isoform balance altered the progesterone-regulated transcriptome: PGR-A/B-equivalent cells exhibited pro-inflammatory gene signatures, while PGR-B-dominant cells demonstrated suppression of inflammatory signaling and altered cell cycle programs. PGR Cut&Run profiling revealed distinct genomic binding patterns associated with each isoform profile. Integration of the PGR cistrome with chromatin interaction maps suggested that these isoforms directly regulate distinct gene subsets involved in inflammation and fibrosis. Mechanistically, ESR1 indirectly activated PGR-A expression, potentially through recruitment of FOXO1 at the distal enhancer, suggesting a non-canonical, enhancer-mediated mechanism of PGR regulation. Distal enhancers regulate the PGR isoform balance and shape the progesterone-responsive transcriptome in human endometrial stromal cells. This enhancer-mediated mechanism expands current models of PGR regulation beyond promoter-level control and may offer potential therapeutic targets to restore normal progesterone responsiveness in conditions marked by PGR isoform imbalance.