Integrated analysis of chromatin accessibility and transcriptome profiles in granulosa cells of sheep with different FecB genotypes

BMPR1B is the major gene regulating ovine fertility, wherein the A746G coding mutation (the FecB mutation) has been shown to markedly increase ovulation rate and lambing number. Granulosa cells (GCs) provide essential support for follicular development and oocyte maturation, directly influencing reproductive performance. However, the epigenetic mechanisms by which FecB genotypes regulate GCs function via chromatin accessibility and specific transcription factors remain largely unknown. In this study, ATAC-seq and RNA-seq were combined to systematically characterize chromatin accessibility and transcriptomic differences between GCs from FecB BB and FecB ++. A total of 9,025 differentially accessible regions (DARs) and 534 differentially expressed genes (DEGs) were identified. Functional enrichment analysis indicated that the TGF-β and PI3K-Akt signaling pathways were implicated in the regulation of GCs proliferation. By integrating chromatin accessibility and expression data, four candidate genes (CYP2R1, PTGFR, FBN1, and HK2) were prioritized, their expression was positively correlated with chromatin openness. Notably, the DARs associated with CYP2R1 and HK2 were located within 2 kb upstream of their transcription start sites, suggesting that regulatory elements in these proximal promoter regions may govern their transcriptional activity. Transcription factor motif analysis revealed a significant enrichment of AP-2 family motifs in open regions near CYP2R1, whereas open regions near HK2 were predicted to be bound by bHLH family transcription factors. Dual-luciferase assays verified that transcription factor TFAP2A directly binds the proximal DAR of CYP2R1 and regulates its transcription. These findings reveal the chromatin landscape and regulatory network in GCs of different FecB genotypes, providing mechanistic insights into how the FecB locus epigenetically governs sheep fertility.