Abstract
Follicular atresia is a crucial factor that affects the egg-laying performance of chickens, and granulosa cells (GCs) are the root cause of follicular atresia. Investigating the fate-determining genes of GCs can help improve egg production and laying period duration in chickens. Notably, transforming growth factor beta 2 (TGFB2) and insulin like growth factor binding protein 5 (IGFBP5) were previously identified as key genes responsible for GC degeneration. In this study, the promoters of TGFB2 and IGFBP5 were identified and subjected to DNA pull-down assays. The pulled-down proteins were then analyzed using mass spectrometry. Additionally, single-cell regulatory network inference and clustering (SCENIC) analysis results from previous single-cell RNA sequencing data were integrated to investigate the upstream transcriptional regulators of TGFB2 and IGFBP5. The results revealed that SMAD family member 3 (SMAD3) is a crucial transcription factor (TF) for IGFBP5, while signal transducer and activator of transcription 2 (STAT2) is a common TF for both TGFB2 and IGFBP5. The regulatory pathway of STAT2/TGFB2/SMAD3/IGFBP5 highlights the significant interaction between the janus kinase (JAK)/STAT and transforming growth factor-β (TGF-β)/SMAD signaling pathways during follicular atresia. Their synergistic regulation leads to GC degeneration, which causes follicular atresia. This study provides new evidence for identifying switch genes, further elucidates the regulatory mechanisms of follicular atresia, and offers new genetic targets for extending the laying period and improving the egg-laying performance.