A self-reinforcing transcriptional loop: <i>ELF5</i> directly activates the <i>STAT5B</i> promoter to orchestrate milk protein synthesis in water buffalo

Signal transducer and activator of transcription 5B (STAT5B) plays a critical role in milk protein synthesis in mammals. However, its transcriptional regulatory mechanisms in buffalo, particularly in Binglangjiang buffalo, a breed characterized by high milk protein content, remain largely unclear. The study isolated and identified the functional STAT5B transcript variant in lactating buffalo mammary gland tissues. Expression analysis revealed that STAT5B was significantly upregulated in lactating buffalo mammary epithelial cells (BuMECs) compared with non-lactating BuMECs, suggesting its potential involvement in lactation regulation. Subcellular localization analysis confirmed that STAT5B was distributed in both the nucleus and cytoplasm of BuMECs, providing a structural basis for its transcriptional regulatory function. Importantly, functional experiments identified a novel positive feedback loop between ELF5 and STAT5B, through which STAT5B promoted casein synthesis in BuMECs. The findings significantly expand our understanding of STAT5B-mediated transcriptional regulation in ruminants and enrich the molecular regulatory network of milk protein synthesis in buffalo. These findings provide important theoretical support and potential molecular targets for genetic improvement of milk protein content in the buffalo dairy industry.