Decoding the Enhancer-Driven Regulatory Network for Lactoferrin Across Species

Lactoferrin (LTF) is a multifunctional protein critical to mammalian milk, playing essential roles in immune regulation, antimicrobial activity and iron absorption. However, the genetic basis driving its mammary specific high expression remain poorly understood. This study reveals the expression regulatory network and evolutionary characteristics of the mammalian LTF gene by integrating multi-omics data and cross-species comparative analysis. Gene expression profiling revealed that LTF gene is predominantly and specifically expressed in glandular tissues, particularly in the salivary and mammary glands. In both human and bovine mammary tissues, LTF gene was primarily expressed in secretory luminal epithelial cells. Correlation analysis in 585 Holstein cows demonstrated a negative correlation was observed with somatic cell. By integrating multi-omics data, we identified three functional enhancers: an upstream enhancer harboring 20 cis-eQTLs and two evolutionarily conserved ruminant-specific enhancer elements. Furthermore, EHF and ELF5 were experimentally validated as key transcription factors targeting these enhancers to regulate the LTF gene. Cross-species analysis further uncovered a human-specific enhancer (chr3:46488833-46489420) driving exceptionally high LTF gene expression, which retained 3.11-fold enhancer activity in MAC-T cells. Finally, by integrating the predicted transcription factors of three enhancer regulatory elements with their expression correlation to LTF gene, expression levels, and binding site enrichment analysis, we constructed a comprehensive cross-species regulatory network for LTF gene. This study investigates the potential factors governing LTF gene expression in the mammary gland, identifying three enhancers and 39 core conserved expressed transcription factors within a high-resolution regulatory network. Our findings provide evolutionary insights into lactation-specific LTF gene regulation and offer functional targets for improving mammary gland health and breeding cattle with enhanced LTF level.