Decoding the enhancer-driven regulatory network for lactoferrin across mammalian species

Lactoferrin (LTF) is a pleiotropic milk protein with essential roles in mucosal immunity, antimicrobial defense, and iron homeostasis, yet the regulatory architecture underlying mammary-enriched LTF expression remains incompletely understood. In this study, multi-omics integration and comparative genomics were used to resolve the regulatory landscape, transcriptional control, and evolutionary diversification of mammalian LTF. Cross-tissue profiling revealed that LTF was highly expressed in glandular tissues, predominantly in the salivary and mammary glands. Single-cell transcriptomic analysis further localized LTF expression mainly to secretory luminal epithelial cells in human and bovine mammary tissues. Correlation analysis of 585 Holstein cows demonstrated that LTF expression was negatively associated with somatic cell score. Multi-omics analysis identified three functional enhancers, including an upstream enhancer harboring 20 cis-expression quantitative trait loci (eQTLs) and two evolutionarily conserved ruminant-specific enhancer elements. Functional validation demonstrated that EHF and ELF5 directly targeted these enhancers and regulated LTF transcription. Cross-species analysis also identified a human enhancer at chr3:46488833-46489420 that contributed to the exceptionally high LTF expression in humans and retained 3.11-fold enhancer activity in bovine MAC-T cells. By integrating transcription factor prediction, expression correlation with LTF, transcription factor abundance, and binding-site enrichment, a high-resolution cross-species regulatory network was constructed, comprising three enhancer elements and 39 conserved core transcription factors expressed across mammalian mammary tissues. These findings establish an evolutionarily diversified regulatory framework for lactation-specific LTF expression and identify candidate regulatory elements and transcription factors that may inform strategies to improve mammary gland health and increase LTF content in dairy cattle.