H3K4me3 CUT&Tag and Transcriptome Analysis Reveal the Epigenetic Regulatory Landscape in Mammary Gland Tissues of Yili Horses at Different Lactation Stages

H3K4me3, a well-established histone modification associated with active promoters, plays a critical role in orchestrating gene expression programs that govern mammary gland development and lactation. In this study, we present the first comprehensive epigenomic profiling of H3K4me3 modifications during mammary gland development in Yili horses using Cleavage Under Targets and Tagmentation (CUT&Tag) and RNA sequencing. Mammary gland tissues were collected from two developmental stages—early lactation and peak lactation. A total of 393 differentially expressed genes (DEGs) were identified between two groups, among which 72 DEGs (54 upregulated H3K4me3 targets and 18 downregulated targets) were directly regulated by H3K4me3. KEGG enrichment analyses revealed that these DEGs were involved in ECM–receptor interaction, focal adhesion, the PI3K-Akt signaling pathway, and the calcium signaling pathway. In these pathways, five genes were identified as potential regulators of mammary gland development. Among these, PTGES, COL1A1, PDGFRB, and RYR1 exhibited consistent upregulation at both the transcriptomic and chromatin levels, whereas PRKAG3 showed significant downregulation. These findings offer novel insights into the epigenetic regulation of lactation in horses and lay a theoretical foundation for improving milk production traits through targeted molecular breeding strategies.