Abstract
BACKGROUND: Heat stress seriously affects the production and health of dairy cows and is a key factor limiting the sustainable development of the dairy industry. DNA methylation serves as an important epigenetic regulatory mechanism closely associated with an animal's response to heat stress. However, the specific molecular mechanism of DNA methylation in cows' heat stress response is not fully understood. RESULTS: In this study, whole genome bisulfite sequencing analysis of blood identified 49861 specific differentially methylated regions corresponding to 7613 differentially methylated genes between spring and summer dairy cows. Among them, 4069 the promoter region of differentially methylated genes were significantly enriched in key biological pathways such as substance transport, reactive oxygen species metabolism, signal transduction, and energy metabolism. By integrating the expression data of 4069 promoter differentially methylated genes, 157 genes were further screened, and their DNA methylation levels were negatively correlated with gene expression. The changes in DNLZ, GNAS, and SMAD5 genes were most significant, and network analysis showed that DNLZ gene has high connectivity in the protein-protein interaction network, indicating its potential key function in heat stress response. Experimental verification shows that under heat stress conditions, the methylation level of CpG islands in the promoter region of DNLZ gene significantly increases, and its methylation level is significantly negatively correlated with gene expression level. The Dual-luciferase reporter assays using constructs containing the DNLZ promoter reporter gene experiment further confirms that promoter methylation significantly inhibits DNLZ transcriptional activity, and the higher the degree of methylation, the stronger the inhibitory effect. CONCLUSIONS: The research results provide new insights into the mechanism of heat stress-related DNA methylation in dairy cows, clarify the key roles of genes such as DNLZ, and provide potential target genes and epigenetic markers for the cultivation of heat-resistant dairy cows.