Molecular regulation of whole genome DNA methylation in heat stress response of dairy cows

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.

Keywords: DMG; DNA methylation; Dairy cows; Heat stress; WGBS.

Fig. 1

The Genome-wide methylation patterns in dairy cows (A). The average ratio of DNA methylation types of dairy cows in spring (B) and summer (C). The whole genome methylation map of dairy cows in spring (D) and summer (E): the outermost circle of the whole genome methylation map is the chromosome length scale; the CpG (purple), CHG (blue), and CHH (green) methylation types of chromosome intervals are arranged from the outside to the inside (darker colors indicate higher methylation levels); the innermost circle represents the number of genes in the corresponding interval (darker colors indicate more genes)

Fig. 2

DMRs and DMGs in the genomes of spring and summer dairy cows: A Distribution statistics of DMRs across different genomic regions; B Volcano plot of DMRs, with blue representing hypomethylated DMRs and red representing hypermethylated DMRs in the summer group relative to the spring group; C Cluster heatmap of DMR methylation for the top 100 promoter DMGs, Color changes represent different degrees of methylation; D PPI network analysis of DMGs in the heat shock protein (HSP) family. Colored nodes indicate core proteins and their direct interactors, white nodes represent indirectly linked proteins. Lines denote functional associations, including experimentally validated interactions and bioinformatics-based predictions

Fig. 3

GO and KEGG pathway enrichment analysis of DMGs in promoter region: A GO term enrichment analysis of DMGs. The y-axis represents the pathway names, and the x-axis represents the Count. B KEGG pathway enrichment analysis of DMGs. The y-axis represents the pathway name, and the x-axis represents the gene number. The color of the dots corresponds to different ranges of P-values, darker color represent smaller P-values, and the size of the dots represents the number of genes

Fig. 4

Identification of key DMRs and DMGs in promoter in dairy cows under heat stress (A) The volcano plot of DEGs; B Venn diagram of promoter DMGs and DEGs; C The methylation levels of DMRs in the promoters of GNAS, DNLZ, and SMAD5; D The expression levels of GNAS, DNLZ, and SMAD5 using TPM (number of transcripts per million reads). E PPI Network interaction of DMEGs with negative regulation of expression by promoter methylation. Note: In (C) and (D), arrows indicate the direction of change, upward arrows indicate an increase, and downward arrows indicate a decrease in summer expression; *** FDR < 0.001

Fig. 5

The DNLZ expression was suppressed as its promoter methylation increased in dairy cows under heat stress. A Predicted CpG island in the DNLZ DMR sequences; B Nucleotide sequence of DNLZ1(part of DNLZ promoter), with 1 to 39 indicating the cytosine numbering of CG sites, and single underlines representing predicted transcription factor binding site; C Detection of mRNA expression levels of DNLZ, MeCP2, DNMT1, DNMT3 A, and DNMT3B. D The methylation level of each CG site in DNLZ1. E The overall methylation level of CpGs in DNLZ1 in dairy cows. Note: * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 6

The DNLZ expression was suppressed with increased methylation of CpG island in its promoter in Mac-T cells under heat stress. A The overall methylation level of CpGs in DNLZ1 in Mac-T cells. B The expression level of DNLZ gene in Mac-T cells after 24 and 48 h of heat stress treatment. C and D The expression levels of four methylases in Mac-T cells after 48 and 72 h of heat stress treatment. * Indicates P < 0.05, ** indicates P < 0.01

Fig. 7

The methylated promoter of DNLZ gene suppressed its expression in vitro. A Schematic diagram of PGL3-DNLZ promoter luciferase reporter plasmid structure. B Partial methylation identification, where Lane 1 ~ Lane 7 are single enzyme cleavage products of DNLZ plasmids and HpaII under different conditions: Lane 1 is 0U + 0 h, Lane 2 is 2U + 0.5 h, Lane 3 is 4U + 0.5 h, Lane 4 is 2U + 1 h, Lane 5 is 4U + 1 h, Lane 6 is 2U + 2 h, Lane 7 is 4U + 2 h, Lane 8 is plasmid, and M is DNA marker. C The relative activity detection results of dual luciferase after complete methylation of PGL3-DNLZ plasmid. D Detection results of dual luciferase relative activity of PGL3-DNLZ plasmid at different M.Sss I enzyme concentrations and treatment times. Note: The complete gel image can be found in Additional file 1: Figure S3. This image represents the full experimental process, with no parts cropped before the electrophoresis experiment