The Whole Genome-wide DNA Methylation Analysis Of Mammary Tissue From Chinese Holstein With Staphylococcus Aureus Mastitis

Staphylococcus aureus(S.aureus)is one of the most common infectious pathogens of mastitis.Due to various factors affect mastitis infection,exploring the regulation mechanism of mastitis from different aspects will be in favor of selective breeding for improving mastitis resistance of dairy cows.DNA methylation,one of the important epigenetic regulatory mechanism,plays vital roles in health and production of dairy cows.This study is aimed to build up the whole genome wide DNA methylation map of S.aureus infected Chinese Holstein and explore its potential regulation mechanism of mastitis caused by S.aureus.DNA extracted from mammary tissues of S.aureus infected and negative control Holstein cows were used for whole genome-wide DNA methylation measurement by Methylation-dependent restriction-site associated DNA sequencing(Methyl-RAD Seq).Methylation sequencing data passed quality control was annotated by SOAP according to bovine reference genome,followed by differently methylation analysis through R package Edge and functional enrichment analysis through Enricher.R.Differently expressed and methylated genes were selected by joint analysis of whole genome-wide methylation result and transcriptome data.Bisulfite Sequencing PCR(BSP)was used to confirm the methylation level of selected differently expressed and methylated genes.The results were as followed:1.Whole genome-wide DNA methylation sites was non-uniform distribution in chromosomes,methylation sites were concentrated in intergenic region and intron of genes.The methylation level of gene body was higher than that of its upstream and downstream,which meant that the methylation level significantly increased before transcriptional start site and decreased after transcriptional termination site.Compared with negative control,9181 differently CCGG methylated sites were found in the mammary tissues of Chinese Holstein with S.aureus-induced mastitis,including 5722 sites with up-regulated methylation level and 2459 sites with down-regulated methylated level(P<0.05,|log2FC|>1).At the same time,1790 differently CCWGG methylated sites were found with 1150 up-regulated sites and 640 down-regulated sites(P<0.05,|log2FC|>1).The total sequencing depth of all methylation sites in same gene was used to represent the gene methylation level,and then the difference analysis found 363 differently CCGG methylated genes(236 up-regulated and 127 dowm-regulated genes)and 301 differently CCGG methylated genes(177 up-regulated and 124 down-regulated genes)(P<0.05,|log2FC|>1).GO and KEGG enrichment analysis indicated that differently CCGG methylated sites were related to immune response to diseases,such as positive regulation of toll-like receptor 4 signaling pathway,Th17 cell differentiation,B cell receptor signaling pathway and many pathways related to immune response to diseases.While the differently CCWGG methylated sites played regulatory roles on metabolism procession,such as galactose metabolism,regulation of lipolysis in adipocyte as well as starch and sucrose metabolism.2.Joint analysis between whole genome-wide DNA methylation and transcriptome data found 526 differently CCGG methylated genes and 124 differently CCWGG methylated genes with significantly different gene expression level(P<0.05).Functional enrichment analysis showed that the differently expressed and CCGG methylated genes were related to cell adhesion,cell activation and other cellular processes as well as the immune response to diseases,revealing their important regulatory roles on immune response to S.aureus-induced mastitis.3.CXCR1,CDH13,EPO and METTL13 were selected according to above analysis,which had regulatory potential on S.aureus-induced mastitis.One CpG island was predicted in promoter region of CDH13,coding region of CXCR1,EPO and METTL13,and 13,16,16 and 33 CpG loci were detected,respectively.The methylation level of CpG site at 519 bp in CXCR1 coding region was significantly lower in the mammary tissues of Chinese Holstein with S.aureus-induced mastitis than that of control group,while the gene expression level of CXCR1 was significantly up-regulated 12 times(P<0.05).The methylation level of CpG site at 519 bp in CXCR1 coding region was significantly correlated with CXCR1 gene expression level(P<0.05).The methylation level of three CpG sites in CDH13 promoter region(-162,-133 and-93)were significantly correlated with CDH13 expression level,that were not significantly different between S.aureus mastitis and control group.In addition,CXCR1 coding region had high methylation level(>90%),but EPO and METTL13 had low methylation level(<5%),which was consistent with whole genome-wide DNA methylation sequencing analysis,verifying the accuracy and reliability of whole genome-wide DNA methylation sequencing result.To sum up,this study built the whole genome-wide DNA methylation map of Chinese Holstein with S.aureus-induced mastitis and selected S.aureus-induced mastitis related differently methylated sites/genes supported by functional analysis,which play important roles in the immune responses to S.aureus-indnctd mastitis.The reduced methylation level of CpG site at 519 bp in CXCR1 coding region causing up-regulated gene expression level could be regulatory mechanism in response to S.aureus-induced mastitis.The results supplied data reference and scientific basis for epigenetic regulation analysis and molecular assisted selection breeding to improve the host resistance of S.aureus-induced mastitis in Chinese Holstein.