DNA Methylation And Gene Expression Regulation Studies Of Neutrophils In Dairy Cows With E.coli Mastitis

Mastitis is the inflammation of the mammary gland caused by various pathogenic bacteria.Escherichia coli(E.coli),as one of the most common environmental pathogens,lead to the occurrence of acute clinical mastitis.Neutrophils are the first effectors of the inflammatory response,which are important defense cells against the invasion of pathogenic bacteria.After the bacteria invade the mammary gland,the neutrophils detect the components of the pathogen through specific receptors and arrive at an infection site to begin phagocytosis.DNA methylation is involved in the occurrence and development of bovine mastitis and related to disease susceptibility.In this study,firstly we sequenced the blood neutrophils of healthy and E.coli infected mastitis half-sib cows using the RNA-seq and RRBS methods;we did the joint analysis of transcriptome and methylation data,selected the target genes associated with immune,inflammation and defense responses;and then by the bisulfite sequencing and Real-time PCR,we detected the methylation levels and expression levels of genes in the samples,screened the methylation sites of regulation gene expression and studied the key regulatory mechanisms of gene methylation.We would excavate the epigenetic markers associated with cow mastitis inflammation basing on the genome-wide DNA methylation dataset.The findings would provide insights into the molecular mechanism of DNA methylation in mastitis infection process and new targets for breeding and treatment of mastitis.The principal results were as follows:(1)The half-sibling matched healthy cows(HC group,n=3)and E.coli infected mastitis cows(MC group,n=3)were selected from 52 candidate samples based on their milk somatic cell counts(SCCs)records,haematological analysis and bacteria identification results.By the RRBS method,we sequenced and analyzed genome-wide DNA methylation levels of bovine peripheral blood neutrophils of HC and MC group cows.The global DNA methylation maps and methylation levels in different genome regions were investigated.A total of 494 differentially methylated regions were observed between the HC and MC groups,of which 61 were up-regulated and 433 were down-regulated in the mastitis cows compared to the healthy mastitis cows.These results demonstrated that the DNA methylation level in the mastitis cows was decreased relative to the healthy cows.Of these DMRs,75 regions were located with gene promoters,75 regions were located with gene exons,344 regions were located with gene introns.69 differentially methylated genes were harbored in the QTLs interrelated with clinical mastitis,somatic cell count and somatic cell score.Moreover,26 differentially methylated miRNAs were also identified in the healthy and mastitis groups.(2)Using RNA-Seq,we compared the transcriptomic landscapes of neutrophil from the healthy and E.coli infected mastitis cows.The expression levels of 1,094 differentially expressed genes(DEGs)were upregulated,and the expression levels of 245 genes were down-regulated in the mastitis cows relative to the healthy cows.A total of 415 genes were located in the clinical mastitis,SCC and SCS QTLs regions.In addition,829 genes were alternatively spliced,exhibiting different splicing patterns.Among these DEGs,140 genes were involved in inflammatory,immune and defense responses.(3)The correlation analysis between the DNA methylation and gene expression data in mastitis cows versus healthy cows were performed.29 genes were found in both RNA-seq and RRBS sequencing data.Of these genes,the methylation levels of 7 genes promoters were negatively correlation with its expression levels.11 genes were differentially methylated in the exon regions.Moreover,the potential target genes for differentially methylated miRNA were predicted.The targets gene of 26 differentially methylated miRNAs captured 84 differentially expressed genes associated with immune,inflammatory,and defense responses in RNA-Seq.(4)CITED2,SLC40A1,LGR4 and NCF4 gene and bta-mir-15 a were screened out for further experimental verification.The methylation levels of CITED2 and SLC40A1 gene promoters were analyzed by bisulfite sequencing.The methylation levels of CITED2 and SLC40A1 genes in HC groups were significantly higher than those in the MC groups,respectively.The expression levels in HC groups were significantly lower than those in the MC groups.These results indicated that the methylation of CITED2 and SLC40A1 gene promoter regions affected gene differential expression and thus played an important role in the infection process of mastitis.A novel splice variant LGR4-TV was identified,which was characterized by the skipping of partial exon 5.The expression levels of LGR4-reference and LGR4-TV were found to be higher in the HC group than that in the MC group.The methylation level and exon 5 inclusion level of LGR4 gene were also found to be higher in the HC group than that in the MC group,which demonstrated that the methylation of LGR4 exon 5 regulated alternative splicing of exon 5.Meanwhile,the methylation levels of bta-miR-15 a promoter in the HC groups were significantly higher than that in the MC groups,the expression levels were the opposite.The target gene CD163 of bta-miR-15 a was identified.The expression level of target gene CD163 in the transcriptome were significantly higher in the HC groups than that in the MC groups.These results suggested that methylation changes of bta-mir-15 a in the promoter region affected the differential expression of bta-mir-15 a,and the differential expression of bta-mir-15 a caused the differential expression of target gene CD163,thus playing a regulatory role in the mastitis.(5)The promoter methylation and expression levels of NCF4 gene were verified in blood neutrophils of healthy and mastitis cows,which was consistent with sequencing results.In addition,we identified a SNP g.18475 A> G in the 3'UTR region of the NCF4 gene.By dual luciferase activity assay and association analysis with cow somatic cells,this SNP may affect the binding ability of NCF4 gene and bta-miR-2426,regulate the expression of NCF4 gene,which ultimately lead to the difference of mastitis resistance traits in Chinese Holstein cattle.In conclusion,this research investigated the DNA methylation and gene expression patterns of blood neutrophils in E.coli mastitis cattle,analyzed the molecular mechanism of DNA methylation in different regions regulation of gene expression,excavated the key genes that play a role in E.coli mastitis,which provide the theoretical foundation for the application of epigenetic marker in E.coli cows.