Genome-wide methylation and transcriptome of blood neutrophils reveal the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-infected mastitis cows

- The methylation levels in the mastitis cows (MCs) were decreased compared with healthy cows (HCs).
- Twenty-nine genes were found in methylation and transcription data, among which seven genes ’ promoter methylation levels were negatively correlated with expression levels, and 11 genes were differentially methylated in the exon regions.
- coli -induced mastitis, thereby helping explain the function of DNA methylation in the pathogenesis of mastitis and provided new target genes and epigenetic markers for mastitis resistance breeding in dairy cattle..
- Mastitis, one of the most prevalent diseases in the dairy cat- tle industry, leads to great economic losses caused by re- duced milk production, discarded milk, early culling, veterinary services, and labor costs [1].
- the disease may cause several deaths per year in the most severe cases [2]..
- The mastitis-causing pathogens induce inflammation re- sponses in the mammary gland.
- They are present in the bloodstream until signaled to an infection site by the body’s chemical cues.
- In response to the inflammation from bovine mastitis, the host secretes corresponding cytokines that cause changes in the regulation of gene expression [3].
- DNA methylation is involved in the occurrence and development of bovine mastitis [8–.
- The present study aimed to analyze the genome-wide DNA methylation patterns of blood neutrophils in healthy and E.
- The average SCC in the milk of MC group was mL, which was significantly higher than HC group mL (P <.
- In the healthy group, the presence of bacteria growth in the culture plate was not observed.
- Genome-wide DNA methylation patterns of bovine blood neutrophils.
- The figure showed that DNA methylation levels of E.
- The DNA methylation levels were higher in the promoter region, decreased dramatically around the transcript start site, and increased sharply toward the gene’s first exon.
- The methylation levels of total methylated and differentially methylated genes in the mastitis cows were decreased compared with healthy cows.
- A total of 75 regions were located in the exons of genes, whereas 344 regions were located in the introns of genes (Fig.
- www.animalgenome.org/cgi-bin/QTLdb/BT/search), we screened differential methylation genes that are located within cattle QTLs and identified a total of 69 differen- tially methylated genes harbored in the QTLs associated with clinical mastitis, somatic cell count and somatic cell score (Additional file 1: Table S2).
- For example, the RBPJ and HERC5 genes are located in the somatic cell score QTL #10439 region of chromosome 6.
- The RBPJ and HERC5 genes may play roles in the mastitis infec- tion via DNA methylation’s influence.
- 1 Basic information of the three healthy and three E.
- The definitionss in the following figures are the same as the first one.
- Twenty-six differentially methylated miRNAs, including 8 up-methylated and 18 down- methylated miRNAs, were identified in the healthy and mastitic groups (Additional file 1: Table S3).
- The 37 differentially expressed genes are located in the QTL regions related to mastitis, and involved in immune, inflammatory, and defense responses, among which 15 genes have protein interactions (Additional file 2: Figure S2B).
- Of these genes, identified gene numbers in the promoter and exon differential methylated regions were 7 and 11, respectively.
- Seven differentially methylated and -expressed genes were associated with im- mune and inflammation responses in the RRBS and RNA- seq sequencing (Table 1), such as the SLC40A1, CITED2, and LGR4 gene.
- The CITED2 and SLC40A1 genes were hypomethylated in the promoter regions and up-regulated mRNA expression.
- The LGR4 gene was hypomethylated in the exon region and down-regulated mRNA expression..
- The targets of 26 miRNAs captured 84 differentially expressed genes in the HC and MC groups (Additional file 2: Figure.
- The BSP sequen- cing results demonstrate that the average methylation levels of the promoter region of the CITED2 gene in.
- The difference was significant, and the methylation rate in the healthy group was higher compared with the mastitis group (P <.
- The average methylation levels of the promoter region of the SLC40A1 gene in healthy and mastitic cows were 98.09 and 85.71%, respectively, which were significantly differ- ent (P <.
- The expression levels of the CITED2 and SLC40A1 genes in mastitis cows were sig- nificantly higher in healthy cows (P <.
- 6 The methylation and expression levels of the CITED2 and SLC40A1 genes in the neutrophils of healthy and mastitic cows.
- Relative mRNA expression level of the CITED2 gene (c) and the SLC40A1 gene (d)..
- The differentially methylated region of the LGR4 gene was located on gene exon 5 in the RRBS sequencing.
- The LGR4 gene exhibited alternative splicing pattern and dif- ferential expression in healthy and mastitic cows in the RNA-seq.
- We ampli- fied the differentially methylated region of the exon5 CpG island in the LGR4 gene, and the amplified fragment con- tained 10 CpG sites.
- The average methylation level of the LGR4 gene exon 5 in the healthy groups was 23.33%, and the average methyla- tion level in the mastitic groups was 12.67%.
- The LGR4 methylation level was decreased in the mastitic groups compared with the healthy groups (P <.
- To identify the presence of alternative splicing phenomenon in the exon 5 region of the LGR4 gene, we amplified the coding region of the LGR4 gene, and se- quenced, and compared with the mRNA sequence of the.
- The alignment results showed that a new transcript, named LGR4-TV, was identified in the blood neutrophils of cows.
- Therefore, exon 5 of the LGR4 gene is considered the al- ternative splicing exon (Fig.
- The relative exon inclusion levels of the LGR4 gene exon 5 were significantly higher in the neutrophil of healthy cows than in those of mastitic cows (P <.
- The results indicated that the higher methylation level of the LGR4 gene exon 5 in the healthy groups may promote the inclusion level of the LGR4 gene alternative splicing exon 5..
- 7 Experimental verification of the LGR4 gene in the neutrophil of healthy and mastitic cows.
- b Methylation levels of the LGR4 gene exon 5 region.
- d The relative exon inclusion levels of the LGR4 gene exon 5Note: In (d), the relative exon inclusion level was calculated by dividing the amount of the inclusion isoform with the amount of the skipping isoform.
- The methylation level of bta-miR-15a in the healthy group was significantly higher than in the mastitis group (P <.
- The mRNA expression of the CD163 gene in blood neutrophils were detected using real-time PCR.
- The expression levels of the CD163 gene in healthy cows were significantly higher than mastitic cows (P <.
- A positive correlation existed be- tween the promoter methylation level of bta-miR-15a and the expression level of the target gene CD163..
- We determined that overall DNA methylation levels of genome were decreased after E.
- We demonstrated that DNA methylation in promoter regions affected the differential expression of immune-related genes, and DNA methylation in the exon regions regulated the alternative splicing pattern of genes.
- coli mastitis infection and identified genes CITED2, SLC40A1, and LGR4 that regulate the differential expression of genes by DNA methylation in the E.
- In the early stage of pathogen infection, sufficient number of neutro- phils, which are rapidly recruited into the mammary gland, increase phagocytosis and prevent establishment of mastitis.
- In the present study, we extracted neutrophils from the milk venous blood and determined that the total amount of neutrophils in mastitic cows were higher com- pared with healthy cows..
- [11] used MeDIP-chip assay to obtain the overall DNA methylation profiles of the bovine peripheral blood lymphocytes from healthy and S.
- The genome DNA methylation maps of healthy and E.
- coli mastitic cows across chromosomes were drawn in the present study.
- The DNA methylation levels in the neutrophil were down-regulated in E.
- The differences between the two research may be due to the differences in the experiment.
- lymphocytes were used in the study on S..
- Different patho- gens can cause different immune responses in the mam- mary gland, which trigger different signaling pathways, thereby causing differences in the gene expression regula- tion of the corresponding pathways [21, 22]..
- some of these play important roles in the course of neutrophils’ recognition of bacteria and moving toward the infection site.
- CD38-deficient neu- trophils are defective in chemotaxis and cannot accumu- late in the infected tissue of mice [28].
- miRNA also plays an important regulatory role in the immune and inflamma- tory responses of cows with mastitis [31, 32].
- In the present study, we examined the differentially methylated miRNAs in healthy and E.
- Of these 26 differentially methylated miRNAs, bta-mir-15a and bta-miR-146a plays an important role in the regula- tion of mammary inflammation [16, 33].
- An association analysis of DNA methylation and gene ex- pression data in the cow blood neutrophils was performed..
- The CITED2, SLC40A1, and LGR4 genes were included in the 29 differentially methylated and expressed genes.
- In addition to the well-known effects of promoter methylation on the silencing of gene expression, DNA methylation is also involved in the regulation of gene al- ternative splicing [25].
- In the present study, a novel splice variant of the LGR4 gene was characterized by the deletion of a 139 bp sequence in exon 5.
- The rela- tive exon inclusion levels, and methylation levels of the LGR4 gene exon 5 were significantly higher in healthy cows than in mastitic cows.
- Therefore, we specu- lated that DNA methylation of the LGR4 gene exon 5 may regulate the alternative splicing of the LGR4 gene, thereby affecting its expression..
- Such studies greatly accelerate the progress in understanding the roles of DNA methylation in the mastitis infection process and providing new target genes and epigenetic markers for mastitis resistance breeding in dairy cattle..
- The differentially methylated genes lo- cated in the quantitative trait locus (QTL) associated with clinical mastitis (CM), somatic cell count, and somatic cell score (SCS) were screened through AnimalQTL (http://.
- The expression levels of the CITED2 and SLC40A1 genes were identified by quantitative real-time PCR (qPCR).
- A new transcript LGR4-TV was identified in the blood neutrophils of cow.
- The relative exon inclusion levels of the LGR4 gene in neutrophils of healthy and mastitis cows were calculated by dividing the amount of inclusion isoform with the amount of the skipping isoform..
- Therefore, the expression levels of the CD163 gene was also identified by quantitative real-time PCR.
- Summary of the differentially methylated genes in the two libraries.
- A total of 494 DMRs including 61 up- methylated genes and 433 down-methylated genes were identified in the healthy and mastitic groups.
- Summary of the differentially methylated genes harboring in the QTLs associated with clinical mastitis, somatic cell count and somatic.
- Summary of the differentially methylated miRNAs in the two libraries.
- A total of 26 differentially methyl- ated miRNAs including 8 up-methylated miRNAs and 18 down- methylated miRNAs were identified in the healthy and mastitic groups..
- Summary of the differentially expressed genes in the two groups.
- down-regulated genes were identified in the healthy and mastitic groups..
- Summary of differentially methylated and expressed genes in the RRBS and RNA-seq sequencing.
- Regulatory network of differentially methylated miRNAs (yellow circles) in the RRBS sequencing and their putative target genes (blue circles) in the RNA-seq.
- The funding bodies had no role in the study design, collection, analysis and interpretation of data, and in the writing of the manuscript..
- The role of DNA methylation in mammalian epigenetics..
- DNA-remethylation around a STAT5-binding enhancer in the alphaS1-casein promoter is associated with abrupt shutdown of alphaS1-casein synthesis during acute mastitis.
- Epigenetic mechanisms contribute to enhanced expression of immune response genes in the liver of cows after experimentally induced Escherichia coli mastitis..
- The alternative role of DNA methylation in splicing regulation.
- DNA methylation landscapes: provocative insights from epigenomics.
- DNA methylation regulates microRNA expression.
- Negative feedback regulation of NF- κ B action by CITED2 in the nucleus.
- Genetic mutations potentially cause two novel NCF1 splice variants up-regulated in the mammary gland, blood and neutrophil of cows infected by Escherichia coli..
- Integrated analysis of mRNAs and long noncoding RNAs in the semen from Holstein bulls with high and low sperm motility

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