Genome-wide Analysis Of Transcriptome And DNA Methylation Regulation In Antiviral Response Of Porcine Peripheral Blood Mononuclear Cells

It is crucial to genetically improve the disease reisistance of pigs for tackling disease challenges which severely hamper the development of pig industry. Disease resistant traits are complex and vulnerable to environmental variations which cannot be thoroughly explained by classical genetics. The development of epigenetic provides us new insights into understanding of genetic regulatory mechanisms underlying disease resistant traits and identification of associated genetic markers. However, rare studies now focus on the epigenetic landscape of pig diseases. Using genome-wide sequencing of transcriptome and methylome of pig peripheral blood mononuclear cells (PBMCs) in response to viral infection, this study explored the changes in gene expression and DNA methylation as well as the role of DNA methylation in regulating immune-related gene. This study aimed to epigenetically reveal the regulatory mechanisms for disease resistance and immune responses, and identify genes or DNA biomarker associated with high immune response, thus providing new evidence for genetic reisistance breeding of pigs.In this study, polyinosinic-polycytidylic acid (Poly I:C), an artificial surrogate for double-stranded RNA virus, was used to stimulate PBMCs ex vivo. We considered 20 ug/ml of Poly I:C and 24 h was the optimal concentration and cultural time by comparative analysis of the expression levels of immue-related genes (IL6、IL8、TNFα、IFNα、IFNγ、1RF3、TLR3). PBMCs were isolated from six piglets at 35 days old (3 individuals of Dapulian and Landrace piglets, respectively). The PBMCs from each individual were divided into two groups, of which one group was treated with Poly I:C with the final concentration of 20 ug/ml, the other group was set as control by adding PBS, and both groups were cultured for 24 h. The differences in transcriptome and DNA methylome between Poly I:C-treated and control PBMCs were analyzed.Comparative analysis of gene expression patterns in two breed pigs identified 218 and 226 differentially expressed genes specific to Dapulian and Landrace pigs, respectively (FDR< 0.05). Functional annotation analysis revealed that more genes specific to Dapulian pig were enriched in immune response-related terms. Compared with the findings of previous studies focusing on antiviral responses in pigs, seven genes (LAG3、DUSP1、MGP、MS4A7、DNM1、CYP1A1, HNF4A) were considered as promising candidate that potentially influence the antiviral immune reponses.This study profiled the porcine PBMC DNA methylome, in which regions with the densities of 51 to 55 CpGs/kb and CpGo/e ratio close to 0.60 tending to exhibit a higher methylation level, methylation level in subtelomeric regions in chromosomes significantly higher compared with non-subtelomeric regions, DNA methylation levels tending to decrease sharply at TSS and to increase dramatically towards gene bodies, and the CGIs showing higher methylation level than CGI shores. Comparative methylome analysis identified 5,827 differentially methylated regions (DMRs), which were associated with 1,890 genes. Of which 68 genes were identified to be differentially methylated and expressed. After Poly I:C stimulation, the PACSIN1 gene was significantly downregulated and the promoter region was significantly hypermethylated. Functional analysis of promoter methylation demonstrated the PACSIN1 gene expression was suppressed by promoter hypermethylation. RNAi knockdown of PACSIN1 revealed significant higher expression of IL6、IL8、TNFα、NECAP2. This result indicated that DNA methylation negatively regaluates the expression of PACSIN1 by decreasing the transcriptional activity and facilitates proinflammatory cytokine production, and thus enhancing antiviral responses.This study established an ex vivo model system in which porcine PBMCs were treated with Poly I:C for mimicking viral infection. The findings revealed genome-wide DNA methylomic and transcriptomic variations involved in antiviral response and the regulatory role of DNA methylation in gene expression. The present study will advance our understanding of epigenetic response to viral infection, and it is expected to constitute a valuable resource for future epigenetic epidemiology study in pigs and for the analyses of tissue specific DNA methylation pattern.