Screening Of Methylated DNA Sites Relating To Hypoxia Adaptation And Gene Expression Of Epigenetic Modifying Enzymes In Pig

Low pressure and low partial pressure of oxygen of plateau affects survival and development of human and animals, so hypoxia response and hypoxia adaptation are always focus of biological and medicine. To deal with hypoxia, the organism will activate and stable HIFs, also needs some epigenetic regulation, DNA methylation is one of the most important epigenetic machnisms. Tibet pigs live at Qinghai-Tibet Plateau, their adaptation to hypoxia makes them precious species resource and object for hypoxia adaptation machnism. We selected four groups of pig as the study object, which were Tibet pig in highland (TH), Yorkshire in highland (YH), Tibet pig in lowland (TL), Yorkshire in lowland (YL). Using methylated DNA immunoprecipitation and subsequent high throughput sequencing (MeDIP-Seq), and combining RNA-Seq results, we filtered key genes related to hypoxia adaptation that were differentially methylated and differentially expressed simultaneously. We conducted clone sequencing and real time PCR to validate these high throughput results. mRNA semi-quantitative and quantitative assay were conducted for five epigenetic modifying enzymes to reveal their expression differences.486M reads was obtained from MeDIP-Seq, and there were about60K peaks for each individual. Then we used MEDIPS package to obtain differentially methylated regions which were annotated in UCSC. Cluster analysis of differentially methylated genes (DMGs) from MeDIP-Seq assay showed that breed affect DNA methylation level much more than altitude. GO and KEGG pathway analysis results showed that, in condition of hypoxia, DMGs between two breeds were related to PPAR pathway and Fatty acid elongation GO term. From normoxia to hypoxia, Tibet pigs changed DNA methylation of hypoxia adaptation related pathway(VEGF and PPAR).We selected five genes that may play important roles in hypoxia adaptation from DMRs that were both differentially methylated and expressed:EPHX2, RXRG, BCKDHB, UBD and GOT2. Clone sequencing and real time quantitative PCR were implemented for these five genes, and validation results showed that RXRG was both differentially methylated and expressed between different groups; although BCKDHB and EPHX2was differentially methylated, there were no significantly difference between four groups; and there were neither significant difference of methylation nor significant difference of expression between different groups. The methylation difference and expression difference of these five genes had the same trend with that resulted from MeDIP-Seq and RNA-Seq.These five epigenetic modifying genes presented tissue specific expression patterns. Dnmtl and Uhrfl expressed highly in heart, lung and pituitary gland, and the other three genes expressed highly in lung, kidney and pituitary gland. In lung, all these genes expressed higher in TH than that in Yorkshire. In kidney, from nomoxia to hypoxia, genes expressed lower in Tibet pigs, but higher in Yorkshire.This research obtained DNA methylation profile of heart using four groups of pigs, then selected and validated five genes whih may play roles in hypoxia adaptation, validation turned out that RXRG was both differentially methylated and expressed between different groups. We also conducted semi-quantitative expression analysis and quantitative expression analysis of Dnmtl, Dnmt3a, Kdm3a, Hdacl and Uhrfl, results suggested that lung and kidney may play important roles of epigenetic regulation to adapt to hypoxia. In lung, in condition of hypoxia, most genes in Tibet pigs maybe were down regulated compared to Yorkshire. In kidney, from nomoxia to hypoxia, hypoxic response or adaptation may induce many genes’ decreased expression. All these results will be useful to reveal the epigentic regulation of hypoxia adaptation and useful to provide suggestions to do research towards hypoxia adaptation related genes.