Aging-related DNA Methylomes In Porcine Skeletal Muscles

The growth and development of skeletal muscle postnatal belongs to the typical quantitative character, and it is regulated by the regulation net that mainly consists of related minorgene. The DNA methylation plays a key role in regulation of these functional genes and the transcription of regulatory element.A total of six healthy Jinhua pigs of two age groups were used in this study.3pigs in180days old and3pigs in seven years old (the average age in2,475days). The longissimus dorsi muscles were sampled rapidly from each carcass and were analyzed for the DNA methylation by using the MeDlP-Seq approach. Moreover, we also investigated the differences of mRNA transcriptomes between these two age stages. This study preliminarily revealed the methylation and transcriptional differences of the Longissimus dorsi in two age stages.I The average diameter of the Longissimus dorsi muscle were significantly (P<0.01) different between the180days and7years groups, the values were (44.85±8.60) μm and (54.89±10.00) μm, respectively; and cross sectional area of180days-aged and7years-aged also were significantly(P<0.01), the values were (1680.96+657.44) μm2and (2544.70+1035.85) μm2, respectively.II The lipoprotein α content of180days aged pigs (103.10mg/L) were higher than that of the7years-aged pigs (94.10mg/L)(P>0.05); the cholesterol of180days aged pigs was31.3%higher than that of7years-aged pigs (P=0.004); the low density lipoprotein of180days aged pigs was37.3%higher than that of the years aged pigs (P=0.03).III A total of46Gb methylated DNA immunoprecipitation sequencing (MeDIP-seq) data were generated from six samples (about7.64Gb data for each sample), of which75%unique reads (-36.56Gb) were uniquely mapped to the pig genome.(i) We found that methylation level across the choromosome negatively correlated with chromosomal length (Pearson’s r=0.633, P=0.0036) and positively correlated with GC content (Pearson’s r=0.787, P=6.49×10-5), SNP density (Pearson’s r=0.549, P=0.0149), gene density (Pearson’s r=0.516, P=0.0236),density of repeat region (Pearson’r=0.0348, P=0.145). and especially with CpGo/e ratio (Pearson’s r=0.931, P=7.41×10-9).We also found the different methylation level presented in the subtelomeric region of each chromasome.(ii) ICP in both young and middle-aged pigs exhibited relativley higher methylation level than HCP and LCP (P<0.01). The majority of methylated CpG Island located in intragenic and intergenic regions, whereas the CpG Island in promoter showed hypomethylated. It demonstrated that ICPs were more susceptible to methylation and this kind of alteration of methylation status in ICP may play important role in modulating relevant gene expression and involved in some biological process such as aging.IV In total9,234DMR were identified which accounted for6.41%of genomic length and28%of genomic CpG number..(i) For promoters, more DMRs were enriched in ICPs when compared with HCPs and LCPs. Distal(D), intermediate (Ⅰ) and proximal (P) regions of promoters contained121(occupied44%),82(occupied30%) and72(occupied26%), respectively. This result reveals the D regions of promoters contained14%more of DMRs than I regions, and18%more of DMRs than P regions. The vast majority of DMRs (5,567DMRs which occupied58.98%of gene bodies) located in intergenic followed by the medial intron (2,378DMRs, occupied25.19%of gene bodies). The first exon contained relatively low DMRs within the gene body. DMRs enriched in CGI (168) and CGI Shore (875).Then, in CGI Shore, there were287(occupied27.52%of all CGI Shore),224(occupied21.47%of all CGI Shore) and270(occupied25.98%of all CGI Shore)(ii) In order to know the change of expression, functional enrichment analysis of differential methylated genes (228genes, P<0.05)was employed. The results revealed a total of52GO and pathways including the pathway of mitogen-activated protein kinase (MAPK), which may have an effect on skeletal muscle development. Based on the results, we can speculate the molecular basis of muscle phenotypic variation between the180days-aged and the7years-aged pigs.V(i) Reliability of chip data:It is revealed that the chip data is reliable and accurate. Repeatability analysis indicated that average coefficient of variation (C. V) within arrays were between3.5%～6.5%for expression profilingof longissimus dorsi muscle. The Box Plot also showed the similar dispersion. These resultes demonstrate that the microarray technique used in this study is accurate and reproducible.(ii) A total of43,663probes (size in60mer) were generated from six samples, of which23,283unique probes were aligned to the pig genome and were mapped to22043genes. There were total4,184differently expressed genes (P<0.05), of which2,012were up-regulated and2,172were down-regulated in the comparision of7yeras vs.180days. And among them3,212(7.36%) genes was unigene.(iii) The top ten Go and pathways like as heat shock fator, transforming growth factor and extracellular matrix,which may regulate growth and development of muscle between180days-aged and7years-aged pigs.VI Of the888differently expressed genes, pomoter region of46genes showed different methylation level, of which16genes showed positively correlation (r=0.759, P=0.0007) between the promoter methylation and mRNA expression level, eg. MYO5C、ALDOC and MYL12B et al.; and30genes showed negtively correlation (r=-0.647, P=0.0001), eg. GPR143、 GRN and NTF32et al. The genes of ALDOC and KCNB1result by BSP also verfied the same trends in the DMR.VII The same GO or pathway was mapped of the difference genes in DMRs and mRNA revealed that there have5GO or pathway, including the circulatory system process, iron ion binding and ATPase activity,etc.These results highlight some possible candidate functional genes that have great influence and more research value for the growth and development of skeletal muscle of porcine, and preliminary revealed the partial molecular interaction mechanism of the gene exprssion and regulation during the growth and development of skeletal muscle with aging, and also reaveld the epigenetic regulation pattern in the formation of macroscopical phenotypic difference in porcine skeletal muscle.