Integration Of Dna Methylome And Transcriptome Analysis Of Prenatal Skeletal Muscles Between Tongcheng And Yorkshire Pigs

Pigs are the main source of meat protein and can also be used as an important animal model for studying human health problems.Skeletal muscle is the most abundant tissue in animals.Therefore,it is of great importance to study the growth and development of pig skeletal muscle for meat production science and human medical science.In this study,Yorkshire(YK)pigs and Tongcheng(TC)pigs with significant differences in growth rate and meat quality were used as the subjects.RNA-seq was used to explore different time-series expression patterns of differentially expressed genes in longissimus dorsi muscles during 40,55,63,70,and 90 days of gestation respectively in TC and YK.The key time point which might lead to the differences of myogenesis between the two pig breeds was suggested.On this basis,the molecular mechanism of skeletal myogenesis in TC and YK pigs was explored.Differential methylation profiles of longissimus dorsi muscles at 55 days of gestation between TC and YK were detected using the whole genome bisulfite sequencing(WGBS),and the regulation of differential DNA methylation on skeletal muscle development in the two pig breeds was analyzed.Integration of transcriptome data and DNA methylome data could find the regulation mechanism of DNA methylation on differentially expressed genes between TC and YK.The main results are as follows: 1.RNA-seq was used to obtain the transcriptome expression profile of the longissimus dorsi muscle of Tongcheng and Yorkshire pigs during the five stages of pregnancy.The reliability of the sequencing results was verified by real-time quantitative PCR(qPCR).A total of 1317 and 691 differentially expressed genes(DEGs)were screened in TC and YK,respectively.STEM(Time-series Expression Miner)was used to analyze the time-series analysis of the development-dependent DEGs respectively in TC and YK.It was found that profiles 0,39,20 and 21 were significantly enriched in both pig breeds,profiles 10,11,13 and 25 were only significantly enriched in TC,and profiles 14 and 15 were only significantly enriched in Yorkshire.GO and KEGG pathway functional annotation and enrichment analysis were performed on the DEGs in TC and YK,respectively.The GO enrichment analysis of DEGs in the common significantly enriched profile 39 found that many significantly enriched GO terms were related to muscle growth and development.GO enrichment analysis of the DEGs in the common significantly enriched profile 0 found that many significantly enriched GO terms were related to cell cycle.Pathway enrichment analysis showed that 8 of the top 10 pathways significantly enriched for DEGs respectively in TC and YK were co-enriched in the two pig breeds,but the enrichment values were different.It indicated that the developmental mechanisms of embryonic longissimus dorsi muscle in the two pig breeds were similar,but they did have some differences to a certain extent.2.1,677 genes showed significantly differential expression between TC and YK at the five identical stages,most of which were found at 55 days of gestion.Functional annotation and enrichment analysis of GO and KEGG pathway were performed on the DEGs between TC and YK.8 significantly enriched pathways,such as Focal adhesion,ECM-receptor interaction,Oxidative phosphorylation etc.were found.Gene interaction network analysis of DEGs between TC and YK found that PTEN,EP300,MYO9 A,CDK14,IRS1,PPP1 CC and some ribosomal protein genes were suggested to be the key candidate genes for elucidating the developmental differences between the two breeds.3.Alternative splicing(AS)events were identified by ASprofile software,and TSS(alternative 5’ first exon)and TTS(alternative 3’ last exon)were found to be the main types of AS events in TC and YK.4.In this study,the genome-wide DNA methylation profile of longissimus dorsi muscle at 55 days of gestion in TC and YK were constructed by WGBS,and it was revealed that CG was the main DNA methylation type in the two pig breeds,only a small propotion of the methylcytosines occured on CHH and CHG sequences.The DNA methylation level in different genomic elements was analyzed,the DNA methylation level in intron and 3’ UTR was higher than the other genomic elements at CG sequence context,but the DNA methylation level was the highest in the CpG island(CGI)at CHG or CHH sequence context.5.211,822 differentially methylated regions(DMRs)were detected between TC and YK,and 12261 differentially methylated genes(DMG)were obtained.Among which,there were 36,082 DMRs corresponding to 5,301 DMGs hypermethylated in TC,and 175,240 DMRs corresponding to 11,746 DMGs hypermethylated in YK.The functional annotation and enrichment analysis of GO and KEGG pathway were performed on the DMGs between TC and YK.It was found that the DMGs were significantly enriched in many GO terms in the three different sequence contexts of CG,CHG and GHH;KEGG pathway enrichment analysis found that DMGs at non-CG sequence context were significantly enriched in many pathways,whereas DMGs in CG sequence context were only significantly enriched in Phosphatidylinositol signaling system.5946 DMRs corresponding to 4578 DMGs were detected in promoter region.GO and KEGG pathway enrichment analysis of these DMGs found that only DMGs in CHH sequence context were significantly enriched in some GO terms,but some GO terms related to muscle development were found among the mainly enriched GO terms at the three sequence contexts;DMGs at all the three sequence contexts were not significantly enriched in any pathway,but Ribosome was the common mainly enriched pathway,which may indicate that DNA methylation in promoter of the genes involved in Ribosome could regulate muscle development.6.Correlation analysis between DNA methylation level and gene expression level at the overall level found that the DNA methylation level was negatively correlated with the level of gene expression at these regions which was 2 kb upstream of gene transcription start site,and the other was closed to the transcription start site of gene body.And the level of DNA methylation was positively correlated with the level of gene expression at the region of the gene body near to transcription termination site.616 genes were found to be both DMGs and DEG,which were the DEGs suggested to be regulated by DNA methylation.Pathway enrichment analysis of the 616 genes revealed that there were five mainly enriched pathways playing an important role in muscle growth and development,they were focal adhesions,Wnt signaling pathway,Apoptosis-multiple species,ECM-receptor interaction and TGF-beta signaling pathway.7.Eukaryotic overexpression vectors of pcDNA3.1(+)-RPL6 and pcDNA3.1(+)-TPPP3 were constructed;the interference fragments of RPL6 and TPPP3 were synthesized and the interference fragments with the best interference efficiency were found.Immunofluorescence was used to detect changes in myotube number and size after interfering with the gene expression of RPL6 and TPPP3.The results showed that the number of myotubes in the two interfering groups was obviously smaller than that of the negative control group,and the size of myotubes became larger in the group of interfering with RPL6.Flow cytometry was used to examine the effect on cell cycle progression after interference with the gene expression of RPL6 and TPPP3.The results showed that RPL6 can regulate the cell cycle of C2C12 cells,interfering with RPL6 lead to the proportion of cell population in G1 phase in the group significantly higher than that in the group of negative control,and the proportion of cell population in S phase and G2 phase was significantly lower than that in the group of negative control.And TPPP3 may not regulate the cell cycle,the proportion of cell population in the group of interference with TPPP3 in all phases of the cell cycle was no significant difference with the group of negative control.Overexpression RPL6 was identified to increased the abundance of PCNA(a proliferation marker)by Immunofluorescence analysis...