Abstract
BackgroundDNA methylation is an epigenetic regulatory form that plays an important role in regulating the gene expression and the tissues development.. However, DNA methylation regulators involved in sheep muscle development remain unclear. To explore the functional importance of genome-scale DNA methylation during sheep muscle growth, this study systematically investigated the genome-wide DNA methylation profiles at key stages of Hu sheep developmental (fetus and adult) using deep whole-genome bisulfite sequencing (WGBS).ResultsOur study found that the expression levels of DNA methyltransferase (DNMT)-related genes were lower in fetal muscle than in the muscle of adults. The methylation levels in the CG context were higher than those in the CHG and CHH contexts, and methylation levels were highest in introns, followed by exons and downstream regions. Subsequently, we identified 48,491, 17, and 135 differentially methylated regions (DMRs) in the CG, CHG, and CHH sequence contexts and 11,522 differentially methylated genes (DMGs). The results of bisulfite sequencing PCR (BSP) correlated well with the WGBS-Seq data. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis revealed that some DMGs were involved in regulating skeletal muscle development and fatty acid metabolism. By combining the WGBS-Seq and previous RNA-Seq data, a total of 159 overlap genes were obtained between differentially expressed genes (DEGs) and DMGs (FPKM > 10 and fold change > 4). Finally, we found that 9 DMGs were likely to be involved in muscle growth and metabolism of Hu sheep.ConclusionsWe systemically studied the global DNA methylation patterns of fetal and adult muscle development in Hu sheep, which provided new insights into a better understanding of the epigenetic regulation of sheep muscle development.
Highlights
DNA methylation is an epigenetic regulatory form that plays an important role in regulating the gene expression and the tissues development
DNA methyltransferase (DNMT) expression levels The expression levels of DNMTs (DNMT1, DNMT3A, and DNMT3B) in Longissimus dorsi (LD) muscle of fetal and adult sheep were first analyzed by Quantitative reverse transcriptionPCR
Genome-wide DNA methylation profiling Global DNA methylation analysis of the LD muscle was performed by whole-genome bisulfite sequencing (WGBS) with 30× genome coverage and > 99% conversion efficiency
Summary
DNMTs expression levels The expression levels of DNMTs (DNMT1, DNMT3A, and DNMT3B) in LD muscle of fetal and adult sheep were first analyzed by Quantitative reverse transcriptionPCR (qRT-PCR). The results showed that the DMRs of ADIPOQ and ITGA1 genes were in distal intergenic and intron respectively, trends of DNA methylation levels of those genes were consistent with those of expression. QRT-PCR results showed that the expression level of the ADIPOQ gene was upregulated with development, Fig. 6 Heat map cluster analysis of differentially methylated regions (DMRs) in different gene functional regions. DIAPH1 and NR4A1 genes have DMRs in intron and distal intergenic besides DMRs in promoter regions, the DNA methylation levels of the DIAPH1 and NR4A1 genes were downregulated at the adult stage, which was the opposite of that observed for their expression levels. The results indicated that DNA methylation in promoter regions of MAPT, DIAPH1, NR4A1, and DLK1 affected their gene expression levels during skeletal muscle development.
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