Genome-Wide DNA Methylation Sequencing Identifies Methylation-Associated Genes Related to Porcine Skeletal Muscle Fiber Diversity

Alterations in skeletal muscle fibers are closely linked to human diseases, yet the mechanisms by which DNA methylation drives muscle fiber diversity remain unclear. Here, we performed whole-genome bisulfite sequencing (WGBS) to generate single-base-resolution DNA methylomes of fast-twitch biceps femoris (Bf) and slow-twitch soleus (Sol) muscles and integrated transcriptomes to reveal associations between DNA methylation and gene expression. The global methylation level in skeletal muscle averaged 4.22%, with context-specific rates of 70.0% (CG), 1.16% (CHG), and 1.12% (CHH). The distribution of methylation levels of the mCs in CG and CHG contexts was similar but distinct from that in the CHH context. Furthermore, 152,286 DMCs and 51,079 DMRs were identified between Bf and Sol muscles. Bf showed higher global methylation (4.54%) than Sol (3.90%), and distinct differences in DNA methylation occurred throughout the genomic regions and gene functional element regions. CG-context DMCs and DMRs were mainly located in CpG islands and introns, while CHG and CHH DMCs and DMRs were exon-enriched. DMR-associated genes were significantly enriched in pathways related to muscle growth and remodeling and disease-related pathways. Moreover, five DMR-linked differentially expressed genes—HSPB6, ATP2A2, TNNI1, HOXA11, and PTPN3—were identified as critical candidate regulators of muscle fiber diversity. In addition, a context-dependent (CG, CHG, CHH) relationship between DNA methylation patterns and gene expression was observed across different genomic regions in the two muscle types. Our findings highlight the essential role of DNA methylation in skeletal muscle fiber diversity and identify potential epigenetic targets and pathways.