Coordinated transcriptional and post-transcriptional epigenetic regulation during skeletal muscle development and growth in pigs

Background: N6-methyladenosine (m⁶A) and DNA 5-methylcytosine (5mC) methylation plays crucial roles in diverse biological processes, including skeletal muscle development and growth. Recent studies unveiled a potential link between these two systems, implicating the potential mechanism of coordinated transcriptional and post-transcriptional regulation in porcine prenatal myogenesis and postnatal skeletal muscle growth.

Methods: Immunofluorescence and co-IP assays were carried out between the 5mC writers and m⁶A writers to investigate the molecular basis underneath. Large-scale in-house transcriptomic data were compiled for applying weighted correlation network analysis (WGCNA) to identify the co-expression patterns of m⁶A and 5mC regulators and their potential role in pig myogenesis. Whole-genome bisulfite sequencing (WGBS) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were performed on the skeletal muscle samples from Landrace pigs at four postnatal growth stages (days 30, 60, 120 and 180).

Results: Significantly correlated expression between 5mC writers and m⁶A writers and co-occurrence of 5mC and m⁶A modification were revealed from public datasets of C2C12 myoblasts. The protein-protein interactions between the DNA methylase and the m⁶A methylase were observed in mouse myoblast cells. Further, by analyzing transcriptome data comprising 81 pig skeletal muscle samples across 27 developmental stages, we identified a 5mC/m⁶A epigenetic module eigengene and decoded its potential functions in pre- or post-transcriptional regulation in postnatal skeletal muscle development and growth of pigs. Following integrative multi-omics analyses on the WGBS methylome data and MeRIP-seq data for both m⁶A and gene expression profiles revealed a genome/transcriptome-wide correlated dynamics and co-occurrence of 5mC and m⁶A modifications as a consequence of 5mC/m⁶A crosstalk in the postnatal myogenesis progress of pigs. Last, we identified a group of myogenesis-related genes collaboratively regulated by both 5mC and m⁶A modifications in postnatal skeletal muscle growth in pigs.

Conclusions: Our study discloses a potential epigenetic mechanism in skeletal muscle development and provides a novel direction for animal breeding and drug development of related human muscle-related diseases.