The myoblast methylome: Multiple types of associations with chromatin and transcription

Epigenetic changes are implicated in repair and physiological responses of postnatal skeletal muscle (SkM) as well as in prenatal embryogenesis. This study examined the epigenomics and transcriptomics of human myoblasts (SkM progenitor cells) vs. those of diverse other cell and tissue types from both global genomic and single-gene perspectives. Using enzymatic methyl-seq (EM-seq) and whole-genome bisulfite sequencing (WGBS) of myoblast DNA and available WGBS profiles of other cell cultures, we determined myoblast differentially methylated regions (DMRs). We also examined several DMRs in reporter gene assays. Among the unusual findings was the positive association of promoter-adjacent hypermethylation in myoblasts with transcription turn-on for some genes (e.g., SIM2 and TWIST1), but at down-modulated levels. In contrast, some genes, like the brain-specific OLIG2, were silent and embedded in repressed chromatin in myoblasts and many diverse cell populations but displayed myoblast-specific hypermethylated promoters. OLIG2 exhibited binding of the myogenic MYOD transcription factor in its vicinity. One OLIG2-downstream MYOD+ site overlapped enhancer chromatin in myotubes. These findings suggest that OLIG2 silencing only by repressive chromatin is insufficient in myoblasts without the addition of DNA hypermethylation. The numerous hypomethylated DMRs at enhancer chromatin in myoblasts were bound by MYOD much more frequently than by CTCF. Combined with previous studies, our results suggest a more prevalent role for MYOD than for CTCF in organizing transcription-enhancing chromatin interactions at hypomethylated regions. We also found that myoblast-hypomethylated enhancer chromatin enriched in H3K36me3 was much more likely to be associated with genes preferentially expressed in myoblasts than were such regions without H3K36me3 enrichment. Therefore, there may be a special role in gene upregulation for the regional combination of DNA hypomethylation with H3K27ac, H3K4me1, and H3K36me3 enrichment. The unusual relationships between epigenetics and gene expression that we observed need further study in the SkM lineage to better understand fine-tuning of muscle differentiation and diseases. Overall design: DNA was purified from non-transformed myoblast primary cultures derived from quadriceps biopsies of control individuals (Tsumagari et al., 2013), namely, CM1 (42-year-old female), CM3 (46-year-old male), and CM6 (45-year-old male). Cell cultures had been carefully generated and passaged in collaboration with Rabi Tawil (Tsumagari et al., 2011). Each batch of myoblasts used for DNA methylation analysis was checked for >90% purity by desmin immunostaining because fibroblast-like contaminating cells can easily outgrow myoblasts and give misleading methylome results (Chandra et al. 2015). WGBS was performed on CM3 using standard protocols (Chen F, Zhang G, et al. 2016). Methylation profiling of CM3, CM1, and CM6 was done using enzymatic methyl-seq (EM-seq) (Vaisvila et al. 2021). This process involved enzymatic oxidation of 5mC and 5hmC (by TET2), providing protection from further deamination by APOBEC3A while unmethylated cytosine are deaminated to uracil. Briefly, 0.2 µg of DNA was used to prepare EM-seq libraries in duplicate. The libraries were then cleaned using NEBNext® sample selection beads, pooled, and diluted to 1.5 nM for sequencing on a NovaSeq 6000(Illumina).