Regulation of proline-directed kinases and the trans-histone code H3K9me3/H4K20me3 during human myogenesis.

We present a system-level analysis of proteome, phosphoproteome and chromatin state of precursors of muscle cells (myoblasts) differentiating into specialized myotubes. Using stable isotope labeling (SILAC) and nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), we found that phosphorylation motifs targeted by the kinases PKC, CDK and MAPK showed increased phosphorylation during myodifferentiation of LHCN-M2 human skeletal myoblast cell line. Drugs known to inhibit these kinases either promoted (PD0325901 and GW8510) or stalled (CHIR99021 and roscovitine) differentiation, resulting in myotube-and myoblast phenotypes, respectively. The proteome especially the myogenic and chromatin-related proteins, including histone methyltransferases correlated with their phenotypes, leading us to quantify histone post-translational modifications (PTMs) and identify two gene-silencing marks, H3K9me3 and H4K20me3, with relative abundances changing in correlation with these phenotypes. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) demonstrated that H3K9me3 is erased from the gene loci of myogenic regulatory factors (MRFs) namely myoD , myoG and myf5 in differentiating myotubes. Together, our work integrating histone PTM, phosphoproteomics and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during myodifferentiation in vitro .