TY - JOUR
T1 - Regulation of proline-directed kinases and the trans-histone code H3K9me3/H4K20me3 during human myogenesis
AU - Bhanu, Natarajan V.
AU - Sidoli, Simone
AU - Yuan, Zuo Fei
AU - Molden, Rosalynn C.
AU - Garcia, Benjamin A.
N1 - Publisher Copyright:
© 2019 Bhanu et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/5/17
Y1 - 2019/5/17
N2 - Wepresent a system-level analysis of proteome, phosphoproteome, and chromatin state of precursors of muscle cells (myoblasts) differentiating into specialized myotubes. Using stable isotope labeling of amino acids in cell culture and nano-liqud chromatography-mass spectrometry/mass spectrometry, we found that phosphorylation motifs targeted by the kinases protein kinase C, cyclin-dependent kinase, and mitogen-activated protein kinase 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 proteomes, especially the myogenic and chromatin-related proteins including histone methyltransferases, correlated with their phenotypes, leading us to quantify histone post-translational modifications and identify two genesilencing marks, H3K9me3 and H4K20me3, with relative abundances changing in correlation with these phenotypes. ChIP- quantitative PCR demonstrated that H3K9me3 is erased from the gene loci of myogenic regulatory factors namely MYOD1, MYOG, and MYF5 in differentiating myotubes. Together, our work integrating histone post-translational modification, phosphoproteomics, and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during myodifferentiation in vitro.
AB - Wepresent a system-level analysis of proteome, phosphoproteome, and chromatin state of precursors of muscle cells (myoblasts) differentiating into specialized myotubes. Using stable isotope labeling of amino acids in cell culture and nano-liqud chromatography-mass spectrometry/mass spectrometry, we found that phosphorylation motifs targeted by the kinases protein kinase C, cyclin-dependent kinase, and mitogen-activated protein kinase 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 proteomes, especially the myogenic and chromatin-related proteins including histone methyltransferases, correlated with their phenotypes, leading us to quantify histone post-translational modifications and identify two genesilencing marks, H3K9me3 and H4K20me3, with relative abundances changing in correlation with these phenotypes. ChIP- quantitative PCR demonstrated that H3K9me3 is erased from the gene loci of myogenic regulatory factors namely MYOD1, MYOG, and MYF5 in differentiating myotubes. Together, our work integrating histone post-translational modification, phosphoproteomics, and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during myodifferentiation in vitro.
UR - http://www.scopus.com/inward/record.url?scp=85066121490&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA118.004977
DO - 10.1074/jbc.RA118.004977
M3 - Article
C2 - 30872405
AN - SCOPUS:85066121490
SN - 0021-9258
VL - 294
SP - 8296
EP - 8308
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 20
ER -