Overexpression of DNA methyltransferase in myoblast cells accelerates myotube formation.
We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation-phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase-transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse-oriented plasmid. At the early stage of transfection of the sense-oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense-oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoD1 was already fully expressed under the growth conditions while, in control cells, MyoD1 was expressed at low levels. This elevated level of MyoD1 transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaII sites in exon 1 through exon 2 of the MyoD1 gene and the expression of the MyoD1 transcript are positively correlated.
Takagi
H
H
Institute for Protein Research, Osaka University, Japan.
Tajima
S
S
Asano
A
A
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D003402:Creatine Kinase / Q000096:biosynthesis; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D007122:Immunoelectrophoresis; D008745:Methylation; D051379:Mice; D009132:Muscles / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D017570:MyoD Protein / Q000235:genetics*; D009218:Myosins / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D014162:Transfection
D018076:DNA, Complementary; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D003596:Cytosine; D004247:DNA; D015254:DNA Modification Methylases; D003402:Creatine Kinase; D009218:Myosins