Induction of endogenous myosin light chain 1 and cardiac alpha-actin expression in L6E9 cells by MyoD1.
Rat L6E9 muscle cells commit to terminal differentiation by forming a large muscle syncitia complete with the expression of a large number of muscle-specific contractile protein genes. To determine whether these cells, which fail to synthesize MLC (myosin light chain) 1 and cardiac alpha-actin, exhibit a deficiency in the expression of muscle determination genes, we measured expression of MyoD1, myogenin, Myf-5, and MRF-4. Results show these cells do not synthesize MyoD1, yet express the other myogenic determination genes. Transient expression of exogenous MyoD1 in these cells is sufficient to activate endogenous MLC 1 and cardiac alpha-actin mRNA synthesis during muscle differentiation. Previously undetected myosin heavy chain (MHC) isoforms (beta-MHC and perinatal MHC) are also transcribed at low levels in L6E9 muscle cells, and in MyoD1-transfected L6E9 cells no change occurs in their expression. Furthermore, treatment with the demethylating agent 5-azacytidine activates expression of the endogenous MyoD1 gene in L6E9 cells and, subsequently, rescues deficiencies in their myogenic biochemical program. These results demonstrate that the endogenous MyoD1 gene in L6E9 cells is not defective and can be functionally activated. Also, the MyoD1 protein plays an essential role, which cannot be compensated by other known muscle determination proteins, in the induction of MLC 1 and cardiac alpha-actin expression.
Muthuchamy
M
M
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Pajak
L
L
Wieczorek
D F
DF
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D017570:MyoD Protein; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats
D000199:Actins; D009124:Muscle Proteins; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D009687:Nuclear Proteins; D009841:Oligonucleotides; D010750:Phosphoproteins; D012333:RNA, Messenger; D009218:Myosins; D001374:Azacitidine