Absence of MEF2 binding to the A/T-rich element in the muscle creatine kinase (MCK) enhancer correlates with lack of early expression of the MCK gene in embryonic mammalian muscle.
During skeletal muscle development, different types of muscle fibers are generated, which express different combinations of muscle-specific gene products. For example, the muscle creatine kinase gene (MCK) is highly expressed in fetal but not embryonic myotubes. We performed transient transfections of CAT reporter constructs, driven by the MCK promoter with variable lengths of 5'-flanking sequence, into primary cultures of embryonic and fetal muscle cells. Reporter activity was observed in fetal but not embryonic muscle cells. We assayed the ability of nuclear extracts prepared from embryonic and fetal muscle and C2C12 myotubes to bind specific regulatory elements in the MCK enhancer. The profile of DNA/protein complexes resulting from electrophoretic mobility shift assays was qualitatively the same with all extracts used when the oligonucleotide probes represented the MCK-E-box, MHox site, CArG-box, and AP2 site. In contrast, no binding activity to the MEF2 site was observed with embryonic nuclear extract. Interestingly, MEF2 mRNAs and proteins were detected in both fetal and embryonic muscle, with the exception of the MEF2D1b isoform, which is restricted to fetal muscle. Furthermore, we found that protein phosphatase inhibitors included in the preparation of embryonic nuclear extracts or added to the medium of transfected embryonic myotubes can restore MEF2 DNA binding activity, as well as reporter activity driven by the MCK promoter and partial transcriptional activation of the endogenous MCK gene. We propose that phosphorylation of MEF2 regulates its activity and represents an important aspect of the mechanism controlling stage-specific transcription during skeletal myogenesis.
Ferrari
S
S
Dipartimento di Scienze Biomediche, Università di Modena, Italy. sferrari@c220.unimo.it
Molinari
S
S
Melchionna
R
R
Cusella-De Angelis
M G
MG
Battini
R
R
De Angelis
L
L
Kelly
R
R
Cossu
G
G
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D064326:MEF2 Transcription Factors; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D019319:Okadaic Acid; D014638:Vanadates; D004247:DNA; D003402:Creatine Kinase; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases