Defective myogenesis in NFB-s mutant associated with a saturable suppression of MYF5 activity.

Abstract

Myogenic cell lines have proved to be useful tools for investigating the molecular mechanisms that control cellular differentiation. NFB-s is a mutant myogenic cell line which fails to differentiate in vitro, and can repress differentiation in normal myogenic cells when fused to form heterokaryons. The NFB-s cell line was used here to study the molecular mechanisms underlying such myogenic repression. Using muscle-specific reporter genes, we show that NFB-s cells fail to activate fully the muscle differentiation program at a transcriptional level, although muscle-specific transcription can be enhanced by regulators of differentiation such as pertussis toxin. Paradoxically we find that the myogenic regulator myf5 is expressed at constitutively high levels in NFB-s cells, and retains DNA binding activity. Expression plasmids encoding NFB-derived myf5 cDNA can rescue the myogenic phenotype in NFB-s cells, demonstrating that a threshold level of positive regulators must be reached before the myogenic program is activated. Thus, the dominant negative phenotype does not appear to result from defective myf5, but is due to a dosage-dependent saturable mechanism that interferes with myf5 function. These studies demonstrate that the stoichiometric ratio of positive and negative regulators is critical for determining the myogenic differentiation state.