INHIBITION OF MYOGENESIS BY MULTIPLE CYCLIN-CDK COMPLEXES - COORDINATE REGULATION OF MYOGENESIS AND CELL-CYCLE ACTIVITY AT THE LEVEL OF E2F

During skeletal myogenesis, cell cycle withdrawal accompanies the expr ession of the contractile phenotype. Here we show that ectopic express ion of each D-type cyclin is sufficient to inhibit the transcriptional activation of the muscle-specific creatine kinase (MCK) gene. In cont rast, ectopic expression of cyclin A or cyclin E inhibits MCK expressi on only when they are co-expressed with their catalytic partner cyclin -dependent kinase 2 (Cdk2). For each of these conditions, myogenic tra nscriptional inhibition is reversed by the ectopic co-expression of th e general Cdk inhibitor p21. Inhibition of MCK expression by cyclins o r cyclin-Cdk combinations correlates with E2F activation, suggesting t hat the inhibition is mediated by the overall Rb-kinase activities of the Cdk complexes. In support of this hypothesis, a hyperactive mutant of Rb was found to partially re verse the inhibition of MCK expressio n by cyclin D1 and by the combination of cyclin A and Cdk2. These data demonstrate that the inhibition of myogenic transcriptional activity is a general feature of overall Cdk activity which is mediated, at lea st in part, by an pocket protein/E2F-dependent pathway, MCK promoter a ctivity is also inhibited by ectopic E2F1 expression, but this inhibit ion is not reversed by the co-expression of p21. Analyses of a series of E2F1 mutants revealed that the transcriptional activation, leucine zipper, basic, and cyclin A/Cdk2-binding domains are dispensable, but the helix-loop-helix region is essential for myogenic inhibition. Thes e data demonstrate that myocyte proliferation and differentiation are coordinated at the level of E2F and that these opposing activities are regulated by different E2F domains.