Molecular control of myogenesis: antagonism between growth and differentiation

Insight into the molecular mechanisms that control establishment of the skeletal muscle phenotype has recently been obtained through cloning of a family of muscle-specific regulatory factors that can activate myogenesis when transfected into non-muscle cells. This family of factors, which includes MyoD, myogenin, myf-5, and MRF4, can bind DNA and transactivate muscle-specific genes in collaboration with ubiquitous cellular factors. Growth factors play an antagonistic role in myogenesis by suppressing the actions of the myogenic regulatory factor family. This review will focus on the regulation and mechanism of action of this family of myogenic regulatory factors and on the central role of peptide growth factors in modulating their expression and biological activities.     

MyoD recruits the cdk9/cyclin T2 complex on myogenic-genes regulatory regions

During skeletal myogenesis, muscle-regulatory factors bHLH and MEF2 promote the expression of muscle-specific genes by recruiting several chromatin-modifying complexes on specific DNA regulatory sequences. A number of MyoD-interacting proteins have been reported, but whether they are recruited to the chromatin of myogenic loci, and the relationship with other chromatin bound proteins is unknown. We show that MyoD recruits cdk9/cyclin T2, together with the histone acetyltransferases p300 and PCAF, and the chromatin remodeling complex SWI/SNF, on promoters and enhancers of muscle-specific genes, and that this event correlates with the acetylation of histone tails, remodeling of chromatin, and phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II at these elements.     

Muscle development in Ciona intestinalis requires the b-HLH myogenic regulatory factor gene Ci-MRF

The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.     

Reversibility of muscle differentiation in the absence of commitment: analysis of a myogenic cell line temperature-sensitive for commitment

The interrelationship between commitment (irreversible withdrawal from the cell cycle) and muscle-specific gene expression was analyzed with the myogenic cell line ts 3b-2, which is temperature sensitive for commitment and cell fusion. The rates of synthesis and levels of accumulation of muscle-specific mRNAs and proteins in the ts 3b-2 cells at permissive and nonpermissive temperatures are comparable, indicating that neither commitment nor cell fusion is required for induction of muscle-specific gene expression. In the absence of commitment, the cells are reversibly withdrawn from the cell cycle during gene induction, and expression of the muscle-specific genes is deinduced upon the switch to growth-stimulating conditions. The deinduction reflects coordinate and preferential cessation of muscle-specific mRNA synthesis, coupled with destabilization of the muscle-specific mRNAs in the cytoplasm, without effect on constitutively expressed housekeeping protein genes. The phenotype of the ts 3b-2 cells demonstrates that commitment and muscle-specific gene expression are both required, but alone are insufficient, to produce the terminally differentiated muscle phenotype.     

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.