TRANSCRIPTIONAL REGULATION OF SKELETAL MUSCLE-SPECIFIC GENES

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Skeletal muscle phenotype is dramatically influenced by the type of motoneuron innervation it receives. The pattern and frequency of nerve- elicited electrical activity modifies the distribution of receptors on the myotube surface and the contractile properties of the myofiber by regulating gene transcription. We previously demonstrated that the MyoD family of transcription factors regulate the expression of genes coding for nicotinic acetylcholine receptor (nAChR) subunits and contractile proteins during muscle differentiation, and that they may account for the down regulation of receptor genes during muscle innervation. Using in situ hybridization, we found that the levels of nAChR and myogenic factor transcripts were lower throughout the innervated myofibril, and that the myogenic factor mRNAs were not co-localized with receptor RNAs at synaptic nuclei. Two days after denervation, all these transcripts accumulated in the myofibril nuclei. In addition, we found that the levels of myogenin protein are higher in nuclei isolated from denervated muscle than innervated muscle, consistent with the idea that the myogenic factors mediate the up-regulation of receptor genes after denervation. Next, we investigated the effects of different stimulation frequencies on the expression of the myogenic factors. Northern blot analysis of RNA isolated from muscle that was either denervated, or denervated and stimulated with extracellular electrodes, showed that the MyoD family of factors is down-regulated by depolarization frequencies typical of fast- or slow-twitch muscle. In contrast, expression of the genes coding for troponin I isoforms, which are proteins involved in conferring the fast and slow contractile properties to muscle, was selectively up-regulated only by the corresponding type of stimulus frequency; denervation down-regulated their expression. These results suggest that the MyoD family does not determine fiber-type specificity and that different factors regulate these properties. We have begun to characterize the regulatory sequences of the myogenin and TnI slow gene to identify the elements that either confer repression or activation of transcription in response to innervation. We found in transgenic mice that 3.7 kb of myogenin gene upstream sequences impart muscle-specific transcription to a CAT reporter gene and partial responses to denervation.