TRANSCRIPTIONAL REGULATION OF MUSCLE-SPECIFIC GENES BY ELECTRICAL ACTIVITY

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Motoneuron innervation dramatically influences the phenotypic properties of skeletal muscle. The pattern and frequency of nerve- elicited electrical activity modifies transcription of nicotinic acetylchol e receptor (nAChR) genes, as well as genes coding for myofibril proteins which determine the contractile properties of the muscle fiber. We have identified muscle genes that are either repressed or selectively stimulated by activity; our goal is to elucidate the molecular mechanisms underlying activity-dependent transcriptional regulation. To this end, we previously demonstrated that myogenin, a muscle-specific factor that regulates transcription of nAChR genes in cultured cells, is repressed by innervation and de-repressed by denervation. Changes in myogenin levels in muscle preceded receptor regulation during development and after denervation, suggesting that regulation of myogenin may lie upstream of receptor. Analysis of a 3.7 kb myogenin upstream region in transgenic mice have shown that these sequences confer muscle-, developmental-, and denervation-specific regulation. We have used myoblast implantation experiments to separate sequences that confer tissue and developmental specificity from those imparting the denervation response. We analyzed the cis-acting elements regulating expression of the troponin I slow (TnIs) gene, a myofibril protein coding gene that is specifically expressed in slow-type muscle and is up-regulated selectively by "slow patterns" of electrical stimulation. A 6.3 kb TnIs fragment, containing the first 2 noncoding exons plus upstream sequences, was found to direct transcription specifically to slow-type muscle. Delineation of this region in cultured C2C12 myocytes demonstrated that 200 bp of upstream sequence are necessary and sufficient to confer muscle- and developmental-specific transcription. Based on these sequences, we have begun to clone, and analyze the expression of, putative trans-acting factors that may regulate TnIs transcription. A cDNA coding for a novel member of the ets-family, with highest homology to PEA-3, has been isolated and sequenced. The functional properties of this protein are currently under investigation. A rat MEF-2 cDNA was also cloned and sequenced, and its expression during development and in different fiber types is currently being studied. Analysis of the interactions of these families of trans-acting factors should help elucidate the molecular mechanisms underlying fiber-type specific regulation of muscle genes by innervation.