TRANSCRIPTIONAL REGULATION OF SYNAPSE-SPECIFIC GENES IN SKELETAL MUSCLE

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The MyoD gene family codes for proteins with a conserved helix-loop helix domain that function as transcription factors which regulate the expression of a battery of skeletal muscle genes during myogenic determination and differentiation. We have found that the interaction of these myogenic factors with nicotinic acetylcholine receptor (nAChR) subunit enhancers may account for the coordinate transcriptional upregulation of receptor genes during muscle differentiation, and the repression of their expression during muscle innervation. We have identified a region in the receptor gamma subunit that confers tissue- and developmental-specific transcription of the chloramphenicol acetyl transferase (M reporter gene to transfected C2Cl2 myocytes and 3T3 fibroblasts. The 56 bp sequence works as an enhancer. Two closely spaced "MyoD binding sites" (or E boxes) are present in the nAChR alpha and gamma subunit enhancers. Utilizing electrophoretic mobility shift assays, in presence of competing sequences, we demonstrate that myogenin and MyoD bind preferentially to the downstream E box present in both enhancers. Point mutations in the MyoD binding sites specifically obliterate transcription of the receptor genes in transfected cells. Regulation of the myogenic factor mRNA levels was studied during innervation, and after denervation of muscle, to determine if the expression of the myogenic factors can account for the repression of nAChR transcription occurring during innervation of skeletal muscle. MyoD, Myogenin and myf-5 mRNA levels were observed to be repressed during perinatal development, whereas Mrf-4 levels begin to increase around birth. The repression of myogenin and MyoD mRNA levels during development is due to innervation, because denervation causes the levels of both transcripts to increase dramatically. Interestingly, the responses of myogenin and MyoD to innervation and denervation precede the changes in nAChR mRNA levels. Electrical activity per se, administered by extracellular electrodes, down-regulates myogenic factor mRNAs. Antibodies to the myogenic factors are being generated and characterized to study the localization and modification of these proteins during muscle innervation. Our results are consistent with the idea that, during myogenic differentiation, myogenin and MyoD coordinately activate transcription of a large number of skeletal muscle genes. But that during innervation, in response to electrical activity, these factors selectively down-regulate a battery of genes that are specifically expressed at the neuromuscular synapse.