Skeletal muscle diversity and plasticity are dramatically influenced by motoneuron depolarization; nerve-derived electrical activity regulates the contractile properties of slow- and fast-twitch muscles by regulating transcription of specific genes. We have used transgenic mice to identify regulatory sequences that direct the fiber-specific transcription of the slow and fast troponin I (TnI) genes which are specifically regulated by selective depolarization frequencies. Mice harboring different chloramphenicol acetyltransferase (CAT) reporter constructs driven by progressive deletions of the rat TnI slow gene revealed that a 128 bp enhancer was sufficient to direct specific expression in slow muscle when linked to the 500bp promoter sequence; mice harboring just the 500bp TnI slow promoter region failed to show transcriptional activity in any tissue. A 144 bp enhancer from the quail TnI fast gene was found to confer transcription specifically in fast muscles when linked to the same 500bp promoter from the TnI slow gene. Interestingly, the rat TnI slow and quail fast fiber-specific enhancers share similar core elements. These results suggest that related trans-acting factors, or the formation of higher-order transcriptional complexes, are used to respond to selective patterns of muscle depolarization. On the other hand, we have identified sequences in the myogenin promoter that cause transcriptional repression in response to activity. Analyses in transgenic mice and muscles injected with myoblasts engineered to express the CAT reporter have revealed a region of approximately 0.5kb involved in the down-regulation of myogenin expression in response to innervation. Factors expressed specifically in denervated muscle were found to bind a 30bp element residing in the 0.5kb fragment. Ets factors have been implicated in coupling extrinsic signals to transcription. A novel ets-related transcription factor, PEF, was cloned and characterized. We found that PEF interacts synergistically with MyoD and myogenin to enhance transcription of muscle genes. Cotransfection with MEF2A showed no cooperativity with PEF. Our results suggest the possible combinatorial interaction between members of the ETS and myogenic BHLH families may mutually contribute to the stringency of binding site selection by these two families resulting in the differential modulation of various muscle specific genes.
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