TRANSCRIPTIONAL REGULATION OF MUSCLE SPECIFIC GENES BY ELECTRICAL ACTIVITY

我们的长期目标是在分子水平上理解马达是如何运动的

Our long-term interest is to understand at a molecular level how motor circuits are established and maintained. We would like to understand how motoneurons are initially matched with their corresponding types of myofibers (targets) during embryonic development, and later, how the contractile properties of the muscle continue to be regulated by motoneuron depolarization. Nerve-elicited activity initiates signal transduction cascades that culminate in the nucleus, where distinct frequencies of depolarization differentially regulate the transcription of genes encoding fast- or slow-twitch contractile proteins and leads to the down-regulation of receptors transcribed at extra-junctional nuclei. We have used the regulation of troponin I slow (TnIs) and fast (TnIf) isoforms as models to study activity-dependent transcriptional regulation; both of these genes are differentially up-regulated by specific depolarization frequencies (100 vs. 10 Hz). Because activity-dependent fiber specification is not observed in cultured muscles, we have used transgenic mice to identify the regulatory sequences that direct TnI transcription to either slow or fast-twitch myofibers. Using this approach, we identified a 128bp rat TnIs slow upstream regulatory element (SURE) and a 144bp quail TnIf fast intronic regulatory element (FIRE) that direct transcription of the chloramphenicol acetyltransferase (CAT) transgene specifically to either slow or fast muscles, respectively. Sequence alignment of the rat and human TnIs SURE with the quail TnIf FIRE identified common DNA motifs, namely two A/T-rich sequences (A/T1 and A/T2) with homology to homeotic protein and MEF2 binding sites, a CACC box, an E box, and a novel motif (GCAGGCA) that we denoted the CAGG box. Point mutations in either the A/T2 site, E box or CAGG box practically abolish the SURE function; mutations in the A/T1 and CACC sites have a lesser effect. Using competitive electrophoretic mobility shift assays with nuclear extracts derived from Sol8 myotubes, we demonstrate specific binding to these motifs and show that the A/T1 and A/T2 bind different factors. Our results demonstrate that the linear arrangement of DNA sequence motifs is conserved in the regulatory elements of the TnI slow and fast genes, and suggest the interaction of multiple protein-DNA complexes are necessary for enhancer function. In the past years, we have investigated the possible role of the myogenic transcription factors MyoD, myogenin and MRF-4 in the regulation of acetylcholine receptors and contractile proteins. We have found that these factors are not expressed selectively at junctions nor specific fiber-types, and that denervation dramatically increases their expression levels (10-100 fold). An upstream region in the myogenin gene of approximately 400 bp has been mapped that is required for its denervation-dependent transcriptional activation. Sites binding immediate early genes present in this region are being investigated.