TRANSCRIPTIONAL REGULATION OF SYNAPSE-SPECIFIC GENES IN SKELETAL MUSCLE

MyoD基因家族编码具有保守螺旋-环螺旋的蛋白质 作为转录因子发挥作用的结构域，调节表达 一组骨骼肌基因在生肌性测定和 差异化。我们已经发现，这些肌源性细胞之间的相互作用 含有烟碱型乙酰胆碱受体(NAChR)亚单位增强剂的因子可能 解释受体基因的协同转录上调 在肌肉分化过程中，及其表达的抑制 在肌肉神经支配过程中。我们已经确定了受体中的一个区域 提供组织和发育特异性转录的伽马亚基 氯霉素乙酰转移酶(M报告基因)的表达 C2Cl2肌细胞和3T3成纤维细胞。56个碱基的序列作为一个 增强剂。存在两个间隔很近的“MyoD结合位点”(或E盒 在nAChRα和伽马亚单位增强剂中。利用电泳法 迁移率变化分析，在存在竞争序列的情况下，我们证明 肌生成素和MyoD优先与下游存在的E盒结合 在两种增强剂中都有。MyoD结合位点的点突变 在转基因细胞中消除受体基因的转录。 在此过程中，我们研究了生肌因子mRNA水平的调节 神经支配，以及肌肉失神经后，以确定是否 肌源性因子的表达可解释nAChR的抑制 转录发生在骨骼肌的神经支配过程中。MyoD， 观察到肌生成素和Myf-5的mRNA水平在 围产期发育，而MRF-4水平在出生前后开始增加。 在发育过程中，肌生成素和MyoD的mRNA水平受到抑制 神经支配，因为去神经支配会导致两种转录物的水平 急剧增加。有趣的是，肌生成素和肌生成素的反应 MyoD对神经支配和去神经支配先于nAChR mRNA的变化 级别。电活动本身，由细胞外给药 电极，下调生肌因子mRNAs。针对该病毒的抗体 肌源性因子正在产生和表征，以研究 这些蛋白质在肌肉神经支配过程中的定位和修饰。 我们的结果与这样的观点是一致的，即在生肌过程中 分化、肌生成素和MyoD协同激活转录 大量的骨骼肌基因。但在神经支配期间，在 对电活动的反应，这些因素选择性地下调a 一组在神经肌肉组织中特异表达的基因 Synapse。

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.