TRANSCRIPTIONAL REGULATION OF MUSCLE-SPECIFIC GENES BY ELECTRICAL ACTIVITY

运动神经元的支配显著影响表型。 骨骼肌的特性。神经的模式和频率- 诱发的电活动改变烟碱乙酰胆醇的转录 E 受体(NAChR)基因，以及肌原纤维蛋白编码基因 这决定了肌肉纤维的收缩特性。我们有 识别的肌肉基因要么被抑制，要么被选择性地 受活性刺激；我们的目标是阐明分子机制 潜在的依赖活性的转录调控。为此， 我们之前证明了肌肉生成素，一种肌肉特异性因子， 调节培养细胞中nAChR基因的转录，被抑制 通过神经支配，通过去神经去抑制。肌生素的变化 在发育和发育过程中，肌肉中的水平先于受体调节 在失神经后，提示肌肉生成素的调节可能 受体的上游。北京地区3.7kb的生肌素基因上游区序列分析 转基因小鼠已经表明，这些序列赋予肌肉-- 发育和去神经特有的调节。我们已经使用了 成肌细胞植入实验以分离授予 组织和发育的特异性来自于那些赋予 去神经反应。我们分析了顺式作用元件的调控 肌原纤维蛋白肌钙蛋白I慢速(TnIS)基因的表达 编码基因，在慢型肌肉中特异表达，是 通过电刺激的“慢模式”选择性地上调。一个 6.3 kb TnIS片段，包含前2个非编码外显子和 上游序列，被发现特异性地将转录引导到 慢型肌肉。培养的C2C12心肌细胞中该区域的定位 证明了200个碱基的上游序列是必要的， 足以赋予肌肉和发育特异的转录。 基于这些序列，我们已经开始克隆，并分析了 可能调节TNI的反式作用因子的表达 抄写。编码Ets-家族一个新成员的cDNA，具有 与PEA-3同源性最高的基因已被分离和测序。这个 该蛋白的功能特性目前正在研究中。 另外，还克隆了大鼠MEF-2基因，并对其进行了序列测定和表达。 在开发过程中，不同类型的纤维目前正在 学习。分析这些反式行为家族之间的相互作用 这些因素应该有助于阐明潜在的分子机制。 神经支配对肌肉基因的纤维型特异性调节。

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.