TRANSCRIPTIONAL REGULATION OF MUSCLE-SPECIFIC GENES BY ELECTRICAL ACTIVITY

电活动对肌肉特异性基因的转录调控

• 批准号: 3778571
• 负责人: A BUONANNO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3778571
• 关键词: acetylcholine; complementary DNA; denervation; electrical property; electrostimulus; gene expression; genetic regulation; genetic transcription; genetically modified animals; in situ hybridization; innervation; laboratory mouse; laboratory rat; molecular cloning; muscle contraction; muscle proteins; myofibrils; myogenesis; nicotinic receptors; nucleic acid sequence; striated muscles; synapses; transcription factor

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.

运动神经元神经支配显着影响表型 骨骼肌的特性。神经的模式和频率 引发的电活动改变烟碱乙酰胆碱的转录 e 受体 (nAChR) 基因，以及编码肌原纤维蛋白的基因 它决定了肌纤维的收缩特性。我们有 确定了被抑制或选择性的肌肉基因 受到活动的刺激；我们的目标是阐明分子机制 潜在的活性依赖性转录调控。为此， 我们之前证明了肌生成素，一种肌肉特异性因子， 调节培养细胞中 nAChR 基因的转录，并被抑制 通过神经支配和去神经支配去抑制。肌生成素的变化 在发育和发育过程中，肌肉中的水平先于受体调节 去神经后，表明肌细胞生成素的调节可能位于 受体的上游。 3.7 kb 肌细胞生成素上游区域的分析 转基因小鼠已表明这些序列赋予肌肉、 发育和去神经特异性调节。我们已经用过 成肌细胞植入实验分离序列，赋予 组织和发育特异性来自那些赋予 去神经反应。 我们分析了顺式作用元件调节 肌钙蛋白 I 慢 (TnIs) 基因（一种肌原纤维蛋白）的表达 特异在慢型肌肉中表达的编码基因 通过电刺激的“缓慢模式”选择性上调。一个 6.3 kb TnIs 片段，包含前 2 个非编码外显子以及 上游序列，被发现可以特异性地指导转录 慢型肌肉。在培养的 C2C12 肌细胞中描绘该区域 证明 200 bp 的上游序列是必要的并且 足以赋予肌肉和发育特异性转录。 基于这些序列，我们已经开始克隆并分析 可能调节 TnIs 的推定反式作用因子的表达 转录。编码 ets 家族新成员的 cDNA， 与 PEA-3 具有最高同源性，已被分离并测序。这 该蛋白质的功能特性目前正在研究中。 还克隆并测序了大鼠 MEF-2 cDNA，及其表达 在开发过程中，目前正在使用不同的纤维类型 研究过。分析这些反式作用家族的相互作用 因素应有助于阐明潜在的分子机制 通过神经支配对肌肉基因进行纤维型特异性调节。