TRANSCRIPTIONAL REGULATION OF SKELETAL MUSCLE-SPECIFIC GENES

骨骼肌特异性基因的转录调控

• 批准号: 3842311
• 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/3842311
• 关键词: acetylcholine; electrical property; electrophoresis; electrostimulus; gene expression; genetic transcription; genetically modified animals; in situ hybridization; innervation; laboratory mouse; muscle proteins; myofibrils; myogenesis; neurogenesis; nicotinic receptors; northern blottings; striated muscles; synapses; transcription factor

Skeletal muscle phenotype is dramatically influenced by the type of motoneuron innervation it receives. The pattern and frequency of nerve- elicited electrical activity modifies the distribution of receptors on the myotube surface and the contractile properties of the myofiber by regulating gene transcription. We previously demonstrated that the MyoD family of transcription factors regulate the expression of genes coding for nicotinic acetylcholine receptor (nAChR) subunits and contractile proteins during muscle differentiation, and that they may account for the down regulation of receptor genes during muscle innervation. Using in situ hybridization, we found that the levels of nAChR and myogenic factor transcripts were lower throughout the innervated myofibril, and that the myogenic factor mRNAs were not co-localized with receptor RNAs at synaptic nuclei. Two days after denervation, all these transcripts accumulated in the myofibril nuclei. In addition, we found that the levels of myogenin protein are higher in nuclei isolated from denervated muscle than innervated muscle, consistent with the idea that the myogenic factors mediate the up-regulation of receptor genes after denervation. Next, we investigated the effects of different stimulation frequencies on the expression of the myogenic factors. Northern blot analysis of RNA isolated from muscle that was either denervated, or denervated and stimulated with extracellular electrodes, showed that the MyoD family of factors is down-regulated by depolarization frequencies typical of fast- or slow-twitch muscle. In contrast, expression of the genes coding for troponin I isoforms, which are proteins involved in conferring the fast and slow contractile properties to muscle, was selectively up-regulated only by the corresponding type of stimulus frequency; denervation down-regulated their expression. These results suggest that the MyoD family does not determine fiber-type specificity and that different factors regulate these properties. We have begun to characterize the regulatory sequences of the myogenin and TnI slow gene to identify the elements that either confer repression or activation of transcription in response to innervation. We found in transgenic mice that 3.7 kb of myogenin gene upstream sequences impart muscle-specific transcription to a CAT reporter gene and partial responses to denervation.

骨骼肌表型受肌肉类型的显着影响 它接受的运动神经元神经支配。神经的模式和频率 引起的电活动改变受体的分布 肌管表面和肌纤维的收缩特性 调节基因转录。我们之前证明了MyoD 转录因子家族调节编码基因的表达 对于烟碱乙酰胆碱受体 (nAChR) 亚基和收缩 肌肉分化过程中的蛋白质，以及它们可能解释 肌肉神经支配过程中受体基因的下调。使用 原位杂交，我们发现nAChR和肌源性的水平 整个受神经支配的肌原纤维的因子转录物较低，并且 生肌因子 mRNA 不与受体 RNA 共定位 在突触核。去神经两天后，所有这些成绩单 积聚在肌原纤维核中。此外，我们发现 从去神经支配的细胞核中分离出的肌细胞生成素蛋白水平较高 肌肉比受神经支配的肌肉更重要，这与以下观点一致： 生肌因子介导受体基因上调 去神经支配。接下来我们研究一下不同刺激的效果 生肌因子表达的频率。北方印迹 分析从去神经支配的肌肉中分离出的 RNA，或者 去神经并用细胞外电极刺激，表明 MyoD 因子家族受去极化频率下调 典型的快肌或慢肌。相比之下，表达 编码肌钙蛋白 I 亚型的基因，这些蛋白质参与 赋予肌肉快速和慢速收缩特性 仅通过相应类型的刺激选择性上调 频率;去神经支配下调了它们的表达。这些结果 表明 MyoD 家族并不决定纤维类型特异性 并且不同的因素调节这些特性。我们已经开始 表征肌细胞生成素和肌钙蛋白 I 慢基因的调控序列 识别抑制或激活的元素 转录响应神经支配。我们在转基因小鼠中发现 3.7 kb 的肌细胞生成素基因上游序列赋予肌肉特异性 CAT 报告基因的转录和部分反应 去神经支配。