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: 5203322
• 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/5203322
• 关键词: denervation; electrophysiology; gene induction /repression; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; innervation; laboratory mouse; molecular cloning; muscle cells; muscle proteins; myogenesis; nicotinic receptors; nucleic acid sequence; striated muscles; tissue /cell culture; transcription factor; troponin

Skeletal muscle diversity and plasticity are dramatically influenced by motoneuron depolarization; nerve-derived electrical activity regulates the contractile properties of slow- and fast-twitch muscles by regulating transcription of specific genes. We have used transgenic mice to identify regulatory sequences that direct the fiber-specific transcription of the slow and fast troponin I (TnI) genes which are specifically regulated by selective depolarization frequencies. Mice harboring different chloramphenicol acetyltransferase (CAT) reporter constructs driven by progressive deletions of the rat TnI slow gene revealed that a 128 bp enhancer was sufficient to direct specific expression in slow muscle when linked to the 500bp promoter sequence; mice harboring just the 500bp TnI slow promoter region failed to show transcriptional activity in any tissue. A 144 bp enhancer from the quail TnI fast gene was found to confer transcription specifically in fast muscles when linked to the same 500bp promoter from the TnI slow gene. Interestingly, the rat TnI slow and quail fast fiber-specific enhancers share similar core elements. These results suggest that related trans-acting factors, or the formation of higher-order transcriptional complexes, are used to respond to selective patterns of muscle depolarization. On the other hand, we have identified sequences in the myogenin promoter that cause transcriptional repression in response to activity. Analyses in transgenic mice and muscles injected with myoblasts engineered to express the CAT reporter have revealed a region of approximately 0.5kb involved in the down-regulation of myogenin expression in response to innervation. Factors expressed specifically in denervated muscle were found to bind a 30bp element residing in the 0.5kb fragment. Ets factors have been implicated in coupling extrinsic signals to transcription. A novel ets-related transcription factor, PEF, was cloned and characterized. We found that PEF interacts synergistically with MyoD and myogenin to enhance transcription of muscle genes. Cotransfection with MEF2A showed no cooperativity with PEF. Our results suggest the possible combinatorial interaction between members of the ETS and myogenic BHLH families may mutually contribute to the stringency of binding site selection by these two families resulting in the differential modulation of various muscle specific genes.

骨骼肌的多样性和可塑性受到 运动神经元去极化;神经源性电活动调节 通过调节慢肌和快肌的收缩特性， 特定基因的转录。我们用转基因小鼠来鉴定 调控序列，指导纤维特异性转录的 慢肌钙蛋白I和快肌钙蛋白I（TnI）基因，其由 选择性去极化频率。小鼠携带不同的 氯霉素乙酰转移酶（CAT）报告基因构建体， 大鼠TnI慢基因的进行性缺失显示， 增强子足以指导慢肌中的特异性表达， 与500 bp启动子序列相连;仅携带500 bp TnI的小鼠 慢启动子区在任何一种细胞中都没有表现出转录活性， 组织. 发现来自鹌鹑TnI快基因的144 bp增强子 当与快速肌肉连接时， TnI慢基因启动子500 bp。有趣的是，老鼠的TnI 和鹌鹑快速纤维特异性增强剂共享相似的核心元件。 这些结果表明，相关的反式作用因子，或形成 高级转录复合物，用于响应 肌肉去极化的选择性模式。另一方面， 在肌细胞生成素启动子中鉴定了引起转录的序列， 对活动的反应。转基因小鼠中的分析， 肌肉注射成肌细胞工程表达CAT报告 已经揭示了一个大约0.5kb的区域参与了 肌细胞生成素表达的下调是对神经支配的反应。 在失神经肌肉中特异性表达的因子被发现与 一个30 bp的元件位于0.5kb的片段中。Ets因素已经被 参与将外部信号与转录偶联。一种新型 ets相关转录因子PEF的克隆和鉴定。我们 发现PEF与MyoD和肌细胞生成素协同作用， 增强肌肉基因的转录。与MEF 2A共转染显示， 与PEF无协同性。我们的研究结果表明， ETS和肌源性BHLH家族成员之间的相互作用可能 相互促进结合位点选择的严格性， 两个家庭导致不同的肌肉调制 特定基因