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: 2575646
• 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/2575646
• 关键词: animal genetic material tag; electrostimulus; gene induction /repression; genetic regulatory element; genetic transcription; genetically modified animals; human genetic material tag; laboratory mouse; laboratory rat; motor neurons; myofibrils; nucleic acid sequence; regulatory gene; striated muscles; troponin

Our long-term interest is to understand at a molecular level how motor circuits are established and maintained. We would like to understand how motoneurons are initially matched with their corresponding types of myofibers (targets) during embryonic development, and later, how the contractile properties of the muscle continue to be regulated by motoneuron depolarization. Nerve-elicited activity initiates signal transduction cascades that culminate in the nucleus, where distinct frequencies of depolarization differentially regulate the transcription of genes encoding fast- or slow-twitch contractile proteins and leads to the down-regulation of receptors transcribed at extra-junctional nuclei. We have used the regulation of troponin I slow (TnIs) and fast (TnIf) isoforms as models to study activity-dependent transcriptional regulation; both of these genes are differentially up-regulated by specific depolarization frequencies (100 vs. 10 Hz). Because activity-dependent fiber specification is not observed in cultured muscles, we have used transgenic mice to identify the regulatory sequences that direct TnI transcription to either slow or fast-twitch myofibers. Using this approach, we identified a 128bp rat TnIs slow upstream regulatory element (SURE) and a 144bp quail TnIf fast intronic regulatory element (FIRE) that direct transcription of the chloramphenicol acetyltransferase (CAT) transgene specifically to either slow or fast muscles, respectively. Sequence alignment of the rat and human TnIs SURE with the quail TnIf FIRE identified common DNA motifs, namely two A/T-rich sequences (A/T1 and A/T2) with homology to homeotic protein and MEF2 binding sites, a CACC box, an E box, and a novel motif (GCAGGCA) that we denoted the CAGG box. Point mutations in either the A/T2 site, E box or CAGG box practically abolish the SURE function; mutations in the A/T1 and CACC sites have a lesser effect. Using competitive electrophoretic mobility shift assays with nuclear extracts derived from Sol8 myotubes, we demonstrate specific binding to these motifs and show that the A/T1 and A/T2 bind different factors. Our results demonstrate that the linear arrangement of DNA sequence motifs is conserved in the regulatory elements of the TnI slow and fast genes, and suggest the interaction of multiple protein-DNA complexes are necessary for enhancer function. In the past years, we have investigated the possible role of the myogenic transcription factors MyoD, myogenin and MRF-4 in the regulation of acetylcholine receptors and contractile proteins. We have found that these factors are not expressed selectively at junctions nor specific fiber-types, and that denervation dramatically increases their expression levels (10-100 fold). An upstream region in the myogenin gene of approximately 400 bp has been mapped that is required for its denervation-dependent transcriptional activation. Sites binding immediate early genes present in this region are being investigated.

我们的长期兴趣是在分子水平上了解马达 建立和维护电路。我们想要了解如何 运动神经元最初与其相应类型的 胚胎发育期间的肌纤维(靶标)，以及后来，如何 肌肉的收缩特性继续受到以下因素的调节 运动神经元去极化。神经诱发活动启动信号 转导级联最终在细胞核中，在那里不同的 去极化频率不同地调节转录 编码快或慢收缩蛋白的基因，并导致 在连接外核转录的受体的下调。 我们使用了肌钙蛋白I慢(TnI)和快(TnIf)的调节 以异构体为模型研究活性依赖转录 调控；这两个基因都被差异地上调了 特定的去极化频率(100对10赫兹)。因为 在培养的细胞中没有观察到活性依赖的纤维规格 肌肉，我们已经使用转基因小鼠来鉴定调控 引导TnI转录为慢或快-抽动的序列 肌纤维。使用这种方法，我们发现了一个128bpTnI缓慢的老鼠 上游调控元件(Sure)和144个碱基对的快速内含子 调控元件(FIRE)，直接转录 氯霉素乙酰转移酶(CAT)转基因对任一种 分别是慢的或快的肌肉。大鼠和大鼠的序列比对 如果火发现了共同的DNA基序， 即两个与同源同源的富含A/T的序列(A/T1和A/T2 蛋白质和MEF2结合位点、CACC盒、E盒和新基序 (GCAGGCA)，我们将其表示为CAGG框。两个基因中的任何一个都存在点突变 A/T2站点、E盒或CAGG盒实际上取消了SURE功能； A/T1和CACC位点突变的影响较小。vbl.使用 核抽提物竞争性电泳率漂移分析 来源于Sol8肌管，我们展示了与这些物质的特异性结合 基序，表明A/T1和A/T2结合了不同的因子。我们的 结果表明，DNA序列基序的线性排列 在TnI慢基因和快基因的调控元件中是保守的， 并提示多个蛋白质-DNA复合体之间的相互作用 增强功能所必需的。在过去的几年里，我们已经 研究了肌源性转录因子的可能作用 MyoD、Mygenin和MRF-4在乙酰胆碱受体和MARF-4调节中的作用 收缩蛋白。我们发现，这些因素没有被表达出来 选择性地在交界处也不是特定的纤维类型，而去神经 显著提高了它们的表达水平(10-100倍)。一条上游 肌生成素基因中约400bp的区域已被定位 是依赖去神经的转录激活所必需的。 存在于该区域的结合早期基因的位点正在被 调查过了。