TRANSCRIPTIONAL REGULATION OF MUSCLE SPECIFIC GENES BY ELECTRICAL ACTIVITY

我们的长期兴趣是在分子水平上了解 建立和维护电路。我们想知道 运动神经元最初与其相应类型的 肌纤维（目标）在胚胎发育过程中，后来，如何 肌肉的收缩特性继续受到 运动神经元去极化 神经诱发活动启动信号 在细胞核中达到顶峰的转导级联， 去极化频率差异调节转录 基因编码的快速或缓慢收缩蛋白，并导致 在连接外核转录的受体的下调。 我们已经使用了肌钙蛋白I慢（TnIs）和快（TnIs）的调节， 异构体作为研究活性依赖性转录的模型 调节;这两个基因都被差异上调 特定的去极化频率（100与10 Hz）。因为 在培养的细胞中未观察到活性依赖性纤维规格 肌肉，我们已经使用转基因小鼠，以确定调节 指导TnI转录慢或快的序列 肌纤维使用这种方法，我们鉴定了一个128 bp的大鼠TnIs慢 上游调控元件（SURE）和一个144 bp的鹌鹑Tactinase快速内含子 调控元件（FIRE），其指导转录 氯霉素乙酰转移酶（CAT）转基因特异性地 慢肌或快肌。大鼠的序列比对， 人类TnIs与鹌鹑TcDNAFIRE确定了共同的DNA基序， 即两个富含A/T的序列（A/T1和A/T2）， 蛋白和MEF 2结合位点、CACC盒、E盒和新基序 （GCAGGCA），我们表示CAGG盒。点突变， A/T2部位、E箱或CAGG箱实际取消SURE功能; A/T1和CACC位点的突变具有较小的影响。使用 用核提取物的竞争性电泳迁移率变动分析 来源于Sol 8肌管，我们证明了特异性结合这些 基序，并显示A/T1和A/T2结合不同的因子。我们 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， 并表明多种蛋白质-DNA复合物的相互作用是 增强子功能所必需的。 在过去的几年里，我们 研究了肌源性转录因子的可能作用， MyoD、肌细胞生成素和MRF-4在乙酰胆碱受体调节中的作用 收缩蛋白我们发现这些因素并没有表现出来 选择性地在连接处，也不是特定的纤维类型， 显著增加其表达水平（10-100倍）。上游 肌细胞生成素基因中大约400 bp的区域已经被定位， 是其失神经依赖性转录激活所必需的。 结合该区域中存在的立即早期基因的位点正在被 研究了

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.