TRANSCRIPTION REGULATION OF SYNAPSE-SPECIFIC GENES IN SKELETAL MUSCLE

肌细胞生成素和MyoD是结合到肌细胞生长因子的调节区的蛋白质。 电池的骨骼肌基因和激活他们的转录过程中 肌肉分化我们发现这两种蛋白质都与 烟碱乙酰胆碱受体（nAChR）α亚单位的增强剂， 受神经支配调控的基因。凝胶阻滞和DNA足迹 用于鉴定增强子结合的肌细胞生成素和MyoD中的区域。 这些区域含有MyoD结合位点的共有序列。的 结合位点的功能作用通过分离肌管来评估 用含有与基因连接的α亚基增强子的构建体 氯霉素乙酰转移酶（CAT）。点突变结合 位点特异性地消除了转染肌肉中CAT的表达 细胞由于nAChR基因受肌细胞生成素和MyoD调节，我们决定 为了研究这些因子的表达在肌原性生长过程中是如何调节的， 分化和神经支配期间。Myogenin和MyoD水平 转录本被发现减少小鼠后肌期间， 围产期，神经支配发生的时间。这种减少是 由于神经支配，由于成年骨骼肌的去神经支配导致 这两种转录本的水平都有显著增加。有趣的是 肌细胞生成素和MyoD对神经支配和去神经支配的反应，先于 nAChR mRNA水平的变化。肌肉“电活动”，在缺乏 神经，被发现下调肌细胞生成素和MyoD的水平 成绩单由于肌细胞生成素和MyoD是反式调节因子， 激活大量骨骼肌基因的表达， 分化，有趣的是，推测他们的镇压 在神经支配过程中可能导致突触的选择性下调， 基因.我们还集中在确定顺式元件，调节 nAChR γ亚基基因的转录。两个区域， 成肌细胞分化过程中γ亚基基因的转录 被确认了身份一个区域增强CAT报告基因的表达 由γ亚基上游调控序列驱动的基因，和另一个 抑制其表达。实验正在进行中，以确定是否 这些调节序列作为经典的增强子和沉默子， 以及它们是否受到与肌细胞生成素或MyoD相关的肌细胞生成因子的调节。

Myogenin and MyoD are proteins that bind to the regulatory regions of a battery of skeletal muscle genes and activate their transcription during muscle differentiation. We found that both proteins interact with the enhancer of the nicotinic acetylcholine receptor (nAChR) alpha subunit, a gene that is regulated by innervation. Gel retardation and DNA footprinting were used to identify the regions in the enhancer bound myogenin and MyoD. The regions contained a consensus sequence for the MyoD binding site. The functional role of the binding sites was assessed by transfecting myotubes with a construct containing the a subunit enhancer linked to the gene chloramphenicol acetyl transferase (CAT). Point mutations in the binding sites specifically obliterated expression of CAT in the transfected muscle cells. Since the nAChR gene is regulated by myogenin and MyoD, we decided to study how the expression of these factors is regulated during myogenic differentiation, and during innervation. The levels of myogenin and MyoD transcripts were found to decrease in mouse hind muscle during the perinatal period, a time when innervation is taking place. This decrease is due to innervation, since denervation of adult skeletal muscle lead to a dramatic increase in the levels of both transcripts. Interestingly, the responses of myogenin and MyoD to innervation and denervation, precede the changes in nAChR mRNA levels. Muscle "electrical activity", in absence of nerve, was found to down-regulate the levels of myogenin and MyoD transcripts. Since myogenin and MyoD are trans-regulatory factors that activate the expression of a large battery of skeletal muscle genes during differentiation, it is interesting to speculate that their repression during innervation could lead to the selective down-regulation of synaptic genes. We have also focused in identifying the cis-elements that regulate transcription of the nAChR gamma subunit gene. Two regions that regulate the transcription of the gamma subunit gene during myoblast differentiation were identified. One region enhances the expression of the CAT reporter gene driven by the gamma subunit upstream regulatory sequence, and another site represses its expression. Experiments are in progress to determine if these regulatory sequences function as classical enhancers and silencers, and if they are regulated by myogenic factors related to myogenin or MyoD.