Regulation of Skeletal Myofiber-Type Gene Expression

脊椎动物肌肉发育的研究为研究复杂的个体发育过程中的基因调控提供了难得的机会。在肌肉发生过程中，发生了组织特异性和发育阶段特异性的基因切换，最终形成了两种主要类型的骨骼肌：快抽动和慢抽动肌纤维。每种肌肉纤维类型的特点是表达独特的收缩蛋白亚型组合。肌纤维类型的建立源于多种因素的相互作用，如细胞谱系、肌纤维所接受的神经类型和循环激素水平。虽然在理解肌肉特异性基因表达的分子机制方面已经取得了很大进展，但对于肌纤维类型特异性基因的差异表达几乎一无所知。韦德博士建议通过对人类肌钙蛋白I基因家族的研究来研究肌纤维型特异性基因表达的调控机制。成体慢纤维TnI基因在胚胎肌肉纤维形成过程中存在差异表达，可作为纤维类型特异性基因表达的发育调控模型。他克隆了人骨骼肌TnI慢基因，并通过体外瞬时表达分析鉴定了其肌肉特异性表达所需的多个顺式作用序列。这些研究已经确定了一种肌肉特异性增强子，它不被生肌调节因子MyoD1反式激活。他将进一步确定肌肉特异性调控元件的特征，并将这些研究扩展到检查肌肉纤维类型特异性基因的活性。通过将TnI基因结构直接导入小鼠和大鼠特定的快、慢抽动肌肉中的新方法，以及通过携带杂交TnI基因结构的转基因小鼠的发展，将在体内研究纤维类型特异性基因表达的机制。这些TnI基因构建体的体内表达将通过CAT活性分析、RNase保护分析以及与肌肉组织横切面的基因特异性探针的原位杂交来监测。这些体外和体内的表达研究将评估特定的顺式作用序列是否可以赋予肌肉纤维类型特定的基因表达，并评估反式作用因子在这一过程中的作用。推测的调节纤维类型特定基因表达的反式作用因子将通过凝胶迁移率变化分析和DNA足迹技术来表征。如果发现了这样的因素，他将从表达文库中克隆具有代表性的cDNA。这些研究将是第一批研究调节肌肉纤维类型发育和维持的分子机制之一。

The study of vertebrate muscle development presents exceptional opportunities for the investigation of gene regulation during complex ontogenetic processes. During myogenesis both tissue- specific and developmental stage-specific gene switching occur, culminating in the formation of the two major types of skeletal muscle: fast-twitch and slow-twitch myofibers. Each of the muscle fiber types is characterized by the expression of unique combinations is contractile protein isoforms. The establishment of muscle fiber type arises from the interplay of factors such as cell lineage, type of innervation the myofiber receives, and the level of circulating hormones. Whereas much progress has been made in understanding the molecular mechanisms responsible for muscle-specific gene expression, almost nothing is known about differential myofiber-type-specific gene expression. Dr Wade proposes to investigate the mechanisms regulating myofiber-type specific gene expression through the study of the human troponin I gene family. The adult slow-fiber TnI gene is differentially expressed during embryonic muscle fiber formation, and can serve as a model for the developmental regulation of fiber-type- specific gene expression. He has cloned the human skeletal muscle TnI slow gene, and has identified multiple cis-acting sequences required for its muscle-specific expression by the use of in vitro transient expression assays. These studies have identified a muscle specific enhancer which is not trans- activated by the myogenic regulatory factor MyoD1. He will further characterize the muscle-specific regulatory elements and extend these studies to examine muscle fiber-type-specific gene activity. The mechanisms underlying fiber-type-specific gene expression will be investigated in vivo by the novel approach of directly introducing TnI gene constructs into specific fast and slow twitch muscles in mice and rats, and by the development of transgenic mice carrying hybrid TnI gene constructs. The in vivo expression of these TnI gene constructs will be monitored by CAT activity assays, RNase protection assay,and by in situ hybridization of gene specific probes to cross sections of muscle tissue. These in vitro and in vivo expression studies will assess whether specific cis-acting sequences can confer muscle fiber-type-specific gene expression and evaluate the role of trans-acting factors in the process. The putative trans-acting factors regulating fiber-type-specific gene expression will be characterized by gel mobility shift assays, and DNA footprinting techniques. If such factors are found, he will clone representative cDNAs from expression libraries. These studies will be among the first to examine the molecular mechanisms regulating muscle fiber type development and maintenance.