Regulation of Skeletal Myofiber-Type Gene Expression

脊椎动物肌肉发育的研究为研究复杂个体发育过程中的基因调控提供了难得的机会。在肌肉形成过程中，组织特异性和发育阶段特异性的基因转换都会发生，最终形成两种主要类型的骨骼肌：快肌纤维和慢肌纤维。每一种肌纤维类型的特点是独特的表达组合是收缩蛋白同种异构体。肌纤维类型的形成源于细胞谱系、肌纤维接受的神经支配类型和循环激素水平等因素的相互作用。尽管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。Wade博士建议通过研究人类肌钙蛋白I基因家族来研究调节肌纤维类型特异性基因表达的机制。成人慢纤维TnI基因在胚胎肌纤维形成过程中存在差异表达，可作为纤维型特异性基因表达发育调控的模型。他克隆了人类骨骼肌TnI慢速基因，并通过体外瞬时表达试验确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，它不被肌生成调节因子MyoD1反式激活。他将进一步表征肌肉特异性调控元件，并将这些研究扩展到检查肌肉纤维类型特异性基因活性。通过将TnI基因构建体直接导入小鼠和大鼠的特定快、慢抽搐肌，以及开发携带杂交TnI基因构建体的转基因小鼠，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活性测定、RNase保护测定和基因特异性探针在肌肉组织横截面的原位杂交来监测。这些体外和体内表达研究将评估特定的顺式作用序列是否能赋予肌肉纤维类型特异性基因表达，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异性基因表达的反式作用因子将通过凝胶迁移位移测定和DNA足迹技术来表征。如果这些因子被发现，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的研究。

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.