Regulation of Skeletal Myofiber Type Gene Expression

这项拟议的研究考察了在哺乳动物骨骼肌组织的胚胎形成、成熟和多样化过程中，协调基因激活受到调控的分子机制。近年来，随着成肌细胞向终末分化肌管的转变，在理解激活肌肉特异性基因的分子机制方面取得了巨大的进展。肌肉发生的一个基本但不太明确的方面是肌肉细胞成熟为特定类型的肌纤维。在过去的NSF支持期间，PI描述了编码人类慢抽动肌钙蛋白I(TnI)亚型的基因，并表明TnI启动子的细胞类型表达涉及多个顺式作用成分；上游肌肉特异性增强子，内含子1内的第二肌肉特异性增强子，以及肌肉特异性最小启动子。PI与合作者一起，证明了TnIS启动子/CAT结构在转基因小鼠中表现出适当的肌肉纤维类型特异性表达，但初步结果表明，转基因(缺乏内含子增强子)在胚胎发育过程中受到异常调控。需要检验的假说是，肌肉发育早期和后期的基因调控需要不同的调控元件，这些调控元件结合不同的反式作用因子组合；上游元件可能在TnIs基因表达的神经依赖限制中发挥主要作用，以减缓肌肉纤维的抽动；下游序列，如内含子1增强子，可能在肌纤维形成的早期阶段发挥主要作用。为了定位纤维类型特异性基因表达的上游序列，PI建立了体内肌肉注射实验，用来研究成年大鼠快、慢抽动肌肉的差异基因表达。这一系统使PI有了独特的机会来理解调节纤维类型专一性的分子机制。PI将使用体内注射试验来描述TnIS基因的纤维类型特定表达所涉及的顺式作用成分。一旦这些顺式元件在体外被狭隘地定义，诱变研究将被用来监测修改这些纤维特异性调节元件的后果。为了研究这些序列元件在肌肉发育和成熟过程中基因表达调控中的作用，将产生更多的转基因小鼠品系。转基因构建体在胚胎和成年小鼠中的表达将通过报告基因活性分析以及Northern印迹、免疫组织化学和原位杂交分析进行监测。接下来将讨论以下问题：1)在成人骨骼肌中限制TnIs基因表达的顺式作用序列和反式作用因素是什么，以及这些顺式作用序列和反式作用因素与调控体外肌肉特异性表达的成分有何关系？2)“肌肉特异性”和“纤维特异性”调控元件如何在体内肌肉发育和成熟过程中对基因表达的时空调控做出贡献？3)上游和内含子增强子是否分别介导了初级和成熟肌纤维中Tnis基因调控的不同方面？

The proposed research examines the molecular mechanisms by which coordinate gene activation is regulated during the embryonic formation, maturation, and diversification of mammalian skeletal muscle tissue. In recent years, tremendous progress has been made towards understanding the molecular mechanisms that activate muscle-specific genes as committed myoblasts undergo the transition to terminally differentiated myotubes. A fundamental, but much less well defined, aspect of myogenesis is the maturation of muscle cells into specific types of myofibers. During the past NSF support period, the PI characterized the gene encoding the human slow-twitch isoform of troponin I (TnIs) and has shown that multiple cis-acting components are involved in the cell type-expression of the TnIs promoter; an upstream muscle- specific enhancer, a second muscle-specific enhancer within intron 1, and a muscle-specific minimal promoter. In conjunction with collaborators, the PI demonstrated that a TnIs promoter/CAT construct exhibits appropriate muscle fiber type-specific expression in transgenic mice, but preliminary results have indicated that the transgene (which lacks the intron enhancer) is aberrantly regulated during embryogenesis. The hypothesis to be tested is that gene regulation during early and late stages of muscle development requires separate regulatory elements which bind different combinations of trans-acting factors; the upstream elements may play a major role in the innervation dependent restriction of TnIs gene expression to slow twitch muscle fibers and the downstream sequences, such as the intron 1 enhancer, may play a major role in earlier stages of myofiber formation. In order to map the upstream sequences conferring fiber type-specific gene expression, the PI established an in vivo muscle injection assay with which to study differential gene expression in fast versus slow twitch muscles of adult rats. This system enables the PI the unique opportunity to un derstand the molecular mechanisms by which fiber type specificity is regulated. The PI will use the in vivo injection assay to delineate cis- acting components involved in the fiber type-specific expression of the TnIs gene. Once such cis-elements are narrowly defined in vitro mutagenesis studies will be used to monitor the consequences of modifying these fiber- specific regulatory elements. Additional lines of transgenic mice will be produced in order to study the role of these sequence elements in the regulation of gene expression during muscle development and maturation. The expression of transgene constructs in embryonic and adult mice will be monitored by reporter gene activity assays along with Northern blot, immunohistochemical, and in situ hybridization analyses. The following questions will then be addressed: 1) What are the cis-acting sequences and trans-acting factors that restrict TnIs gene expression to slow-twitch fibers in adult skeletal muscle, and how might these be related to the components governing muscle-specific expression in vitro? 2) How do "muscle-specific" and "fiber-specific" regulatory elements contribute to the spatial and temporal control of gene expression during muscle development and maturation in vivo? 3) Do the upstream and intron enhancers mediate separate aspects of TnIs gene regulation in primary versus mature myofibers?