Regulation of Skeletal Myofiber Type Gene Expression

骨骼肌纤维类型基因表达的调节

• 批准号: 9405895
• 负责人: Robert Wade
• 金额: $ 30.5万
• 依托单位: University of Maryland at Baltimore
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-15 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405895&HistoricalAwards=false
• 关键词: Regulation; Skeletal; Myofiber; Type; Gene

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?

拟议的研究探讨了协调基因激活在胚胎形成，成熟和哺乳动物骨骼肌组织的多样化过程中受到调节的分子机制。 近年来，随着定向成肌细胞向终末分化肌管的转变，对激活肌肉特异性基因的分子机制的理解取得了巨大进展。 肌发生的一个基本的但不太明确的方面是肌细胞成熟为特定类型的肌纤维。 在过去的NSF支持期间，PI表征了编码肌钙蛋白I（TnIs）的人慢收缩亚型的基因，并表明多个顺式作用组分参与TnIs启动子的细胞类型表达;上游肌肉特异性增强子，内含子1内的第二个肌肉特异性增强子和肌肉特异性最小启动子。 PI与合作者一起证明，TnIs启动子/CAT构建体在转基因小鼠中表现出适当的肌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）在胚胎发生过程中受到异常调节。 有待检验的假设是，在肌肉发育的早期和晚期阶段的基因调节需要结合反式作用因子的不同组合的单独的调节元件;上游元件可能在依赖于神经支配的TnIs基因表达对慢颤肌纤维的限制中起主要作用，而下游序列，如内含子1增强子，可能在肌纤维形成的早期阶段起主要作用。 为了绘制赋予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试验，用于研究成年大鼠快肌与慢肌中的差异基因表达。 该系统使PI有独特的机会了解调节纤维类型特异性的分子机制。 PI将使用体内注射试验来描述参与TnIs基因的纤维类型特异性表达的顺式作用组分。 一旦这样的顺式元件被狭义地定义，体外诱变研究将用于监测修饰这些纤维特异性调控元件的结果。 为了研究这些序列元件在肌肉发育和成熟过程中对基因表达的调节作用，将产生额外的转基因小鼠品系。 将通过报告基因活性测定沿着北方印迹、免疫组织化学和原位杂交分析来监测转基因构建体在胚胎和成年小鼠中的表达。 然后将解决以下问题：1）限制TnIs基因表达的顺式作用序列和反式作用因子是什么，以及这些如何与控制肌肉特异性表达的成分在体外？ 2)在体内肌肉发育和成熟过程中，“肌肉特异性”和“纤维特异性”调控元件如何在空间和时间上控制基因表达？ 3)上游增强子和内含子增强子介导初级肌纤维和成熟肌纤维中TnIs基因调控的不同方面吗？