Exercise Hypertrophy and Control of Myosin Induction

描述(申请人提供)：肌球蛋白重链(MyHC)是骨骼肌纤维表型和功能的典型标志，因为它是肌肉纤维最大卸载缩短速度(Vmax)的主要决定因素，目前，关于机械过载(MOV)的综合生物学反应如何导致MyHC基因的纤维类型转移和转录调控改变，人们知之甚少。为此，我们的DNA-蛋白质相互作用和转基因缺失/突变研究定义了一个最小的BetaMyHC启动子(-293/120)，它模仿内源BetaMyHC基因在发育过程中和对MOV的响应的表达模式。这些研究还确定了一个顺式作用元件(富含βA/T；-269/-258)，它是骨骼肌缓慢表达所必需的，并在这个最小的BetaMyHC启动子的MOV反应中发挥潜在作用。在MOV肌肉cDNA文库的酵母1-杂交筛选中，我们发现名义转录增强因子-/(NTEF-1)特异性地与BetaMyHC A/T富含元件结合，尽管先前观察到TEF蛋白仅与肌肉-CAT(MCAT)元件相互作用。我们发现，TEF蛋白可以反式激活肌肉细胞中的BetaMyHC启动子和BetaA/-T富含/胸苷激酶启动子。我们还表明，TEF蛋白结合延伸到MEF2和A/T丰富元件的子集，以及回文mt位点，只有在使用MOV核提取液时才会丰富。重要的是，由于TEF蛋白调节众多的肌肉基因，我们预计我们的发现将对基础和肥大条件下横纹肌和平滑肌基因网络的调节以及所有TEF表达细胞的基因调节具有全球相关性。因此，这项资助的主要目的是通过实现以下目的来破译TEF蛋白在调节骨骼肌表型中的生理作用：1)研究针对横纹肌的转基因TEF-1蛋白过度表达的表型后果；2)通过酵母2-杂交和经典蛋白质组学分离和研究肌肉特异性TEF相互作用蛋白；3)通过建立转基因敲除小鼠来证实NTEF-1在体内的功能。Northern和Western分析将评估TEF-1蛋白过度表达或缺失对整个肌肉表型的影响，这将与整体肌肉和单纤维功能分析相关。这项工作有望确定潜在的治疗蛋白质靶点，旨在为疾病、太空飞行或长时间卧床引起的机械负荷变化引起的肌肉表型改变和衰弱功能丧失提供对策。

DESCRIPTION (provided by applicant): Myosin heavy chain (MyHC) is a quintessential marker of skeletal muscle fiber phenotype and function since it is a major determinate of muscle fiber maximum unloaded velocity of shortening (Vmax), Presently, little is known about how the integrative biological response to mechanical overload (MOV) leads to fiber-type shifts and altered transcriptional regulation of MyHC genes. Towards this end, our DNA-protein interaction and transgenic deletion/mutagenesis studies have defined a minimal betaMyHC promoter (-293/+120) that mimics the expression pattern of the endogenous betaMyHC gene during development and in response to MOV. These studies also identified a cis-acting element (beta A/T-rich; -269/-258) that is required for slow skeletal muscle expression and that serves a potential role in MOV-responsiveness of this minimal betaMyHC promoter. In a yeast 1-hybrid screen of an MOV muscle cDNA library we found that nominal transcription enhancer factor-/(NTEF-1) specifically binds the betaMyHC A/T-rich element despite previous observations that TEF proteins interacted solely at muscle-CAT (MCAT) elements. We showed that TEF proteins can transactivate a betaMyHC promoter and a betaA/-T-rich/thymidine kinase promoter in muscle cells. We also show that TEF protein binding extends to a subset of MEF2 and A/T-rich elements, and the palindromic Mt site, which was enriched only when using MOV nuclear extracts. Importantly, since TEF proteins regulate numerous muscle genes, we expect that our findings will have global relevance to the regulation of striated and smooth muscle gene networks under basal and hypertrophic conditions, and gene regulation in all TEF expressing cells. Thus, the major focus of this grant is to decipher the physiological role of the TEF proteins in regulating skeletal muscle phenotype by performing the following aims: 1) to study the phenotypic consequences of transgenic over expression of TEF-1 proteins targeted to striated muscle, 2) to isolate and study muscle specific TEF interacting proteins by yeast 2-hybrid and classical proteomics, and 3) to confirm the in vivo function of NTEF-1 by generating transgenic knock-out mice. Northern and western analyses will assess the impact of TEF-1 protein over expression or deletion on whole muscle phenotype, which will be correlated to whole muscle and single fiber functional analysis. This work is expected to identify potential protein targets for therapies aimed at providing countermeasures against altered muscle phenotype and debilitating loss of function induced by altered mechanical loads resulting from disease, space flight or extended bed rest.