Transcriptional and epigenetic control of skeletal muscle mass

原理：自噬是一种蛋白质水解途径，线粒体等细胞器可以被运送到溶酶体中进行再循环。包括我们在内的最近研究已经证明了自噬对维持骨骼肌质量和功能的重要性。在过去的五年里，在NSERC的资助下，我在麦吉尔大学建立了一个前沿的研究项目，致力于研究自噬在骨骼肌质量和功能调节中的重要性。这个项目的结果是在高评价的期刊上发表了七篇文章，并成功培养了几名研究生和本科生。该程序还揭示了骨骼肌的自噬持续数天，这需要通过转录和表观遗传网络长期上调自噬相关基因的表达。人们对这些网络的性质知之甚少。为了填补这一基础细胞生物学知识的空白，我想建立一个研究项目，重点研究转录因子EB （TFEB）和转录信号传导和激活因子1 （STAT1）以及微rna （miRNAs）在骨骼肌自噬调控中的作用。这个项目将是我长期研究目标的重要组成部分，全面确定自噬在骨骼肌功能中的调节作用，并将为HQP提供一个良好的环境，以培训最新的分子生物学技术。

Rationale: Autophagy is a proteolytic pathway whereby organelles such as mitochondria can be delivered to the lysosome for recycling. Recent studies including ours have demonstrated the importance of autophagy to the maintenance of skeletal muscle mass and function. Over the past five years, with funding from NSERC, I established a leading-edge research program at McGill University dedicated to study the importance of autophagy in the regulation of skeletal muscle mass and function. This program resulted in the publication of seven articles in highly-rated journals and in the successful training of several graduate and undergraduate students. This program also revealed that autophagy in skeletal muscles persists for days and that this requires prolonged upregulation of autophagy-related gene expression by transcriptional and epigenetic networks. Little is known about the nature of these networks. To fill this gap in basic cell biology knowledge, I would like to establish a research program that focuses on identifying the roles of transcription factor EB (TFEB) and signal transducer and activator of transcription 1 (STAT1) as well as micro RNAs (miRNAs) in the regulation of skeletal muscle autophagy. This program would be an essential element in my long-term research objective, which is to comprehensively identify the regulatory roles of autophagy in skeletal muscle function, and will provide an excellent environment for HQP to train in the latest advances in molecular biological techniques. Objective 1: To identify the regulatory roles of TFEB and STAT1 in skeletal muscle autophagy and mass, we will generate new transgenic mouse models in which the expression of TFEB and Stat1 is selectively deleted in skeletal muscles. We will cross breed TFEB-floxed (TFEBf/f) mice and Stat1-floxed (Stat1f/f) mice with mice expressing Cre recombinate-ER driven by human skeletal Actin promoter (HSA-ER). Muscle-selective deletion of TFEB and Stat1 will be accomplished by 1 week of daily Tamoxifen injection. These mice will be exposed to two stimuli which elicit muscle atrophy, namely, acute starvation (24h) and hind limb denervation (14 days). In both models, we will measure changes in skeletal muscle mass, extent of autophagy, autophagy-related gene expression and mitochondrial function. Objective 2: To identify the regulatory roles of microRNAs in skeletal muscle autophagy and mass, we will focus on several miRNAs that are known to regulate autophagy in cancer cells. We will evaluate the expression of these miRNAs in skeletal muscles in response to acute starvation and denervation. We will then employ miRNA mimics and inhibitors to evaluate the importance of these miRNAs in regulating autophagy in cultured skeletal myoblasts. Finally, we will identify the putative targets of these miRNAs by performing pull down assays of biotinylated miRNA mimics followed by RNA sequencing of miRNA-mRNA complexes.