Transcriptional and epigenetic control of skeletal muscle mass

原理：自噬是一种蛋白质分解途径，线粒体等细胞器可以被运送到溶酶体进行循环利用。最近的研究，包括我们的研究，已经证明了自噬对于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研究项目，致力于研究自噬在调节骨骼肌质量和功能中的重要性。这一计划的结果是在高评价的期刊上发表了七篇文章，并成功地培训了几名研究生和本科生。该程序还揭示了骨骼肌中的自噬持续数天，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，我想建立一个研究计划，重点确定转录因子EB(TFEB)、信号转导和转录激活因子1(STAT1)以及微RNAs(MiRNAs)在调节骨骼肌自噬中的作用。这项计划将是我长期研究目标的重要组成部分，我的长期研究目标是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，以培训分子生物学技术的最新进展。 目的：建立选择性缺失TFEB和STAT1基因的转基因小鼠模型，探讨TFEB和STAT1在骨骼肌自噬和肌肉质量中的调控作用。我们将TFEBf/f(TFEBf/f)和STAT1/f(Stat1f/f)小鼠与人骨骼肌动蛋白启动子(HSA-ER)驱动表达Cre重组ER的小鼠杂交。肌肉选择性删除TFEB和STAT1将通过每天注射他莫昔芬1周来完成。这些小鼠将暴露在两种刺激下，导致肌肉萎缩，即急性饥饿(24小时)和后肢失神经(14天)。在这两个模型中，我们将测量骨骼肌质量、自噬程度、自噬相关基因表达和线粒体功能的变化。 目的：为了确定microRNAs在骨骼肌自噬和质量中的调节作用，我们将重点介绍几个已知调节癌细胞自噬的miRNAs。我们将评估这些miRNAs在骨骼肌中的表达，以应对急性饥饿和失神经。然后，我们将使用miRNA模拟物和抑制剂来评估这些miRNAs在调节培养的骨骼肌母细胞自噬中的重要性。最后，我们将通过对生物素化的miRNA模拟物进行下拉分析，然后对miRNA-mRNA复合体进行RNA测序，来确定这些miRNAs的假定靶标。

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.