Transcriptional and epigenetic control of skeletal muscle mass

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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.