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Global Mef2 Target Gene Analysis in Skeletal and Cardiac Muscle

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Global Mef2 Target Gene Analysis in Skeletal and Cardiac Muscle GLOBAL MEF2 TARGET GENE ANALYSIS IN SKELETAL AND CARDIAC MUSCLE STEPHANIE ELIZABETH WALES A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO FEBRUARY 2016 © Stephanie Wales 2016 ABSTRACT A loss of muscle mass or function occurs in many genetic and acquired pathologies such as heart disease, sarcopenia and cachexia which are predominantly found among the rapidly increasing elderly population. Developing effective treatments relies on understanding the genetic networks that control these disease pathways. Transcription factors occupy an essential position as regulators of gene expression. Myocyte enhancer factor 2 (MEF2) is an important transcription factor in striated muscle development in the embryo, skeletal muscle maintenance in the adult and cardiomyocyte survival and hypertrophy in the progression to heart failure. We sought to identify common MEF2 target genes in these two types of striated muscles using chromatin immunoprecipitation and next generation sequencing (ChIP-seq) and transcriptome profiling (RNA-seq). Using a cell culture model of skeletal muscle (C2C12) and primary cardiomyocytes we found 294 common MEF2A binding sites within both cell types. Individually MEF2A was recruited to approximately 2700 and 1600 DNA sequences in skeletal and cardiac muscle, respectively. Two genes were chosen for further study: DUSP6 and Hspb7. DUSP6, an ERK1/2 specific phosphatase, was negatively regulated by MEF2 in a p38MAPK dependent manner in striated muscle. Furthermore siRNA mediated gene silencing showed that MEF2D in particular was responsible for repressing DUSP6 during C2C12 myoblast differentiation. Using a p38 pharmacological inhibitor (SB 203580) we observed that MEF2D must be phosphorylated by p38 to repress DUSP6. This established a unique model whereby MAPK signaling results in repression of a MAPK phosphatase. The second MEF2 target gene studied was Hspb7, a small heat shock protein that is highly expressed in striated muscle. Using a combination of bioinformatic and biochemical analysis we found that AP-1 can inhibit Hspb7 transcription, in contrast to MEF2 which activates it. Additionally, the glucocorticoid receptor (GR) regulates Hspb7 in a manner dependent on the presence of MEF2. We also demonstrate an in vivo role for Hspb7 in autophagy which has significant implications in skeletal muscle wasting. Overall we found that MEF2A regulates distinct gene networks in skeletal and cardiac muscle, yet important shared target genes such as DUSP6 and Hspb7 also illustrate that MEF2A regulates some common gene programs that are critical to striated muscle health. ii ACKNOWLEDGEMENTS This was a lot of fun but also a lot of hard work! I couldn’t have done it without my parents, Genevieve and Stephen, who taught me to value education and encouraged me to pursue science and learning (and still do!). I wouldn’t be who I am without their love. To my husband, Mike, for keeping me laughing even when things got (seemingly) overwhelming. Also to Amanda, Al, Mike Sr., Heather, Leah and Kristyn: your encouragement and friendship have been so important to me! I would like to thank my supervisor, Dr. McDermott, for training me in all of the different aspects of science and academia. I would also like to thank Dr. Blais for teaching me how to do bioinformatic analyses and my committee members Dr. Scheid and Dr. Cheung for giving me direction during my PhD. There is no way I could have made it through six years without great lab members – Thank you for your support and criticism over the years. In particular I’d like to thank Nezeka for training me from the beginning and helping me talk through my problems. Also, thank-you Sara for being so great to work with on all of our collaborations. iii TABLE OF CONTENTS ABSTRACT ............................................................................................................. ii ACKNOWLEDGEMENTS .................................................................................. iii TABLE OF CONTENTS ...................................................................................... iv LIST OF TABLES ................................................................................................. vi LIST OF FIGURES .............................................................................................. vii LIST OF ABBREVIATIONS ............................................................................... ix CHAPTER I: Literature Review ............................................................................ 1 1. Development of striated muscle ............................................................................................................ 1 1.1 Early development of the mesodermal lineages .............................................................................. 1 1.2 Skeletal myogenesis ........................................................................................................................ 2 1.3 Cardiogenesis .................................................................................................................................. 7 1.4 Striated muscle contraction ............................................................................................................. 9 2. The role and regulation of MEF2 ........................................................................................................ 11 2.1 Overview of MEF2 ....................................................................................................................... 11 2.2 Role of MEF2 during vertebrate development and adult tissue maintenance ............................... 13 2.3 MEF2 protein:protein interactions ................................................................................................ 15 2.4 MEF2 and chromatin remodelling ................................................................................................ 17 2.5 Regulation of MEF2 by post-translational modifications ............................................................. 22 2.6 MEF2 and miRNA gene silencing ................................................................................................ 23 3. Therapeutic relevance of MEF2 in skeletal and cardiac muscle disease ............................................ 24 3.1 Metabolic disease .......................................................................................................................... 24 3.1a: Diabetes ..................................................................................................................................... 25 3.1b: Pathways regulating muscle atrophy ......................................................................................... 28 3.2 Role of MEF2 in skeletal muscle regeneration ............................................................................. 33 3.3 Role of MEF2 in cell death and muscle atrophy ........................................................................... 37 3.4 Cardiac hypertrophy ...................................................................................................................... 39 4. Summary of Literature Review ........................................................................................................... 42 iv CHAPTER II: Statement of Purpose ...................................................................43 CHAPTER III: Global MEF2 target gene analysis in cardiac and skeletal muscle reveals novel regulation of DUSP6 by p38MAPK-MEF2 signaling .....44 CHAPTER IV: Regulation of Hspb7 by MEF2 and AP-1 in muscle atrophy .73 CHAPTER V: Summary of Dissertation ...........................................................106 CHAPTER VI: Future Directions and Conclusions .........................................108 REFERENCES .....................................................................................................111 APPENDIX ...........................................................................................................149 Expanded Material and Methods .......................................................................................................... 149 Bioinformatic Analysis ......................................................................................................................... 156 v LIST OF TABLES Chapter I: Literature Review Table 1. MEF2 and human disease................................................................................................34 Chapter IV: Regulation of Hspb7 by MEF2 and AP-1 in muscle atrophy Table S1. MEF2A target genes that contain AP-1 consensus sequences within the enriched DNA fragment.......................................................................................................................................104 Table S2. siRNA oligonucleotides..............................................................................................105 Table S3. Primers used in qRT-PCR and ChIP-qPCR................................................................105 vi LIST OF FIGURES Chapter I: Literature Review Figure 1. Mesoderm specification during embryogenesis...............................................................2 Figure 2. Somite specification.........................................................................................................3 Figure 3. Embryonic myogenesis....................................................................................................4
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