THE TBX20-CASZ1 INTERACTION PROVIDES MECHANISTIC INSIGHT FOR DILATED CARDIOMYOPATHY PATHOGENESIS Leslie M Kennedy A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Cell and Developmental Biology in the Biology Department. Chapel Hill 2017 Approved by: Mark Peifer Joan Taylor Victoria Bautch Steve Crews Frank Conlon © 2017 Leslie M Kennedy ALL RIGHTS RESERVED ii ABSTRACT Leslie M Kennedy: The TBX20-CASZ1 Interaction Provides Mechanistic Insight for Dilated Cardiomyopathy Pathogenesis (Under the direction of Frank Conlon) Heart disease is the leading cause of death in the U.S. and throughout the world. It claims more lives than all types of cancers combined, and constitutes a significant financial burden on the global society. Such grim statistics necessitate serious interventions and a better understanding of the genetic underpinnings that predispose to the cardiovascular pathologies that ultimately lead to heart failure and death. A primary method of combating the high rates of morbidity and mortality in those with heart disease, is early detection. In this thesis dissertation, I present the first account of an endogenous interaction between the essential cardiac transcription factors TBX20 and CASZ1. This is the first account of an unbiased affinity purification of TBX20 from cardiac tissue, with subsequent identification of TBX20 transcriptional network components by tandem mass spectrometry. I go on to show, that when this interaction is disrupted specifically in the cardiac environment, animals mutant for these two factors display cardiovascular defects that phenocopy the defects seen in human patients with the heart disease subtype dilated cardiomyopathy. I also found that a specific DCM mutation that maps to the T-box of TBX20 disrupts the interaction with CASZ1, and disruption in the combined transcriptional activity of TBX20 and CASZ1. As the leading type of cardiomyopathy in the young, and the leading cause of heart transplantation, dilated cardiomyopathy, it is critical that the field identifies treatable mechanisms that lead to DCM. iii ACKNOWLEDGEMENTS First, thanks to my Heavenly Father for His grace and favor. In the midst of the struggles, His love has made all the difference. Thanks to the Conlon lab for their support and training throughout this process. This has been an excellent lab for becoming a scientist, and learning to communicate science articulately and effectively. Special thanks to Frank for allowing me to join his lab family. Frank has been a productive and insightful mentor from whom I’ve learned a lot over the years. Thanks to Joan Taylor, Steve Crews, Mark Peifer, and Vicki Bautch for serving on my thesis committee. I cannot imagine how difficult it must be to balance functioning in so many different roles, while still giving students like me undivided attention and invaluable feedback during committee meetings. I understand their time is valuable, and I appreciate their lending a fraction of it to help me succeed. I would not be in graduate school or completing my thesis dissertation without the guidance and support of the Biological and Biomedical Sciences Program, the Initiative for Maximizing Student Diversity Program, the Initiative for Minority Excellence, and the Postdoctoral Readiness to Obtain Professorial Success Program. I have had the great honor of working with Ashalla Freeman, Kathy Woods, Sibby Anderson-Thompkins, and countless more who have made it their goal to help me succeed and celebrate with me as I do. Their programs have made my tenure here at UNC an extraordinarily awesome adventure. Huge thanks to my friends and family, who have supported me endlessly throughout my career. My family has been so incredibly helpful in so many ways. Just knowing that they were in my corner, calling to check on my daughter and I, praying for me, and routing for me iv even when I didn’t realize it. Having a support system has been an incredible blessing for which I am incredibly thankful. I could not have hand-picked a better family of which to be a part. Sincere appreciation goes to my big sister and brother who have been great role models in so many ways. My brother has traveled good part of the most interesting parts of the world, and is filled with passion and knowledge of things that are far beyond my understanding. My sister accomplished so many things in her career while raising my 3 beautiful niece and nephews with her husband. Both my sister and brother have conquered the world in my eyes, while staying humble and true to their family and friends. They are exemplary individuals I am truly honored to know and love. Thanks to my incredible mother, always had a way of making me feel as if no person in the entire universe was as smart as I was or could do the things I did. That type of unwavering belief is incredibly impactful. Because of that example you set for me, I know how to show my daughter that I believe she can accomplish absolutely anything, without any doubt in my mind. Thanks to my mother for showing me what it means to truly believe in someone. To me, that is one of the most important lessons you taught me. To my dear sweet daughter…who’s beautiful spirit encourages me to be better. My daughter, who is currently only 4 years old, will bring me a blanket if she even thinks I might be cold, tells me I should rest when my eyes look tired, shares her very last cookie if she sees that I have eaten all mine. My daughter is the kindest, sweetest, and most compassionate person I know – even to people she does not know. I’m so blessed to be her mother, and pray that I cultivate her to be the truest and best version of herself that she could possibly be. Thus far, I am beyond impressed with the beautiful, intelligent, empathetic person that she already is. Abundant gratitude to my ace. He has been present, and a lot of times having someone be truly present is everything. He has been an integral part of my life’s journey, v especially the journey to completing my doctoral degree. His encouragement, his wisdom, his prayers, his unwillingness to allow me to settle or to quit, he has been such a critical part of my development as a scientist, as a professional, and as a person. The fact that I could never thank him certainly will not prevent me from trying to do so. vi TABLE OF CONTENTS LIST OF FIGURES………………………………….…………………………………………………….................ix LIST OF TABLES..……………………………………………………………………………………………..…….....xi LIST OF ABBREVIATIONS…………………………………………………………………………………………xii CHAPTER 1: INTRODUCTION…………………………………………………………………………….……….1 Global Health………………………………………….……………………………………………….……….1 Heart Disease………………………………………….……………………………………………….……….2 Dilated Cardiomyopathy………………………….……………………………………………….………..3 Dilated Cardiomyopathy Causative Mutations…………………………………………….……….5 Essential Roles of TBX20 in Development…………………………………………….…………….7 Genetic and Molecular Interactions in Heart Disease Pathogenesis……………….……..10 Dissertation Goals…………………………………………….……………………………………………..12 References…………………………………………….…………………………………………………………18 CHAPTER 2: DUAL TBX20 AND CASZ1 HAPLOINSUFFICIENCY IS CAUSATIVE TO DILATED CARDIOMYOPATHY……………………………………………………..………………………26 Preface…………….……………………….……………..……………………….……………..……………..26 Introduction………………………………………….……………………………………………….….……26 Methods………………………………………….……………………………………………….……….…….28 Results………………………………………….……………………………………………….……….………33 Discussion………………………………………….……………………………………………….………….42 Supplemental Materials and Methods……………………………………………………………….60 References………………………………….……………………………………………….………………….84 CHAPTER 3: DISCUSSION………………………………………….……………………………………..………95 vii Developmental Requirement for Protein-Protein Interactions……..…….………………..95 Generating Pluripotent Material for Isolating Endogenous TBX20 Complexes………95 CASZ1 is a Novel TBX20 Interaction Partner……………..…….………………………………..98 Early Detection as a DCM Intervention……………………………………………………………..99 TBX20-CASZ1 Compound Heterozygosity Leads to Dilated Cardiomyopathy………102 Protein Changes in TBX20-CASZ1 Mutant Mice……………..…….……………………….…103 Compensatory and Decompensatory Cardiac Remodeling in Response to TBX20-CASZ1 Heterozygosity……………..…………….………….…………………………….104 References………………………………………….…………………………………..…….………………106 APPENDIX: A GRO/TLE-NURD COREPRESSOR COMPLEX FACILITATES TBX20-DEPENDENT TRANSCRIPTIONAL REPRESSION ……………………111 Preface………………………………………….……………………………………………….………………111 Introduction………………………………………….……………………………………………….………111 Methods………………………………………….……………………………………………….…………….114 Results………………………………………….……………………………………………….………………122 Conclusions………………………………………….……………………………………………….……….132 References………………………………………….……………………………………………….…..……148 viii LIST OF FIGURES Figure 1.1 Myocardial effects of extensive concentric versus eccentric cardiomyocyte growth………………………………………………………………………………………..……….15 Figure 1.2 TBX20 mutations in human cardiovascular disease…………..………..………………….16 Figure 2.1 The TBX20Avi-BirA system for isolation of the TBX20 interactome………………..47 Figure 2.2. Endogenous TBX20 interactome…………………………………………………………………48 Figure 2.3 Double heterozygous hearts undergo pathological remodeling………………………..49 Figure 2.4. TBX20 and CASZ1 interact through their DNA binding domains…………………….51 Figure 2.5. TBX20F256I impairs the TBX20-CASZ1 interaction…………………………………………53 Figure 2.6. Proteomic analysis of mouse hearts with DCM reveals activation of complement cascades and decreased protein abundance