Aryl Hydrocarbon Receptor-Mediated Regulation of Gene Expression during Cardiomyocyte Differentiation A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) In the Department of Environmental Health of the College of Medicine 2015 by Qin Wang M.S., University of Cincinnati, 2015 M.S., Tsinghua University, 2008 B.S., Jilin University, 2005 B.A., Jilin University, 2005 Committee Chair: Alvaro Puga, Ph.D. Professor Department of Environmental Health University of Cincinnati Committee: Dr. Susan Kasper Dr. Ying Xia Dr. Michael Borchers Dr. Peter Stambrook ABSTRACT The focus of this dissertation is on the identification of novel cardiac specific genes regulated by the aryl hydrocarbon receptor (AHR) and the mechanisms through which TCDD exposure induces cardiotoxicity, primarily regarding the dual roles of the receptor in both regulating cardiomyocyte differentiation and in mediating TCDD-caused cardiotoxicity. The AHR is a ligand-activated transcription factor that belongs to the basic-region-helix-loop-helix PER/ARNT/SIM (bHLH-PAS) superfamily of transcription factors. AHR has a wide range of ligands with the prototypical ligand being the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Exposure to TCDD causes toxicity in multiple organs and organisms including the heart that is primarily mediated through AHR. The binding of TCDD to AHR leads to the translocation of the receptor from the cytosol to the nucleus where the receptor heterodimerizes with Aryl Hydrocarbon Receptor Translocator (ARNT) and consequently binds as a heterodimer to DNA, resulting the regulation of expression for hundreds of downstream genes. A battery of genes that have been extensively studied function in the metabolism of xenobiotics, i.e. cytochrome P450, family1 (Cyp1). Beyond the role in xenobiotic metabolism, the Ah receptor has an important role in basic physiologic processes such as cardiovascular development. The goal of this dissertation is to characterize the developmental role of the receptor as well as the consequences of developmental exposure to TCDD during cardiomyocyte differentiation. Chapter I gives a brief background on the chemical properties and toxicology of TCDD with a focus on the cardiotoxicity. The structure and molecular action of AHR as well as its role in cardiovascular development and homeostasis are described. In chapter II, we used next generation sequencing to analyze temporal trajectories of TCDD-dependent global gene expression in differentiating cells expressing the Ah receptor. We found hundreds of genes deregulated by AHR/TCDD axis, including those that regulate multiple signaling pathways involved in cardiac and neural morphogenesis and differentiation. The deregulated genes also include dozens of genes encoding homeobox transcription factors and Polycomb and trithorax group proteins, which are essential regulators of cardiomyogenesis. In chapter III, we investigated I whether these cardiac specific genes regulated by AHR had a developmental window of sensitivity to TCDD exposure. Interestingly, we found that cardiomyocyte contractility was an AHR-dependent TCDD target solely during the early period of differentiation between days 0 and 3. Within the critical time window, TCDD disrupted the concerted expression of genes involved in the TGFβ/BMP2/4 and WNT signaling pathways, significantly suppressed the autocrine secretion of upstream regulators including BMP4, WNT3a and WNT5a, and elevated the secretion of Activin A. TCDD treatment also causes mitochondrial dysfunction, including altered mitochondrial copy number, mitochondria ultrastructural stress, and damage. AHR activation by TCDD during early ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and causes the loss of contractility in the resulting cardiomyocyte lineage. The results presented throughout this work show the AHR functions related both to normal cardiomyocyte development and cardiac toxicological endpoints, and illustrate the ability of this important transcription factor to regulate the expression of a large number of genes in the context of cardiomyocyte differentiation. II III ACKNOWLEDGEMENTS My sincerest gratitude goes to my supervisor Dr. Alvaro Puga. I am truly grateful to him for the opportunity to study and work in his lab. He has taught me how to think scientifically, critically and independently. He believed in me all those years ago, and always encouraged me to grow as an independent scientist. He has given me tremendous freedom to conduct experiments and to try different ideas and projects. I am forever grateful for his patience and constant guidance over the whole term of my Ph.D. studies. I would not be here if it wasn’t him. I am indebted to all of the members of my dissertation committee including Drs. Ying Xia, Michael Borchers, Susan Kasper, and Peter Stambrook. Every question they brought up in committee meetings was always inspirational to me. Their dedication, insight, criticisms and encouragement have greatly helped me on my road to a Ph.D. Many thanks also go to my Biostatistics supervisor Dr. Marepalli B. Rao, for his encouragement and tireless assistance in statistical analysis of my research data. I would like to extend thanks to the classmates and lab members who have helped me in countless ways over those years. Thank you to Dr. Chia-I Ko for both her friendship and willingness to help me in whenever I need her, from experimental techniques and discussions to career pathway suggestions. She is always there to share what she knows with me. Thanks to Vinicius Carreira for his assistance in pathological experiments. Thanks as well to Yunxia Fan, for her logistic assistance over those years, which helped my research keep going smoothly. Many thanks go to Andrew Vonhandorf and Matthew De Gannes for both their friendship and critical reading of this dissertation. I would also like to thank previous lab members including Ying Wang, Drs. Jerald Lee Ovesen, John Recheid, Hisaka Kurita and Francisco Sanchez-Martin, for their support and the wonderful moment when we worked together. Many thanks are given to those collaborators who have contributed in obtaining data presented in this dissertation: Saikumar Karyala, Dr. Xiang Zhang for next generation sequencing experiments, Drs. Mario Medvedovic and Jing Chen for their statistical analysis of the sequencing data. IV I would like to acknowledge the contribution of all the faculty members and students in the Department of Environmental Health. The stimulating and critical environment created by everybody is truly enjoyable to work here. Many thanks for their generous help and kindness. Thank you to many of my friends, past and present, for their patience to listen to my complains, invaluable perspective, and pertinent suggestions during the hard times of my graduate studies. I really appreciate their accompany and the share of their life experiences. My deepest gratitude goes to my parents and my sister, for their unconditional love and endless support. My parents made great efforts to give me and my sister good education. They always guide me whenever I lose sight of the goal. When I lost faith and confidence in my ability to carry on, they did not. They are the driving force for me to bravely keep going forward and pursue my career goals. V TABLE OF CONTENTS ABSTRACT……………………………………………………………………………..………………….I ACKNOWLEDGEMENTS………………………………………….……………………………………IV TABLE OF CONTENTS………………...…………………………………………………………………1 ABBREVIATIONS……………………………………………………………………………………...…3 LIST OF FIGURES………………………………………………………………………………………...6 LIST OF TABLES………………………………………………………………………………………….8 CHAPTER I Introduction 1.1 TCDD properties, creation, human exposure and pleiotropic toxicity………………………...……….9 1.2 The aryl hydrocarbon receptor signaling pathway…………………………………………..………..13 1.3 Physiological roles of AHR…………………………………………………………………………...15 1.4 The role of AHR in cardiovascular development and disease……………………………………...…19 1.5 Mouse embryonic stem cells and differentiation ………………………………………………..……20 1.6 Cardiomyocyte differentiation from mouse ESCs…………………………………………..……...…22 1.7 Purification and enrichment of ESC-derived cardiomyocytes………………………………………..23 1.8 Characteristics of cardiomyocytes derived from pluripotent ESCs………………………………...…24 1.9 ESCs as an in vitro model for toxicological studies…………………………………………….…….26 CHAPTER II Disruption of Aryl Hydrocarbon Receptor Homeostatic Levels during Embryonic Stem Cell Differentiation Derails the Expression of Homeobox Transcription Factors that Control Cardiomyogenesis Abstract……………………………………………………………………………………………………37 2.1 Introduction……………………………………………………...…………………………………….38 1 2.2 Materials and Methods……………………………………………………………………………..….40 2.3 Results…………………………………………………………………………………………………46 2.4 Discussion……………………………………………………………………………………………..51 2.5 Conclusions…………………...…………………………………………………………………….…55 CHAPTER III Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals during the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions Abstract……………………………………………………………………………………………………69 3.1 Introduction……………………………………………………………………………………………70 3.2 Materials and Methods…………………………………………………...……………………………72 3.3 Results…………………………………………………………………………………………………76 3.4 Discussion………………………………………………………………………..……………………82 3.5 Conclusions………………………………………………………………………………………..…..85