THE REGULATORY ROLE AND ENVIRONMENTAL SENSITIVITY OF DNA METHYLATION IN NEURODEVELOPMENT Marisol Resendiz Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Program of Medical Neuroscience, Indiana University August 2017 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Doctoral Committee June 1, 2017 _____________________________________ Feng C. Zhou, Ph.D, Chair _____________________________________ Xiaoming Jin, Ph.D _____________________________________ Debomoy K. Lahiri, Ph.D _____________________________________ Charles R. Goodlett, Ph.D _____________________________________ Richard Day, Ph.D ii ©2017 Marisol Resendiz iii DEDICATION I dedicate this dissertation to my father, who challenged me to live a life of impact and conquer the limits of my own expectations. Your dedication and relentless pursuit of truth have been my guiding light and I am forever grateful. iv ACKNOWLEDGEMENTS I wish to thank my advisor Dr. Feng Zhou for his patience and guidance during my doctoral training. I thank him for allowing me the opportunity to hone my strengths and for consistently pushing me to improve technically and analytically. I also wish to thank the other members of my research committee, Dr. Xiaoming Jin, Dr. Charles Goodlett, Dr. Richard Day, and Dr. Debomoy Lahiri. I sincerely appreciate their time, support, and constructive critique of my research throughout my training. I thank all the people who contributed to my scientific work, particularly Sherry Lo, Yuanyuan Chen, Nailcan Ozturk, Darryl Watkins, and Darren Chang. I thank Sherry for her assistance with methyl-sensitive restriction enzyme assays and for her guidance and assistance with genomic analysis, pyrosequencing, and early embryonic assays. I thank Yuanyuan Chen for her assistance with immunohistochemistry and confocal microscopy. I thank Nailcan Ozturk for his training in the FASD animal model and his assistance in immunohistochemistry, morphometric analysis, and Western Blotting. I thank Darryl Watkins for his assistance with the animal model. Finally, I thank Darren Chang for his assistance with global methylation assays and quantitative PCR. I would also like to thank Feng Yang, Lijun Ni, and Dr. Charles Anthony for their technical instruction during the early portion of my training. I also wish to acknowledge our collaborators Dr. Jill Reiter, Kerry Sanders, and Ron Unkel and I thank Dr. Chrisine Czackowski for her hard work in procuring the NIAAA training grant 2T32AA007462-31, which financially supported my training for four years. I wish to thank the administrative staff of the Department of Anatomy and v Cell Biology and the Stark Neuroscience Research Institute, especially Kate McMillan, Marthe Cadet-Lorgeat and Nastassia Belton. I also thank Latasha Gilson and Mary Harden for their assistance with my training grant. I am grateful to all the people that provided moral support and professional guidance over the course of my study. I thank my mentors Dr. Graciela Unguez, Dr. Mozella Garcia, Dr. Sherry Greene, Dr. James Williams, and Dr. Paul Ardafyio. I am also indebted to my colleagues Rena Meadows, Grace Santa-Cruz Chavez, Kristin Hollister, Karl Koehler, Tina Wang, Carlos Rodriguez and Angelina Hernandez. Finally, I thank my family and friends for their unwavering support and encouragement throughout this journey. vi PREFACE In 1809 Jean-Baptiste Lamarck described a theory once held by Hippocrates and Aristotle which ascribed evolutionary adaptation as a process that occurred directly from one generation to the next- that physiological adaptations acquired during an organism’s lifetime in response to environmental pressures were passed immediately to offspring. Short lived, the notion was widely discredited in part by experiments executed by August Weismann which asserted that germ cells alone could account for inheritance of traits and were incapable of being affected by somatic cells and thus, environmental pressure. Charles Darwin’s theories of evolution and natural selection, along with Gregor Mendel’s work on inheritance effectively replaced Lamarckism by the early 20th century and have endured as the principle mechanisms for evolutionary adaptation and inheritance of traits. The emergence of epigenetics, or the study of stably heritable phenotypes resulting from chromosomal changes without alterations to the DNA sequence, has prompted a re-examination of Lamarckian theory and challenged the long-held belief that genetics bear the sole weight of organismal physiology, functionality, and adaptation. Though the heritability of epigenetic modifications remains a subject of contention among experts and limits the evolutionary scope of the mechanism, decades of past and emerging research have illuminated very clear and biologically relevant roles for epigenetics which rival, or, more accurately, complement the principles of genetics in guiding organismal physiology. vii Marisol Resendiz THE REGULATORY ROLE AND ENVIRONMENTAL SENSITIVITY OF DNA METHYLATION IN NEURODEVELOPMENT The emerging field of epigenetics is expanding our understanding of how biological diversity is generated in the face of genetic limitations. One epigenetic mechanism in particular, DNA methylation, has demonstrated a dynamic range during neural development. Here, we provide evidence that DNA methylation occurs as a cell- unique program aiding in the regulation of neurodevelopmental gene expression. DNA methylation has demonstrated sensitivity to external inputs ranging from stress to chemical exposure and dietary factors. To explore DNA methylation as a means of communicating early-life stress to the brain, we utilized a mouse model of fetal alcohol spectrum disorders (FASD). FASD presents a range of neurodevelopmental deficits and is a leading cause of neurodevelopmental disabilities in the United States. Predicated on the knowledge of alcohol's teratogenic role in brain development, we describe that the normal pattern of cortical DNA methylation and epigenetic correlates is similarly impacted by prenatal alcohol exposure. Due to the biochemical interaction of alcohol metabolism and the pathways regulating DNA methylation synthesis, we further investigated whether dietary manipulation could normalize the cortical DNA methylation program and aid in the protection of FASD characteristics. We found that the alcohol- sensitive DNA methylation landscape is dually capable of registering dietary intervention, demonstrating normalization of disease-related patterns in the cortex and viii improved neurodevelopmental gene expression and morphology. Finally, we investigated the DNA methylation landscape in a crucial corticodevelopmental gene to more accurately define the breadth and scope of the environmental impacts at the nucleotide level. We found that alcohol and dietary supplementation are selective for regions associated with transcriptional control. Collectively, the evidence supports that DNA methylation plays a regulatory role in development and that its sensitivity to external inputs is dynamic and detectable at the smallest genomic level. Importantly, DNA methylation landscapes are adaptable and thus bear diagnostic and therapeutic potential. Feng C. Zhou, Ph.D., Chair ix TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... xiv LIST OF FIGURES ...........................................................................................................xv LIST OF ABBREVIATIONS ........................................................................................ xviii DISSERTATION OUTLINE...............................................................................................1 CHAPTER 1: CELL-UNIQUE DNA METHYLATION DYNAMICS IN THE NORMALLY-DEVELOPING BRAIN ...............................................................................3 1.1 INTRODUCTION .........................................................................................................3 1.1.1 Structural Regulation Conveys Biological Heterogeneity ..........................................3 1.1.2 Epigenetic Modifications ............................................................................................3 1.1.3 DNA Methylation .......................................................................................................4 1.1.4 DNA Methylation Dynamics ......................................................................................7 1.1.5 Role of DNA Methylation in Neural Development……. .........................................10 1.1.6 Developmental DNA Methylation Programs in the Brain… ....................................14 1.1.7 Research Aims…. .....................................................................................................16 1.2 MATERIALS AND METHODS….. ...........................................................................18 1.2.1 Animals…. ................................................................................................................18 1.2.2 Immunohistochemistry….. .......................................................................................18 1.2.3 Confocal Microscopy…. ...........................................................................................22 1.2.4 Quantification of 5mC and 5hmC using molecular enzymatic assays….. ...............22 1.2.5