© 2016 Ying Li ALL RIGHTS RESERVED GENE REGULATION DURING CENTRAL NERVOUS SYSTEM DEVELOPMENT AND POST-INJURY REGENERATION By YING LI A dissertation submitted to the Graduate School-New Brunswick Rutgers, the State University of New Jersey and The Graduate School of Biomedical Sciences In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Biomedical Engineering Written under the direction of Li Cai, Ph.D. And approved by ________________________________________ ________________________________________ ________________________________________ ________________________________________ New Brunswick, New Jersey January 2016 ABSTRACT OF THE DISSERTATION Gene Regulation during Central Nervous System Development and Post-Injury Regeneration By Ying Li Dissertation Director: Li Cai, Ph.D. Central nervous system (CNS) development and post-injury neurogenesis require accurate coordination of neural stem cell proliferation, progenitor cell differentiation, neuron, glia migration and maturation, and synapse formation between axons and dendrites. Such systems with high complexity require strict temporal and spatial control via several levels of regulation, in which the transcription regulation is one of the most critical steps. The developmental and injury-repair process involves over 18,000 genes, for majority of which the molecular mechanism governing their transcription remains largely unknown. In an attempt to address this question, four projects were conducted focusing on two levels of transcription regulation: i.e., chromatin modification, and the interaction of cis-acting regulatory sequences with trans-acting protein factors. Computational methods were adopted to analyze the sequences of the cis-elements and ii make predictions for their interacting transcription factors (TFs). The functional roles of these cis- and trans-elements were further determined in vivo and in vitro. The following findings are presented: 1) the function of DNA topoisomerase II beta (Top2b) in proper laminar formation and cell survival during retinal development; 2) the development of computational method for identifying gene regulatory networks involving enhancers and master TFs that are important in retinal cell differentiation; 3) the mechanism of Notch1 regulation in neural stem/progenitor cells via the interaction between Nkx6.1 and a CNS specific enhancer CR2 during the development of the spinal cord interneurons; and 4) the role of CR2 in aNSC activation after injury. Findings from this dissertation provide new insights into the molecular mechanisms underlying transcription regulation during CNS development and post-injury neurogenesis. They can also serve as a basis for future development of gene therapies and regenerative medicine for neurological disorders including spinal cord injury. iii ACKNOWLEDGEMENTS I would like to first thank my mentor, Dr. Li Cai, for his guidance, support and excellent training. I won’t be able to make all the progress and achievements without his generous support. I would also like to thank my committee members, Dr. Martin Grumet, Dr. Bonnie Firestein, Dr. Roko Rasin and Dr. Kelvin Kwan, for their helpful advices on my research, presentation and manuscripts. I also want to thank all the previous and current Cai lab members, for their help with the experiments and discussions. Finally, I would like to thank my parents, who are the greatest parents in the world and have always been supportive. I am truly grateful to be around with so many nice people and have the opportunity to study here at Rutgers. iv TABLE OF CONTENTS ABSTRACT ---------------------------------------------------------------------------------------------------- ii ACKNOWLEDGEMENTS --------------------------------------------------------------------------------- iv TABLE OF CONTENTS ------------------------------------------------------------------------------------ v LIST OF TABLES -------------------------------------------------------------------------------------------- ix LIST OF FIGURES------------------------------------------------------------------------------------------ xi LIST OF CHARTS ---------------------------------------------------------------------------------------- xiv ABBREIVATIONS --------------------------------------------------------------------------------------------x ACKNOWLEDGEMENT OF SOURCE MATERIAL -------------------------------------------------xi Chapter I. Introduction ------------------------------------------------------------------------------------- 1 1. Transcriptional regulation of gene expression ---------------------------------------- 2 2. Development of the retina and spinal cord -------------------------------------------- 4 3. Gene regulation and activation of adult neural stem cells after spinal cord injury --------------------------------------------------------------------------------------------- 5 Chapter II. Topoisomerase II beta is required for proper retinal development and survival of postmitotic cells ------------------------------------------------------------------------------------------ 7 1. Prologue ---------------------------------------------------------------------------------------- 7 2. Abstract ---------------------------------------------------------------------------------------- 10 3. Introduction ------------------------------------------------------------------------------------ 11 4. Materials and Methods --------------------------------------------------------------------- 14 5. Results ----------------------------------------------------------------------------------------- 17 v 1) Top2b expression is only present in postmitotic cells during mouse retinal development ------------------------------------------------------------------ 17 2) Top2b deficiency does not affect early mouse embryonic neurogenesis but causes morphological defects in the postnatal eye --------------------- 19 3) Top2b deletion leads to defects in retinal lamination------------------------- 22 4) Top2b deficiency causes delayed differentiation of ganglion, horizontal cells and affects their survival ----------------------------------------------------- 24 5) Absence of Top2b leads to defects in Müller glia development ----------- 25 6) Lack of Top2b affects the differentiation/maturation of photoreceptor cells --------------------------------------------------------------------------------------- 27 7) Top2b deletion increases retinal cell death ------------------------------------- 29 8) Top2b deletion impairs transcription of genes associated with cell survival and neurological system development ------------------------------- 32 6. Discussion ------------------------------------------------------------------------------------- 33 7. Acknowledgements ------------------------------------------------------------------------- 39 8. Supplemental Materials -------------------------------------------------------------------- 40 Chapter III. Transcriptional Regulatory network for Retinal Development ------------------- 49 1. Prologue --------------------------------------------------------------------------------------- 49 2. Abstract ---------------------------------------------------------------------------------------- 51 3. Introduction ------------------------------------------------------------------------------------ 52 4. Methods and Results 1) Retina-specific enhancers ---------------------------------------------------------- 54 2) Trans-acting factor binding sites on retina-specific enhancers ------------ 56 3) A motif containing Pou3f2 binding sites ------------------------------------------ 57 vi 4) Key trans-acting factors involved in transcriptional regulatory networks of retinal development ---------------------------------------------------------------------- 59 5) Generation of transcription regulatory network for early retinal development ------------------------------------------------------------------------------- 65 5. Discussion ------------------------------------------------------------------------------------- 70 6. Acknowledgements ------------------------------------------------------------------------- 71 7. Supplemental Materials -------------------------------------------------------------------- 72 Chapter IV. Nkx6.1 Regulates Notch1 Expression during Interneuron Development ----- 76 1. Prologue --------------------------------------------------------------------------------------- 76 2. Abstract ---------------------------------------------------------------------------------------- 78 3. Introduction ------------------------------------------------------------------------------------ 79 4. Materials and Methods --------------------------------------------------------------------- 81 5. Results 1) CR2 activity is in neural stem/progenitor cells and diminishes at the end of spinal cord neurogenesis. ----------------------------------------------------------- 86 2) CR2 activity is restricted to specific neural progenitor domains. ---------- 90 3) CR2 activity persists in the adult neural stem cells. -------------------------- 91 4) A fragment of 139bp is required for CR2 activity. ----------------------------- 94 5) Nkx6.1 regulates CR2 activity. ----------------------------------------------------- 96 6) Nkx6.1 regulates expression of Notch1 and neurogenesis-related genes. ------------------------------------------------------------------------------------------------- 99 6. Discussion -----------------------------------------------------------------------------------