Regulatory logic of cellular diversity in the nervous system Michael Closser Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2018 © 2018 Michael Closser All Rights Reserved Abstract Regulatory logic of cellular diversity in the nervous system Michael Closser During nervous system development, thousands of distinct cell types are generated and assembled into complex circuits that control all aspects of animal cognition and behavior. Understanding what these diverse cells are, how they are generated, and what they do in the context of circuits and behavior form the fundamental efforts of the field of neuroscience. In this thesis, I investigate how the genomic organization of regulatory elements informs specific patterns of gene expression in the nervous system. In particular, I examine how distinct combinations of transcription factors interpret information encoded in the genome to control global gene expression programs in a cell-type-specific manner. In Chapter Two, I describe the establishment of a developmentally inspired transcriptional programming system to generate spinal and cranial motor neurons directly from mouse embryonic stem cells. Programmed motor neurons acquire general characteristics that mirror their in vivo counterparts, providing a robust system for studying cell fate specification in the nervous system. Combinatorial expression of cell-type-specific programming factors informs context-dependent enhancer binding and acquisition of appropriate cell-type-specific molecular and functional properties. In Chapter Three, I take advantage of this robust, experimentally accessible system to probe the chromatin-level organization and regulatory principles controlling specificity of motor neuron gene expression programs. Motor neuron genes are controlled by multiple distantly distributed enhancer constellations stretched across large regulatory domains. Using this motor neuron specification model, I discovered a unique regulatory organization controlling gene expression in the nervous system, whereby neuronal genes are controlled from uniquely complex regulatory domains acting over large distances. In Chapter Four, I extrapolate on the insights gained from studying motor neurons at a single point in time to investigate the dynamics of the regulatory environment during neuronal maturation. We demonstrate that enhancers are highly dynamic even after postmitotic specification. The dynamic nature of enhancers is dependent on combinatorial binding with new transcriptional cofactors. Overall, my results suggest that neuronal gene expression programs within a single cell type are regulated in a highly dynamic fashion by a complex set of enhancers. I propose that during development the immense cellular complexity of the nervous system is established and maintained by correspondingly complex repertoire of enhancers. Table of Contents List of Figures ......................................................................................................................................... iii Acknowledgments .................................................................................................................................. iv CHAPTER 1: Introduction ....................................................................................................................... 1 A. Regulatory control of cell identity and diversity in the nervous system ........................................ 2 1. Fundamental concepts of cellular identity and diversity in the nervous system 2. Generation of generic motor neuron identity in the mammalian spinal cord 3. Dynamic combinatorial expression of transcription factors in motor neurons 4. Combinatorial expression of motor neuron programming factors in other neuronal cells 5. Regulatory control of postmitotic neuron identity 6. Lessons learned from a simple nervous system B. Enhancer control of gene expression ......................................................................................... 14 1. A brief overview of mammalian enhancers 2. Transcription factor binding at enhancers 3. Dynamic activation of enhancers during development 4. Transcription factor control of long-range enhancer-promoter interactions 5. Topological domains and insulated neighborhoods C. Questions, Challenges and Approach ........................................................................................ 26 D. Figures ........................................................................................................................................ 31 CHAPTER 2: Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity ........................................................................................................................... 38 A. Introduction ................................................................................................................................. 39 B. Results ........................................................................................................................................ 41 1. Ngn2, Isl1 and Lhx3 program spinal motor neuron fate 2. Ngn2, Isl1 and Phox2a program cranial motor neuron fate 3. Functional characterization of induced NIL and NIP neurons 4. Expression profiles of motor neuron programming 5. Motor neuron programming bypasses neural progenitor stages 6. Isl1 genome binding is dependent on programming partners 7. Cell-specific Isl binding correlates with gene expression 8. Sequence motifs explain differential Isl binding 9. Lhx3 and Phox2a co-occupy Isl-binding sites C. Discussion .................................................................................................................................. 50 D. Figures ........................................................................................................................................ 53 CHAPTER 3: Neuronal gene expression is controlled by constellations of enhancers distributed across large regulatory domains ......................................................................................................... 64 A. Introduction ................................................................................................................................. 65 B. Results ........................................................................................................................................ 68 1. An integrated approach to map in vivo regulatory regions in motor neurons 2. Dynamic reorganization of the active enhancer landscape in motor neurons 3. Mapping high-resolution enhancer-promoter interactions in motor neurons 4. IL mediated enhancer-promoter connectivity directly control MN expression program 5. Constellations of distributed enhancers control the expression of motor neuron genes 6. Enhancer constellations are distributed across large regulatory regions 7. Independent regulation from enhancer constellations 8. Gene regulatory organization in the nervous system C. Discussion .................................................................................................................................. 82 i 1. Programming factors control the postmitotic expression program in motor neurons 2. Distributed enhancer constellations in motor neurons 3. Regulatory complexity in the nervous system D. Figures ........................................................................................................................................ 88 CHAPTER 4: Expression of terminal effector genes in mammalian neurons is maintained by a dynamic relay of transient enhancers ................................................................................................. 99 A. Introduction ............................................................................................................................... 100 B. Results ...................................................................................................................................... 102 1. Dynamic changes in the genomic regulatory landscape in postmitotic motor neurons 2. Divergent binding of Ngn2 and Isl1 to stage-specific enhancers 3. Isl1 rapidly relocates to Onecut1-bound sites following Lhx3 downregulation 4. Stage-specific enhancers maintain expression of postmitotic motor neuron genes 5. Transient Isl1-bound enhancers control stable motor neuron gene expression 6. Isl1 is recruited to stage-specific enhancers through interactions with Onecut1 or Lhx3 7. Isl1 and p300 are preferentially recruited to clusters of Onecut1 or Lhx3 C. Discussion ................................................................................................................................ 111 1. Outwardly stable gene expression in a dynamic regulatory environment 2. Indirect recruitment of Isl1 to enhancers through protein-protein interactions 3. Isl1 operates as an integrator TF, functionally integrating binding patterns of anchor TFs D. Figures ...................................................................................................................................... 115 CHAPTER 5: General discussion and future perspectives ............................................................. 124 A. Cell- and stage-specific combinatorial binding of transcription factors B. Klf factors as general activators of enhancer-promoter engagement C. Enhancer-promoter specificity D. A dynamic relay