FUNCTION OF OLIGODENDROCYTE TRANSCRIPTION FACTOR 1 (OLIG1) AND ITS POST- TRANSLATIONAL MODIFICATION BY JINXIANG DAI M.S., Institute of Neuroscience, Shanghai Jiao Tong University, Shanghai, China, 2009 B.S., Shanghai Jiao Tong University, Shanghai, China, 2006 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cell Biology, Stem Cell and Development Program 2015 This thesis for the Doctor of Philosophy degree by Jinxiang Dai has been approved for the Cell Biology, Stem Cell and Development Program by Linda Barlow, Chair Wendy B. Macklin, Advisor Bruce Appel David Clouthier Brad Bendiak Aaron Johnson Date: 8 /14/2015 ii Dai, Jinxiang (Ph.D., Cell Biology, Stem Cell and Development) Function of Oligodendrocyte Transcription Factor 1 (Olig1) and its Post-Translational Modification Thesis directed by Professor Wendy B. Macklin ABSTRACT Oligodendrocytes and myelin are essential for maintaining the structural integrity and function of the axons in the central nervous system (CNS). The progressive decline of remyelination in multiple sclerosis is partially due to the failure of oligodendrocyte differentiation within chronic lesions. Oligodendrocyte specific bHLH transcription factors, Olig1 and Olig2, are expressed exclusively by oligodendrocytes and important for development. While the role of Olig2 in the lineage is well established, the role of Olig1 is still unclear. Understanding the function and regulation of Olig1 in oligodendrocyte development will provide mechanistic insight into the differentiation failure of oligodendrocytes in chronic demyelinated lesions. The function of Olig1 in oligodendrocyte differentiation and developmental myelination in brain was analyzed. Both oligodendrocyte progenitor cell commitment and oligodendrocyte differentiation were impaired in the corpus callosum of Olig1 null mice, resulting in hypomyelination throughout adulthood in the brain. As seen in previous studies with this mouse line, while there was an early myelination deficit in the spinal cord, essentially full recovery with normal spinal cord myelination was seen. We demonstrate a unique role for Olig1 in promoting oligodendrocyte progenitor cell commitment, differentiation and subsequent myelination primarily in brain, but not spinal cord. Subcellular translocation of Olig1 is observed during both oligodendrocyte development and remyelination. Translocation of Olig1 seems to be critical for remyelination, but the mechanism regulating this process has not been established. We identified three functional nuclear export i sequences (NES) localized in the bHLH domain and one specific acetylation site at Lysine 150 (human Olig1) in NES1. Acetylation of Olig1 decreased its chromatin association, increased its interaction with Id2 (inhibitor of DNA binding 2) and facilitated its retention in the cytoplasm of mature oligodendrocytes. We establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm, and is required for oligodendrocyte maturation. In summary, these results solve the discrepancy of Olig1 function in oligodendrocyte development by unraveling its brain-specific role in multiple stages of early oligodendrocyte differentiation but not myelinogenesis. This work also provides mechanistic insight into the finely tuned regulation of Olig1 translocation and function by acetylation. The form and content of this abstract are approved. I recommend its publication. Approved: Wendy B. Macklin ii DEDICATION to my wife Jing Wang and my mom Liqin Wang iii ACKNOWLEDGEMENTS I would like to thank my family for their support throughout the years in the USA. Specifically, I would like to my wife, Jing, whose never-ending love encourage me through this long lasting process of graduate study with all the highs and lows. I would like to thank my supervisor, Dr. Wendy Macklin, for her tremendous support in many years in the lab. She has provided amazing guidance and I am very lucky to have been in her lab. She always gives me freedom to pursue any interesting scientific questions. Just as one of her kids, she watch me through the process of learning how to speaking in English, written in English, doing research and presenting my work in public. I appreciate for the time she had spent on editing my Chinese style English and tolerating many troubles I made for the lab. As a senior scientist, your persistent devotion to research is always inspiring to me. I always enjoy the time discussing science with you and have you pulling out the printed papers from your secret drawers. I feel as a spoiled lucky graduate student in Wendy’s lab for many years, and I cannot expect a better mentor than Wendy. I would like to thank all the members of Macklin lab, past and present, for their efforts to make the lab as a big familiar. Here are my brothers and sisters, who care about each other and work together to make the lab as the best place to stay. Specifically, I would like to thank Hilary Sachs for sharing her knowledge and intelligence about any aspect of science. I would thank Katie Bercury for helping through all stages of my graduate training. Without you, my English may still poor, and my papers may be still in my hand as awful written manuscripts. As my best friend you take care of so much of my lab life to make sure I will survive. I would like to thank Marnie Preston for her patience to listen to me and help me with a lot molecular cloning techniques. I thank rest of the lab for their always support , patience and encouragement. iv I would like to thank my committee members for their constant support, especially my chair Dr. Linda Barlow. I still remember my first rotation in her lab, and how she would be able to understand what I mean in poor English. Her enthusiasm for science really encourages me for doing research, and her unwavering support always helps me get through the graduate training. v TABLE OF CONTENTS CHAPTER l. INTRODUCTION------------------------------------------------------------------------------------------------------------1 Temporal waves of OPC specification and maintenance--------------------------------------------------------3 Extracellular cues guiding oligodendrocyte differentiation and myelination------------------------------4 The many faces of BMP and Wnt: Dual mode regulation of OPCs differentiation--------------------4 Growth factors: OPC maintenance and myelinogenesis-----------------------------------------------------5 Extracellular ligands: non-axonal cues leading the way-----------------------------------------------------7 Axonal ligands: local determinants-------------------------------------------------------------------------------8 Neuronal activity: old concept and new evidence-----------------------------------------------------------10 Intrinsic factors regulate oligodendrocyte differentiation and myelination------------------------------11 Transcriptional regulation-----------------------------------------------------------------------------------------12 Epigenetic regulation of oligodendrocyte development---------------------------------------------------15 Post-transcriptional regulation of oligodendrocyte differentiation: non-coding RNAs------------18 Interplay between the extrinsic signalings and transcriptional regulation of oligodendrocyte development--------------------------------------------------------------------------------------20 Intracellular signaling transduction: the bridge from cell surface to nucleus-------------------------21 Post-translational modification of transcription factors---------------------------------------------------23 Dynamic epigenetic regulation: converging on the chromatin-------------------------------------------26 Olig1 and Olig2: molecular and functional divergence--------------------------------------------------------28 II. OLIG1 FUNCTION IS REQUIRED FOR OLIGODENDROCYTE DIFFERENTIATION IN THE MOUSE BRAIN-----------------------------------------------------------------------------------------------------31 Abstract-------------------------------------------------------------------------------------------------------------------31 Introduction--------------------------------------------------------------------------------------------------------------31 Results---------------------------------------------------------------------------------------------------------------------33 vi OPC production, differentiation and early morphogenesis were reduced in Olig1 null mice----33 Oligodendrocyte differentiation was reduced, but not the initiation of axon wrapping in P8 Olig1 null mice-----------------------------------------------------------------------------------38 Oligodendrocyte deficit persists in the brain of adult Olig1 null mice----------------------------------39 Developmental recovery and normal myelination in the spinal cord of adult Olig1 null mice--------------------------------------------------------------------------------------------------------46 Expression and subcellular location of Olig1 is highly coordinated with oligodendrocyte morphogenesis and the onset of myelination------------------------------------------47 Olig2 expression was not altered in Olig1 null brain--------------------------------------------------------50 Olig1 is important for initial oligodendrocyte differentiation in vitro----------------------------------52 Discussion----------------------------------------------------------------------------------------------------------------56