Characterization of NPAS1, a transcription factor that regulates tyrosine hydroxylase expression during dopaminergic neuron development

CHARACTERIZATION OF NPAS1, A TRANSCRIPTION FACTOR THAT REGULATES TYROSINE HYDROXYLASE EXPRESSION DURING DOPAMINERGIC NEURON DEVELOPMENT TEH HUI LENG CHRISTINA NATIONAL UNIVERSITY OF SINGAPORE 2006 CHARACTERIZATION OF NPAS1, A TRANSCRIPTION FACTOR THAT REGULATES TYROSINE HYDROXYLASE EXPRESSION DURING DOPAMINERGIC NEURON DEVELOPMENT TEH HUI LENG CHRISTINA (B.Sc(Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I would like to express my thanks to the following people for being around with me during the course of my postgraduate studies: my supervisor, A/P Lim Tit Meng, for his guidance and giving me the opportunity to pursue my research interest. I am also grateful to him for sending me overseas for conference and attachment and thereby allowing me to broaden my horizons and learning to be more independent. And not forgetting his encouragements and trust in me during my depression when things just not seem to get going, my parents and sisters for always being so supportive of me and making me to be what I am today, Dr Lim Kah Leong, who is forever so concerned about my project. Thank you so much for believing that I can research and all the encouragement that you have given me. You know, your pat on my shoulder made me regain the confidence that I once lost and the will to finish my project, Dr Low Boon Chuan and Dr Ng Huck Hui for their excellent advice and suggestions for my research. Special thanks to Dr Ng for being so participative and willing to share his expertise with me, Prof Wang Shu, my thesis committee member who is always so helpful to my request, Dr Chan Woon Khiong for always being so kind with his reagents, without which I not think I can ever get my experiments going, Dear, my husband who is willing to sacrifice his evenings and weekends to accompany me in the laboratory. Thank you so much for all your love and patience, dear! Yan Tie and Allan for their excellent technical support and advice, without which I may have to spend double the time to finish certain experiments, Junjie and Kevin, my lab mates who are always there to help me out whenever I am stressed out. I am really thankful having you two around, without which the laboratory would have been unbearable, Haiyan, my good sister who never fails to brighten my day with her smile. You are always looking out for me and helping me. Thank you, my friend! Serena, Carol and Xueli for braving through this tough period with me, And all those who have helped me in one way or another. i TABLE OF CONTENTS Acknowledgements i Table of Contents ii List of Figures x List of Tables xiv List of Abbreviations xv Summary xviii CHAPTER ONE – Introduction 1.1) Introduction to Dopaminergic Neurons 1.2) Determination of Cell Fates in the Central Nervous System 1.3) Factors Important for Specification of Dopaminergic Neuron 1.3.1) Sonic Hedgehog 1.3.2) Fibroblast Growth Factor (FGF8) 1.3.3) Transforming Growth Factor-beta 1.4) Transcription Factors in the Control of Dopaminergic Neuron Development 1.4.1) Nurr1 10 1.4.2) Homeobox transcription factors – Lmx1b, Pitx3 and Engrailed and 12 1.5) Dopamine and Associated Pathologies 13 1.5.1) Schizophrenia 13 1.5.2) Parkinson’s Disease 14 1.6) Treatment for Parkinson’s Disease 14 1.7) Role of bHLH Transcription Factors During Development 16 1.7.1) Mechanism of bHLH/PAS protein signaling 19 ii 1.7.2) Members of the bHLH/PAS Protein Family 20 1.7.2.1) Circadian rhythms proteins – Clock, NPAS2 and BMAL1 20 1.7.2.2) Aryl hydrocarbon receptor (AhR) 22 1.7.2.3) Hypoxia Inducible Factor (HIF) 25 1.7.2.4) Single minded Proteins 28 1.7.2.5) NXF (LE-PAS) 30 1.7.2.6) Trachealess and Dysfunction 31 1.7.2.7) ARNT 32 1.7.2.8) Neuronal PAS domain protein 34 1.8) Overview of Experiment 34 CHAPTER TWO – General Materials and Methods 37 2.1) Cell Culture 37 2.2) Transfection of Mammalian Cells 37 2.3) Quantification of Nucleic Acids 37 2.4) Gel Electrophoresis and Purification of DNA 37 2.4.1) Agarose Gel Electrophoresis 37 2.4.2) Isolation and Purification of DNA Fragment by Agarose Gel 38 2.5) Conventional Polymerase Chain Reaction (PCR) 38 2.6) Purification of PCR Product 38 2.7) Enzymatic Manipulations of DNA 38 2.7.1) Endonuclease Restriction Digestion 38 2.7.2) Dephosphorylation of DNA Ends 39 iii 2.7.3) A-tailing of DNA 39 2.7.4) Ligation 39 2.8) Growth, Preparation and Transformation of Competent E.coli Cells 39 2.8.1) Growth of Bacteria in Liquid and Solid Media 39 2.8.2) Preparation of Competent E.coli Cells 40 2.8.3) Transformation of Bacterial Competent Cells 40 2.9) Pure Plasmid Isolation Using Affinity Matrix 41 2.10) DNA Sequencing 41 2.11) Isolation of Proteins from Mammalian Cells 41 2.12) Western Blot Analysis 41 2.13) Flow Cytometry 42 2.14) Dual Luciferase Assay 43 CHAPTER THREE – Identification of Genes Up-regulated Upon MN9D Neuronal Differentiation 44 3.1) Introduction 45 3.2) Materials and Methods 46 3.2.1) Cell Culture 46 3.2.2) Differentiating Agent 46 3.2.3) Construction of cDNA PCR Subtractive Library 46 3.2.4) Colony PCR Screening 47 3.2.5) Identification of Subtractive Library Genes 47 3.2.6) Construction of cDNA Expression Array and Analysis 48 3.3) Results 51 iv 3.3.1) Comparison of Efficiency of Differentiation of MN9D Cells by BDNF, GDNF and n-butyric acid 51 3.3.2) n-Butyric Acid Induces Differentiation of MN9D Cells 3.3.3) cDNA Subtractive Library Identifies Different Classes of Genes Upregulated During MN9D Differentiation 52 52 3.3.4) Microarray Results Confirmed the Up-regulation of Genes From Subtractive Library 53 3.3.5) Casein kinase 2β, SOD1, Hsc70, Syndecan I, Prion protein and PARP Expression Levels During MN9D Differentiation 54 3.4) Discussion 64 3.4.1) Choice of Cell Line 64 3.4.2) Increased Translational Activity during Neuronal Differentiation 64 3.4.3) Casin kinase 2β, SOD1, Prion protein, PARP and Neuritogenesis 65 3.4.4) Hsc70 and Cell Cycle Arrest in Neuronal Cells 67 3.4.5) Syndecan I and Shaping of Morphogen Gradient 67 3.5) Conclusion 68 CHAPTER FOUR – Developmental Expression of NPAS1 in Mouse Embryo 69 4.1) Introduction 70 4.2) Materials and Methods 72 4.2.1) Isolation of RNA from Different Stages of Differentiated MN9D Cells 72 4.2.2) Dissection of Mouse Embryos 72 4.2.3) Construction of cDNAs for Real-time PCR 72 4.2.4) Real-time PCR 73 4.2.5) Extraction of Nuclear and Cytosolic Protein Fractions 74 v 4.3) Results 74 4.3.1) Characterization of mNPAS1 Antiserum 74 4.3.2) NPAS1 Transcript and Protein Level Increased During Early MN9D Differentiation 75 4.3.3) NPAS1 Protein Level Does not Change at Later Stages of MN9D Differentiation 76 4.3.4) NPAS1 is Widely Expressed in the Mouse Embryo 77 4.3.5) Expression of NPAS1 Transcript and Protein Level in the DM and VM 77 4.4) Discussion 84 4.4.1) Dynamic Changes in NPAS1 Expression 84 4.4.2) NPAS1 Begins Expression during Middle Embryonic Stage 84 4.4.3) NPAS1 May Play a Role during Ventral Midbrain Development 85 CHAPTER FIVE – Characterization of Neuronal PAS Domain Protein Functional Domains 86 5.1) Introduction 87 5.2) Materials and Methods 90 5.2.1) Construction of Plasmids 90 5.2.2) Immunohistochemistry 96 5.2.3) Immunoprecipitation 96 5.2.4) Culture and Transformation of Yeast Cells 96 5.2.5) Beta-galactosidase Assay in Yeast 97 5.2.6) Chloramphenicol Acetyltransferase (CAT) and β-gal Assay from Mammalian Cells 97 5.3) Results 98 vi 5.3.1) Cloning of Full Length NPAS1 98 5.3.2) Co-expression ARNT or ARNT2 with NPAS1 Causes NPAS1 Nuclear Translocation 98 5.3.3) N-terminus of NPAS1 is Excluded from the Nucleus 100 5.3.4) Leptomycin B Has No Effect on Subcellular Localization of NPAS1 100 5.3.5) Mutation of NES within NPAS1 Localizes it to the Nucleus 101 5.3.6) NPAS1 and ARNT Interact with Each Other Via Their N-termini 102 5.3.7) Transcriptional Property of ARNT Is Not Required for Nuclear Localization of NPAS1 102 5.3.8) NPAS1 Does Not Contain Any Transactivation Domain 103 5.3.9) NPAS1 Harbors at Least Two Distinct, Non-overlapping Repression Domains 104 5.3.10) Dominant Repression by NPAS1 of ARNT or ARNT2 Transactivation Function 105 5.4) Discussion 135 5.4.1) Mechanism for nuclear localization of NPAS1 135 5.4.2) Partial trans-repression by NPAS1 137 CHAPTER SIX – NPAS1 Down-regulates Tyrosine Hydroxylase Level 140 6.1) Introduction 141 6.2) Materials and Methods 144 6.2.1) Materials 144 6.2.2) Construction of Plasmids 144 6.2.3) Transfection of siRNA 144 6.2.4) Chromatin Immunoprecipitation 145 6.3) Results 146 vii 6.3.1) Over-expression of NPAS1 Promotes G0 Cell Cycle Arrest in MN9D Cells 146 6.3.2) Changes of Tyrosine Hydroxylase Level during MN9D Differentiation 146 6.3.3) Tyrosine Hydroxylase Level Decreased During Late Prenatal Stages in the Ventral Midbrain 147 6.3.4) Effect of Over-expression of NPAS1 on TH Level 148 6.3.5) Effect of NPAS1 Silencing on TH Level 148 6.3.6) NPAS1 Has a Repressive Effect on TH Promoter 149 6.3.7) NPAS1 Binds to TH Promoter In-vivo 149 6.4) Discussion 159 6.4.1) Differentiation of DA Neuron is Accompanied by a Decrease in TH level 159 6.4.2) Mechanism for Transcriptional Repression of TH by NPAS1 160 6.4.3) NPAS1 Negatively Regulates TH Transcription during Midbrain Development 161 CHAPTER SEVEN – Future Work and Final Conclusion 164 7.1) Summary of Work 164 7.2) Future Work 164 7.2.1) Competition between NPAS1 and HIF for ARNT 165 7.2.2) Expression of NPAS1 and TH in Dopaminergic Neurons 165 7.2.3) Functional Redundancy of NPAS1 and NPAS3 on TH Expression 166 7.2.4) Regulation of NPAS1 Promoter 167 7.2.5) Role of NPAS1 on TH Level during Prenatal Development 167 7.2.6) Mining of Data from the Subtractive Library 167 7.3) Final Conclusion 168 viii Kearns, C. 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Neurology 43(5): 986-91. 199 [...]... cellular localization of NPAS1_GFP in MN9D cells 112 5.5 Effect of ARNT and ARNT2 on cellular localization of GFP_NPAS1 in MN9D cells 113 5.6 Effect of ARNT and ARNT2 on cellular localization of GFP in MN9D cells 114 5.7 Effect of ARNT and ARNT2 on cellular localization of NPAS1_GFP in HeLa cells 115 5.8 Effect of ARNT and ARNT2 on cellular localization of GFP in HeLa cells 116 5.9 Effect of ARNT and ARNT2... significant lack of motivation Without treatment, schizophrenia affects the ability to think clearly, manage emotions, and interact appropriately with other people The hypothesis that abnormal regulation of ventral striatal, specifically nucleus accumbens (NAC), dopaminergic transmission represents a neuronal hallmark of schizophrenia has been substantiated in recent years Most etiologic scenarios agree that. .. differentiating dopamine neurons appear at approximately E10-10.5 in the mouse (Lauder and Bloom, 1974) The cessation of proliferation is ensued by upregulation of general and specific dopaminergic markers such as TH Newly formed neurons migrate into medial and lateral positions to form the A8 -A1 0 areas, and also begin to initiate target innervation Several transcription factors controlling these developmental... for Nuclear Localization of NPAS1 127 5.20 Absence of transactivation domain in NPAS1 using the yeast system 129 5.21 Absence of transactivation domain in NPAS1 in the mammalian system 130 5.22 Identification of repression domains in NPAS1 when fused to a strong activator in a yeast system 131 5.23 Identification of repression domains in NPAS1 using the mammalian system 132 5.24 Identification of repression... be first detected at about embryonic day (E) 11.5 in the mouse Most of the midbrain dopaminergic neurons that lie in the substantia nigra and ventral tegmental area are positioned in the vicinity of two essential signaling centers for the DV and AP axis, that is, the floor plate and the isthmic organizer, respectively Many studies on dopaminergic neuron development have shown that dopaminergic progenitors... TGF-β is neutralized between E6 and E10, that is, after the critical period of phenotype induction and acquisition of the TH phenotype (occurring at E4-E7), a significant loss of THpositive neurons is again observed, suggesting that TGF-β acts as a survival-promoting molecule at later stages of development (Farkas et al., 2003) 1.4) Transcription Factors in the Control of Dopaminergic Neuron Development. .. of dopamine and are particularly effective at alleviating the akinesia and rigidity during early stages of the disease Unfortunately, these drugs are only able to control the symptoms of the disease but not on-going cell death Therefore, as the disease progress, less dopaminergic 14 Introduction neurons are available to synthesize dopamine and the effectiveness of such treatment decreases Another approach... its enzymatic and non-enzymatic catabolism (Halliwell, 1992) Dysfunction of the midbrain dopaminergic neurons have been implicated in neuropsychiatric and neurodegenerative disorders including schizophrenia and Parkinson’s disease 1.5.1) Schizophrenia Schizophrenia is a severe brain disease that interferes with normal brain and mental function—it can trigger hallucinations, delusions, paranoia, and significant... fragments of NPAS1 overlapping at the unique NdeI restriction site 108 xiv LIST OF ABBREVIATIONS 2D A aa amp A- P Ahr ALS ARNT bHLH BDNF BMP BSA CaCl2 C C-terminus CKIIβ CNS CAT cDNA DA DBD dATP DNA DTT E En EPO ECL E.coli EDTA EtBR FACS FGF GDNF G g GFP GAP4 HA HIF1 HCl hr HRE Hsc70 2 dimensional adenine amino acid ampicillin anterior-posterior arylhydrocarbon receptor amyotrophic lateral sclerosis arylhydrocarbon... disease that affects more than 2% of the population over 65 years of age PD is characterized at a pathological level by a progressive degeneration and loss of: (1) nigrostriatal and mesolimbic dopaminergic neurons (Hassler, 1938; Hirsch et al, 1998) leading to tremor, rigidity and hypokinesia, the classical symptoms of the disease (Carlsson, 1993; Hornykiewicz, 1993); (2) noradrenergic neurons of the . NATIONAL UNIVERSITY OF SINGAPORE 2006 CHARACTERIZATION OF NPAS1, A TRANSCRIPTION FACTOR THAT REGULATES TYROSINE HYDROXYLASE EXPRESSION DURING DOPAMINERGIC NEURON DEVELOPMENT. CHARACTERIZATION OF NPAS1, A TRANSCRIPTION FACTOR THAT REGULATES TYROSINE HYDROXYLASE EXPRESSION DURING DOPAMINERGIC NEURON DEVELOPMENT TEH HUI LENG CHRISTINA . LIST OF ABBREVIATIONS 2D 2 dimensional A adenine aa amino acid amp ampicillin A- P anterior-posterior Ahr arylhydrocarbon receptor ALS amyotrophic lateral sclerosis ARNT arylhydrocarbon