Dual Roles for Transcription Factor Zic3 in Regulating
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DUAL ROLES FOR TRANSCRIPTION FACTOR ZIC3 IN REGULATING EMBRYONIC STEM CELL PLURIPOTENCY AND DIFFERENTIATION LINDA LIM SHUSHAN B.Sc (Honours) The University of Melbourne, 2003 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering NATIONAL UNIVERSITY OF SINGAPORE August 2008 ACKNOWLEDGEMENTS It is a pleasure to thank the many people who made this thesis possible. My deepest thanks goes to Dr Lawrence Stanton for his invaluable guidance and steadfast belief in me. His mentorship has encouraged creativity, flexibility and room to grow, and these past 4 years have been an amazingly inspiring and enriching experience for me. I would also like to thank my thesis advisory committee, Dr Paul Robson and Dr Chan Woon Khiong, for their critical feedback along the way. I am especially grateful to Jonathan Loh for numerous open discussions and exchange of ideas in the development of the Zic3 project. My thanks goes to Li Pin who taught me the critical foundations of stem cell culture, and to Hoi Aina and Wong Kee Yew who have been so generous in their sharing of technical experience. Special appreciation goes to Lim Yiting and Tahira Allapitchay whose work I have referred to in this thesis, and to Rory Johnson for his feedback and comments. I also wish to thank everyone in the GIS Stem Cell group for stimulating discussions and fun companionship. Finally and most importantly, I thank my parents, who have been encouraging, supportive and incredibly giving at every turn of the corner. I am grateful far beyond what words can express for the depth of their grace, their understanding, and the genuine interest they consistently take in my work. I am immeasurably blessed to have them as my parents. Their love has made all the difference in my life - and it is to them I dedicate this thesis. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS II TABLE OF CONTENTS III ABSTRACT VII LIST OF FIGURES X LIST OF TABLES XIII ABBREVIATIONS XIV CHAPTER 1: Introduction........................................................................................ 1 1.1 Derivation of embryonic stem cells 2 1.2 Regulation of embryonic stem cells 4 1.2.1 The key properties of ES cells 4 1.2.2 Extrinsic signalling pathways maintaining ES cell pluripotency 6 1.3 Transcriptional networks in ES cells 9 1.3.1 Regulation of transcription networks 10 1.3.2 Oct4, Nanog and Sox2 are key regulators of transcription in ES cells 15 1.3.2.1 Oct4 21 1.3.2.2 Sox2 23 1.3.2.3 Nanog 25 1.3.3 Identifying genes that contribute to stem cell pluripotency 27 1.4 Properties of zinc finger transcription factor Zic3 29 1.4.1 The Zic gene family 29 1.4.2 Discovery of Zic3 and its general expression domains during development 34 1.4.3 Biochemical pathways involving Zic3 34 1.5 Role of Zic3 in early embryonic development 37 1.5.1 The embryonic midline 37 1.5.1.1 Breaking bilateral symmetry 37 1.5.1.2 Asymmetric Gene Expression: Reinforcement of Left-Right Polarity 39 1.5.2 Zic3 in the development of the embryonic midline 41 1.5.3 The Zic3-null mouse model 42 1.5.4 Zic3 mutations result in X-linked heterotaxy 44 1.6 Role of Zic3 in neural development 46 1.7 Experimental approach and study rationale 50 CHAPTER 2: Methods & Materials ....................................................................... 53 2.1 Molecular biology techniques 54 2.1.1 Cloning 54 2.1.2 Transformation of chemically competent cells 54 2.1.3 PCR analysis of transformants 55 2.1.4 Isolation of plasmid DNA from bacteria 55 2.1.5 Preparation of bacterial stocks 55 iii 2.1.6 Isolation of genomic DNA from cell lines 56 2.2 Cell culture 56 2.2.1 Mouse ES cell culture 56 2.2.2 Human ES cell culture 57 2.2.3 Isolation, expansion, and mitotic inactivation of MEF cells 58 2.2.4 Maintenance of HEK293T cells 59 2.2.5 Cryopreservation of cell lines 59 2.2.6 Thawing of cell lines 60 2.3 ES Cell-based assays 60 2.3.1 RNA interference (siRNA) 60 2.3.2 RNA interference (shRNA) 61 2.3.3 Rescue of RNAi knockdown 61 2.3.4 Secondary ES-colony replating assay 63 2.3.5 Reprogramming assays 63 2.3.5.1 Viral packaging of reprogramming factors 63 2.3.5.2 Viral infection of fibroblast cells 64 2.4 Establishment of clonal cell lines 65 2.4.1 Clonal Zic3 knockdown lines 65 2.4.2 Clonal Zic3-inducible ES cells 65 2.5 ES cell differentiation protocols 66 2.5.1 Retinoic acid differentiation 66 2.5.2 DMSO and HMBA differentiation 67 2.5.3 Neural differentiation of ES cells 67 2.6 Gene expression analysis 68 2.6.1 RNA extraction 68 2.6.2 cDNA synthesis 68 2.6.3 Quantitative real-time PCR 69 2.7 Protein expression analysis 69 2.7.1 Cell lysis and protein quantitation 69 2.7.2 SDS-PAGE 70 2.7.3 Protein detection and chemiluminescence detection 71 2.7.4 Immunocytochemistry 72 2.8 Custom production of Zic3 antibodies 73 2.9 Chromatin Immunoprecipitation (ChIP) 74 2.9.1 ChIP protocol 74 2.9.2 Quantitative PCR for ChIP enrichment 78 2.9.3 ChIP-chip assays, data processing, and statistical analysis 79 2.10 Luciferase reporter assays 80 2.10.1 Nanog promoter assays 80 2.10.2 Zic3 ChIP-identified promoter assays 80 2.11 Co-immunoprecipitation experiments 81 2.12 Gene Expression arrays 81 2.12.1 Illumina mouse arrays 81 2.12.2 Statistical analysis of microarray data 82 2.12.3 Functional annotations using the Panther database 82 CHAPTER 3: Zic3 is involved in transcriptional regulation of ES cell pluripotency................................................................................................................ 84 3.1 Introduction 85 3.2 Results 87 3.2.1 Zic3 expression is associated with ES cell pluripotency 87 iv 3.2.2 Zic3 is regulated by Oct4, Nanog and Sox2 90 3.2.3 Zic3 RNA interference in ES cells 93 3.2.3.1 Loss of Zic3 leads to ES cell differentiation 93 3.2.3.2 Specificity of Zic3 knockdown 97 3.2.4 Zic3 clonal knockdown lines express endoderm lineage markers 100 3.2.4.1 Zic3 clonal knockdown lines 100 3.2.4.2 Endoderm genes are upregulated in Zic3 clonal knockdown lines 108 3.2.4.2 Endoderm protein expression is upregulated in Zic3 clonal knockdown lines 109 3.2.5 Zic2 is able to partially compensate for the function of Zic3 109 3.3 Discussion 117 3.3.1 Zic3 expression is associated with the key regulators of pluripotency in ES cells. 117 3.3.2 Zic3 functions downstream of Oct4, Nanog and Sox2 and is positively regulated by these factors. 117 3.3.4 Zic2 works in concert with Zic3 to reduce endodermal specification in ES cells 122 3.4 Summary 123 CHAPTER 4: Zic3 interacts with Sox2 in ES cells.............................................. 124 4.1 Introduction 125 4.2 Results 126 4.2.1 Zic3 interacts with Sox2 in embryonic stem cells 126 4.2.2 Zic3 shares regulatory pathways with Sox2 in ES cells 129 4.2.3 Zic3 and Sox2 co-occupy physical binding sites in mouse ES cells 134 4.3 Discussion 140 4.3.1 Zic3 and Sox2 regulate a common set of pathways in ES cells 140 4.3.2 Zic3 and Sox2 are interacting partners in ES cells 142 4.4 Summary 145 CHAPTER 5: Zic3 is a regulator of lineage specification during ES cell differentiation........................................................................................................... 146 5.1 Introduction 147 5.2 Results 148 5.2.1 Zic3 regulates the promoters of lineage-specific genes 148 5.2.2 Zic3 binds to promoters of mesoderm, ectoderm and early developmental genes 155 5.2.3 Zic3 overexpression increases mesoderm and ectoderm specification 159 5.2.3.1 Zic3-inducible overexpression cell lines 159 5.2.3.2 Zic3 overexpression leads to upregulation of ectodermal and mesodermal lineage markers 159 5.2.4 Zic3 upregulates neurogenesis during ES cell neural derivation 163 5.3.1 Zic3 is a regulator of lineage-specific pathways 168 5.3.2 Zic3 enhances neurogenesis during ES cell differentiation 172 5.4 Summary 174 CHAPTER 6: Discussion and future directions................................................... 175 6.1 How does Zic3 maintain ES cell pluripotency? 176 6.2 Does cellular context determine activator or repressor functions of Zic3? 178 6.3 Is Zic3 able to reprogram differentiated cells to pluripotency? 182 6.4 Does Zic3 interact with Sox2 to confer neurogenic potential on ES cells? 184 6.5 Concluding remarks 186 BIBLIOGRAPHY .................................................................................................... 188 v APPENDICES…………………………………………………………….………..201 Appendix 1 - Primers for ChIP-PCR assay ............................................................202 Appendix 2 - FDR Analysis: ChIP-PCR results for Zic3/Sox2 common targets .....205 Appendix 3 - Luciferase cloning primers for Zic3 chip-chip validation....................206 Appendix 4 - GFP fluorescence in mES cells transfected with the pSUPER-GFP shRNA vector ...................................................................................207 Appendix 5 - Zic3 ChIP target gene and their associated promoter regions in mouse ES cells ................................................................................208 Appendix 6 - Sox2 ChIP target gene and their associated promoter regions in mouse ES cells. ...............................................................................214 Appendix 7 - Zic5 and Zic2 are transcribed by a divergent promoter....................243 Appendix 8 - Zic3 shares regulatory pathways with Oct4 & Nanog in ES cells.......244 Appendix 9 - Reprogramming assay with Oct4, Sox2, Klf4, C-Myc and Zic3. .......245 Appendix 10 - Zic3 is required for maintenance of pluripotency in embryonic stem cells ..................................................................................................246 vi ABSTRACT The transcription factors Oct4, Nanog and Sox2 are key regulatory players in embryonic stem (ES) cell biology.