Genomic Analysis of Ribosomal Dna and Its Application to The
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GENOMIC ANALYSIS OF RIBOSOMAL DNA AND ITS APPLICATION TO THE INVESTIGATION OF DISEASE PATHOGENESIS by GABRIEL ETIENNE ZENTNER Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Peter C. Scacheri, Ph.D. Department of Genetics CASE WESTERN RESERVE UNIVERSITY January 2012 Gabriel Etienne Zentner Doctor of Philosophy Guangbin Luo Peter Scacheri Helen Salz Derek Abbott 10-7-2011 To Stephanie, for everything. 1 Table of Contents List of tables 5 List of figures 6 Acknowledgements 8 List of abbreviations 10 Abstract 14 Chapter 1: Background and Significance 16 Overview 17 Structure and transcription of rDNA 17 Organization of the mammalian rDNA repeat 17 Cytological features and chromatin structure of rDNA 20 Nucleosome occupancy of rDNA 21 Transcription of rRNA 23 Cell type-specific regulation of rRNA transcription 24 Epigenetic regulation of rDNA 25 CpG methylation 25 Core histone modifications 27 H1: the linker histone weighs in on rRNA transcription 27 Histone variants 29 H2A.Z 29 H3.3 31 Nucleosome positioning 31 TTF-I sets the stage for epigenetic regulation of rDNA 31 Formation of heterochromatin at rDNA 32 Noncoding RNA transcripts epigenetically regulate rDNA 34 Establishment of active rDNA chromatin 35 Replication timing of rDNA repeats 40 Consequences of dysregulated ribosome biogenesis 41 Crosstalk between ribosome biogenesis and p53 41 Ribopathies: human diseases of ribosome biogenesis 43 The Minute mutations 45 General phenomena related to impaired ribosome biogenesis 46 CHD proteins, CHD7, and CHARGE syndrome 47 CHD proteins 47 CHD7 and CHARGE syndrome 48 Summary and research aims 53 Chapter 2: Integrative genomic analysis of human ribosomal DNA 56 2 Abstract 57 Introduction 58 Results 60 Alignment of high-throughput sequencing data to rDNA 60 Distribution of histone modifications at rDNA 61 Cell type-specificity of histone marks at rDNA 66 Chromatin accessibility and transcription at rDNA 76 Nucleosome occupancy of rDNA 79 ChIP-seq analysis of Pol I chromatin association 79 ChIP-seq analysis of UBF chromatin association 80 The insulator-binding protein CTCF associates with rDNA 87 Discussion 92 Materials and methods 97 Chapter 3: CHD7 functions in the nucleolus as a positive regulator of rRNA biogenesis 105 Abstract 106 Introduction 108 Results 111 CHD7 associates with rDNA 111 CHD7 is dually localized to the nucleoplasm and nucleolus 112 CHD7 influences the levels of the 45S pre-rRNA transcript 116 Depletion of CHD7 reduces cell proliferation and protein synthesis 119 CHD7 antagonizes DNA methylation at active rDNA repeats 122 CHARGE-relevant tissues from Chd7 gene-trap mice show reduced pre-rRNA levels 126 CHD7 promotes rDNA association of the Treacher Collins syndrome protein, treacle 128 Discussion 133 Materials and methods 139 Chapter 4: Investigation into dysregulated ribosome biogenesis as a shared pathogenic component of human haploinsufficiency syndromes 146 Abstract 147 Introduction 149 Results 152 Discussion 157 3 Chapter 5: Discussion and Future Directions 160 Summary 161 Genomic analysis of rDNA 161 CHD7 positively regulates rRNA synthesis 163 Discussion and future directions 164 How does CHD7 promote rRNA biogenesis? 164 Is dysregulated rRNA transcription a pathogenic component of CHARGE syndrome? 167 Relevance of dysregulated rRNA transcription to CHARGE syndrome 167 Relevance of dysregulated nucleoplasmic transcription to CHARGE syndrome 168 A dual-function model for CHD7 and its relevance to CHARGE syndrome 173 rDNA copy number, CpG methylation, and phenotypic variability in CHARGE syndrome 173 rDNA copy number variation 175 CpG methylation 175 Implications of variable rDNA copy number and CpG methylation for phenotypic variability in CHARGE syndrome 176 Dissecting nucleoplasmic and nucleolar functions of CHD7 177 Testing the requirements for a nucleoplasmic function of CHD7 in the mouse 177 Separating the functions of CHD7 using patient- specific iPSCs 179 Investigating CHD7 nucleolar targeting via nucleolar protein interactions 181 CHD7 and rRNA biogenesis: a connection to cancer? 183 Further applications of rDNA genomics 184 Condition-dependent alterations in rDNA chromatin structure 184 Distinguishing active and inactive rDNA repeats 188 Large-scale analysis of protein occupancy at rDNA 190 Appendix: Detailed chromatin immunoprecipitation protocol 192 Bibliography 199 4 List of Tables Chapter 1 Table 1-1. Histone modifications and variants associated with rDNA 28 Chapter 2 Table 2-1. Correlation coefficients for pairwise comparisons 74 Table 2-2. CTCF consensus motifs within human and mouse rDNA 93 Table 2-3. ChIP-PCR primers used in Chapter 2 103 Chapter 3 Table 3-1. qRT-PCR primers used in Chapter 3 144 Table 3-2. ChIP-PCR primers used in Chapter 3 145 Chapter 4 Table 4-1. List of transcription factors and chromatin-associated proteins associated with haploinsufficient congenital anomaly syndromes 154 Chapter 5 Table 5-1. GO biological processes and mouse phenotypes associated with CHD7-bound active and poised enhancers in mESCs 171 5 List of Figures Chapter 1 Figure 1-1. Structure of the mammalian rDNA repeat 19 Figure 1-2. NoRC-dependent silencing of rDNA 36 Figure 1-3. Structure of CHD7 51 Chapter 2 Figure 2-1. Comparison of input samples from K562 cells 62 Figure 2-2. Distribution of histone modifications at rDNA in K562 cells 64 Figure 2-3. H3K4me1 ChIP-PCR in K562 cells 65 Figure 2-4. Normalized tag density scores for histone modifications 67 Figure 2-5. Correlation heatmaps of pairwise comparisons between median signals for histone modifications at rDNA 68 Figure 2-6. Distribution of histone modifications at rDNA in HUVECs 70 Figure 2-7. Distribution of histone modifications at rDNA in H1-hESCs 71 Figure 2-8. Distribution of histone modifications at rDNA in NHEKs 72 Figure 2-9. Comparison of rDNA histone marks across multiple cell types 73 Figure 2-10. Chromatin accessibility, transcription, and nucleosome occupancy at rDNA 77 Figure 2-11. ChIP-seq analysis of Pol I and UBF rDNA association 81 Figure 2-12. UBF associates with nucleoplasmic chromatin 83 Figure 2-13. Analysis of nucleoplasmic UBF peaks 85 Figure 2-14. UBF regulates nucleoplasmic gene transcription 88 6 Figure 2-15. CTCF is associated with human rDNA 90 Figure 2-16. CTCF binds to mouse rDNA 91 Chapter 3 Figure 3-1. CHD7 binds to rDNA 113 Figure 3-2. CHD7 localizes to the nucleoplasm and nucleolus 114 Figure 3-3. CHD7 positively regulates rRNA biogenesis 117 Figure 3-4. CHD7 knockdown does not affect protein levels of known regulators of rRNA transcription 120 Figure 3-5. Loss of CHD7 impairs cell proliferation and protein synthesis 123 Figure 3-6. CHD7 is associated with active rDNA repeats and counteracts rDNA promoter methylation 127 Figure 3-7. Pre-rRNA levels are reduced in CHARGE-relevant tissues from Chd7 gene-trap embryos 129 Figure 3-8. CHD7 promotes association of treacle with rDNA 131 Figure 3-9. CHD7 physically interacts with treacle 132 Chapter 5 Figure 5-1. CHD7 associates with active and poised mESC enhancers 170 Figure 5-2. A model for dual functions of CHD7 174 Figure 5-3. CHD7 expression in HCV-induced HCCs 185 Figure 5-4. CHD7 expression in ovarian cancers 186 Figure 5-5. CHD7 expression in gliomas 187 7 Acknowledgements First and foremost, I thank my thesis advisor, Dr. Peter Scacheri, who has given me the freedom to pursue my own scientific interests and provided me with strong conceptual and experimental training with which to pursue my scientific goals. My training has provided me with the skills to ask and answer my own scientific questions, take scientific risks, and face the challenges of science with energy and enthusiasm. I thank my thesis committee members, Dr. Guangbin Luo, Dr. Helen Salz, Dr. Steven Sanders, and Dr. Derek Abbott for their patience and support throughout my graduate career. I am indebted to my collaborators, Dr. Donna Martin and Dr. Maria Hatzoglou, without whom substantial portions of this work would not have been possible. I also thank Dr. Paul Tesar for his advice and encouragement in the later phases of my graduate work. None of this work would have been possible without the members of the Scacheri lab, both past and present. I am particularly indebted to Michael Schnetz for his advice on choosing the right path through graduate school and his patience in helping me get set up in the lab. I am especially grateful for the friendship I have developed with Stephanie Balow, my "lab sister" and fellow Star Wars geek. Every member of the Scacheri lab, past and present, has contributed to my scientific development and I am truly grateful for all they have done for me. I am also indebted to the administrative staff of the Department of Genetics for their assistance throughout my graduate career. 8 I received invaluable support and encouragement, both scientific and otherwise, from many dear friends near and far, including Jason Heaney, Lorrie Rice, Brian Cobb, Spike Murphy, and Neal Evans. I thank my family, who have been unwavering in their support during my graduate career. They have always encouraged my educational endeavors and challenged me to reach my fullest potential, and it is in no small part because of them that I have completed this work. Last, and certainly not least, I am indebted to Stephanie Doerner for her constant support and encouragement throughout my graduate career. It is no exaggeration to say that, without her, none of this would have been possible. 9 List of abbreviations ac acetyl ActD