GENETIC ANALYSIS OF CHROMOSOME REPLICATION TIMING: AN AUTOSOMAL LOCUS THAT CONTROLS CHROMOSOME-WIDE REPLICATION TIMING AND MONO-ALLELIC EXPRESSION by Eric Paul Stoffregen A DISSERTATION Presented to the Molecular and Medical Genetics Department and the Oregon Health & Science University School of Medicine In partial fulfillment of the requirements for the degree of Doctor of Philosophy July 2011 School of Medicine Oregon Health & Science University ___________________________________ CERTIFICATE OF APPROVAL ___________________________________ This is to certify that the Ph.D. dissertation of Eric Paul Stoffregen has been approved ______________________________________ Mathew Thayer, Ph.D., Mentor ______________________________________ Rosalie Sears, Ph.D., Committee member ______________________________________ R. Michael Liskay, Ph.D., Committee member ______________________________________ Soren Impey, Ph.D., Committee member ______________________________________ R. Stephen Lloyd, Ph.D., Committee member TABLE OF CONTENTS LIST OF FIGURES AND TABLES ...................................................................... vi LIST OF ABBREVIATIONS ................................................................................ xi ACKNOWLEDGEMENTS .................................................................................. xv ABSTRACT ..................................................................................................... xviii CHAPTER ONE: Introduction ............................................................................ 1 Preface .............................................................................................................. 2 Genomic Instability in Cancer ............................................................................ 4 DNA Replication and Chromosome Condensation ........................................... 6 Cell Cycle Checkpoints ................................................................................... 12 DRT/DMC: a mechanism for genomic instability ............................................. 15 X-chromosome inactivation as a model for DRT/DMC .................................... 19 XCI properties on autosomes .......................................................................... 25 CHAPTER TWO: An Autosomal Locus that Controls Chromosome-wide Replication Timing and Mono-Allelic Expression ..................... 33 Abstract ........................................................................................................... 34 i Introduction ..................................................................................................... 35 Results ............................................................................................................ 37 ‘Alternative partner’ analysis ........................................................................ 37 Chromosome 6 deletions display DRT ......................................................... 40 Mono-allelic Expression and DRT ................................................................ 43 Asynchronous replication on Chromosome 6 ............................................... 47 Disruption of mono-allelic expression .......................................................... 49 Discussion ....................................................................................................... 50 Materials and Methods .................................................................................... 53 Cell culture ................................................................................................... 53 DNA Fluorescence in situ hybridization (FISH) ............................................ 54 Centromeric, BAC, and Fosmid probes .................................................... 54 RNA-DNA FISH ........................................................................................... 56 Replication timing assay .............................................................................. 57 PCR and expression analysis ...................................................................... 58 Acknowledgements ......................................................................................... 58 Erratum ........................................................................................................... 59 Figures and Tables.......................................................................................... 60 CHAPTER THREE: A rAAV-mediated targeted deletion on chromosome 6 .............................................................................. 77 Introduction ..................................................................................................... 78 ii Results ............................................................................................................ 82 Generation of a rAAV-targeting vector for chromosome 6 deletions ............ 82 rAAV-targeted deletions on chromosome 6 ................................................. 82 Replication timing of rAAV-targeted deletions on chromosome 6. ............... 83 Discussion ....................................................................................................... 84 Methods .......................................................................................................... 86 Cell culture ................................................................................................... 86 Vector construction ...................................................................................... 86 Production and titering of rAAV .................................................................... 87 Viral infection ............................................................................................... 89 Identifying targeted deletions ....................................................................... 89 PCR analysis and generation of homologous arms ..................................... 90 DNA FISH .................................................................................................... 91 Centromeric and Fosmid probes ............................................................... 92 Replication timing assay .............................................................................. 93 Figures ............................................................................................................ 94 CHAPTER FOUR: Conclusions ....................................................................... 99 Summary ....................................................................................................... 100 Future Studies ............................................................................................... 103 Genetic analysis ......................................................................................... 103 ASAR6 characterization ............................................................................. 111 Mechanistic models ................................................................................... 111 iii Conclusions ................................................................................................... 115 APPENDIX I: Supplemental Data: An Autosomal Locus that Controls Chromosome-wide Replication Timing and Mono-Allelic Expression .................................................................................. 120 Introduction ................................................................................................... 121 Chromosome engineering ............................................................................. 122 Balanced Translocations display DRT/DMC ................................................. 123 DRT/DMC and Genomic Instability................................................................ 124 ‘Alternative Partner’ analysis ......................................................................... 125 Chromosome 6 Alternative Partners .......................................................... 126 Chromosome 10 Alternative Partners ........................................................ 129 Replication Timing Assay .............................................................................. 130 Engineering Deletions in Chromosome 6 ...................................................... 133 Chromosome 6 Proximal Deletions ............................................................ 133 Chromosome 6 Distal Deletions ................................................................. 136 Affymetrix Genotyping, Copy Number, and Loss of Heterozygosity ........... 138 Replication timing of chromosome 6 deletions ........................................... 140 Characterization and Mono-allelic expression of FHLOST ............................ 142 RNA-DNA FISH for expression of ASAR6, FUT9, MANEA and FHL5OST 142 Asynchronous replication timing on chromosome 6 ...................................... 143 Expression of MANEA, FHL5, FUT9, and ASAR6 in primary tissues ............ 145 iv Figures and Tables........................................................................................ 147 Erratum ......................................................................................................... 228 APPENDIX II: Author Contributions .............................................................. 229 Author Contributions by Figure ...................................................................... 230 REFERENCES ................................................................................................. 236 v LIST OF FIGURES AND