Building a Model for Mapping Gene Tic Variation Affecting
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BUILDING A MODEL FOR MAPPING GENETIC VARIATION AFFECTING GENE EXPRESSION by Peter Daniel Lee Department of Human Genetics, McGill University, Montreal February 2005 A thesis submitted to Mc Gill University in partial fulfillment of the requirements of the degree ofPh.D. ©Peter Daniel Lee, 2005 Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 978-0-494-21668-2 Our file Notre référence ISBN: 978-0-494-21668-2 NOTICE: AVIS: The author has granted a non L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ~. PREFACE 1 At the start of this degree, DNA microarrays were still emerging on the scientific stage. Since then much has changed. Initial microarray studies inspired the imagination - we imagined that we were viewing the global machinery of the cell for the first time. Would it be possible to understand the entire gene network? As the technology evolved, this enthusiasm has been followed by the realization that statistical evaluations were needed. This has not replaced the questions raised during the initial phase of inspiration but rather made us aware ofhow challenging it will be to answer such questions convincingly, even at aIl. Over the course of this thesis l observed the evolution of a technology, a process whereby multiple disciplines interacted to develop a common vocabulary. Many questions remain. l have addressed a small fraction of the field in my thesis; the scope of this experiment is already vast beyond the imagination. Along the way, a great number of analytical tools were developed and applied. The following chapters coyer my own process of evolution in the understanding and application of these techniques to address biological questions. Along the way l have received help from many sources. l present a partiallist of names to acknowledge the individuals who, without their support, this degree would not have been possible: my supervisors Thomas Hudson, Michael Hallett, members ofmy advisory committee, Marcel Behr, Patricia Tonin; at the McGill University and Genome Quebec Centre for Innovation, Rob Sladek, Jaroslav Novak, Celia Greenwood, Tomi Pastinen, Bing Ge, Bob Nadon, Pierre Lepage, Jamie Engert, Chon Loredo-Osti, Jenny Koulis, 2 Andrei Verner, Tibor van Rooij, Donna Sinnett, Sebastien Brunet, Vincenzo Forgetta, Janet Faith, Sebastien de Grandpre; members of the McGill Centre for Bioinformatics, Marina Takane, Greg Finak, Francois Pepin, Michelle Scott, Ernesto Iacucci, Trevor Bruen, Rachel Bevan, Kaleigh Smith; members of the MUHC Emil Skamene, Serge Mostowy; the Human Genetics Student Society, in particular, Emily Manderson, Judith Caron, Caroline Gallant and Vanessa Sancho, co-organizers of the Bioinformatics Research Day; the laboratory of Alan Peterson; the Animal Facility at the Montreal General Hospital Research Institute; Laura Benner, Fran Langton at the Dept ofHuman Genetics; Leon Glass, and Michael Mackey at the Centre for Nonlinear Dynamics; Pablo Tamayo, Todd Golub, Bing Ren, David DeGraffe at the WhiteheadlMIT Center for Genome Research and Michael Rebhan at Astra-Zeneca who provided stimulating discussions at the start of this degree; Spyro Mousses at TGEN and Hilmi Ozcelik at the University of Toronto for continued encouragement along the way; Prof Charles J. Lumsden of the University of Toronto who first made me aware ofbioinformatics and the importance of imagination in science; Jerome Holmes, Johanne O'Malley and the staff of Thomson House for unwavering their support, the Post Graduate Student Society, an example of graduate student governance beyond compare - Thomson House and the PGSS represent environments for interdisciplinary interaction, applied learning, and are among the highest benefits of pursuing graduate studies at McGill. Thanks to those who read and edited this thesis, Tom Hudson, Rob Sladek, Jamie Engert, Anny Fortin, Celia Greenwood and Pierre Lepage, who provided the French translation of the abstracto Special thanks to Monica Herman for her help in times of need. There are many others, Cathy Neighbor, Paul Shoniker, Julia Shiu, Martha Shiu. l thank you aIl. 3 For my sister, Mary-Esther Lee and in memory ofmy mom, Esther Kuo Wah Lee, my dad, Rev. David Yiu Shan Lee, and my brother, John David Lee. 4 r--' TABLE OF CONTENTS PREFACE 2 TABLE OF CONTENTS 5 CONTRIBUTIONS OF AUTHORS 7 ABBREVIATIONS 10 ABSTRACT (ENGLISH) 12 RÉSUMÉ (FRANÇAIS) 14 INTRODUCTION 16 PART 1. BIOLOGICAL MODELS 16 PART 2. GENETICS OF GENE REGULATION 19 PART 3. MICROARRAy TECHNOLOGY 26 PART 4. INBRED MICE 30 .~ PART 5. INTEGRATIVE APPROACHES 32 PART 6. THESIS OBJECTIVES 35 CHAPTER 1-UNDERSTANDING THE SYSTEM: CONTROLS IN MICROARRAY EXPERIMENTS 36 CONTROL GENES AND VARIABILITY: ABSENCE OF UBIQUITOUS REFERENCE TRANSCRIPTS IN DIVERSE MAMMALIAN EXPRESSION STUDIES 39 ABSTRACT 40 INTRODUCTION 41 METRODS 42 RESULTS AND DISCUSSION 44 FIGURE LEGENDS 57 CHAPTER 2 - APPLICATION TO EXPERIMENTAL SYSTEMS: GENE EXPRESSION ANALYSIS OF INBRED MOUSE STRAINS 66 TISSUE-SPECIFIC DIFFERENCES IN BASAL GENE EXPRESSION BETWEEN AlJ AND C57BL/6J INBRED MOUSE STRAINS. 70 ABSTRACT 71 INTRODUCTION 72 MATERIALS AND METRODS 75 RESULTS 78 DISCUSSION 80 FIGURE LEGENDS 97 5 CHAPTER 3 - INTEGRATIVE APPROACHES TO DETECTING CIS- REGULATORY VARIATION ACROSS THE MOUSE GENOME 113 ENRICHED DETECTION OF GENES WITH ALLELE-SPECIFIC EXPRESSION DIFFERENCES BY EXPRESSION PROFILING IN RECOMBINANT CONGENIC STRAINS 116 ABSTRACT 117 INTRODUCTION 118 MATERIAL AND METHODS 122 RESULTS 125 DISCUSSION 129 FIGURE LEGENDS 141 GENERAL DISCUSSION 154 CONCLUSIONS 167 REFERENCES 170 APPENDIX A. REVIEW ARTICLE - LA PUCE À ADN EN MÉDECINE ET EN SCIENCE. 188 APPENDIX B. LABORATORY AND ANIMAL USE ETHICS APPROV ALS 189 6 CONTRIBUTIONS OF AUTHORS Mouse Work AIJ and C57BL/6J mice as weIl as the Recombinant Congenic Panel were provided by Dr Emil Skamene and Dr Anny Fortin. Genotyping data for the RCS panel were provided following a Material Transfer Agreement with Emerillon Therapeutics. Housing, sacrificing and tissue dissections were performed by staff at the MUHC Mouse FaciHty managed by Jean-Marie Chavannes. RNA extractions were performed by technicians Scott Gurd and Yannick Fortin, Dr Rob Sladek, graduate student Sarav Sundararajan and myself. Microarray Hybridizations Microarray hybridizations were performed by Dr Rob Sladek, Yannick Fortin, Daniel Vincent and Arek Siwoski in the Chip Facility at the Mc Gill University and Genome Quebec Innovation Centre. Bioinformatics and Infrastructure l handled aIl programming for analysis of microarray data including parsing of data files using Perl, statistical analysis in SAS and R, incorporation of information from other biological databases, constructing a database for the ReS project and design of web interfaces for analyzing data. Bing Ge provided the positions of Affymetrix oligonucleotides and microsatellites from the RCS project, aligned to the UCSC mouse genome assembly. Tibor van Rooji assisted me in setting up databases and with Perl 7 programming. Peter Nowacki set up the lab's first server and infrastructure of critical importance to my work from 1999 to 2002. Marina Takane, M.Sc. graduate in the laboratory of Dr Mike Hallett, was involved in the early part of the RCS project in formulating proposaIs to search for trans-regulatory relationships, and was involved in designing programs to assign DSO to genes. Greg Finàk, Ph.D. candidate in the laboratory of Drs Morag Park and Mike Hallett, assisted me with statistics programming in R. Ernesto Iacucci, M.Sc. candidate in the laboratory ofDrs Hans Zingg and Mike Hallett, provided advice on the use of the Gene Ontology database. The McGill Centre for Bioinformatics provided critical infrastructure in the form of servers, databases, and computational power. AlI computationaIly intensive analyses were performed on this infrastructure from 2002-2005. Francois