On Human Chromosome 7
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PREFERENTIAL ALLELIC EXPRESSION OF GENETIC INFORMATION ON HUMAN CHROMOSOME 7 LAYLA KATIRAEE A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Layla Katiraee 2008 PREFERENTIAL ALLELIC EXPRESSION OF GENETIC INFORMATION ON HUMAN CHROMOSOME 7 LAYLA KATIRAEE Doctor of Philosophy Department of Molecular Genetics University of Toronto 2008 ABSTRACT Genes are typically expressed in equal amounts from both parentally inherited chromosomes. However, recent studies have demonstrated that genes can be preferentially transcribed from a locus. Non-random preferential expression of alleles can occur in a parent-of-origin pattern, known as imprinting, where epigenetic factors regulate their transcription. Alternatively, it can occur in a haplotype-specific pattern, where cis-acting polymorphisms in regulatory regions are thought to underlie the phenomenon. Both forms of unequal allelic expression have been associated with human disease. Consequently, it is important to identify genes subject to unequal allelic expression and characterize mechanisms that regulate differential transcription. This thesis presents the results of a screen for unequal allelic expression where approximately 50 murine transcripts homologous to genes on human chromosome 7 were analyzed. Human chromosome 7 was selected due to its association with several human disorders that show parent-of-origin effects. The screen identified non-imprinted ii preferential allelic expression in numerous transcripts and demonstrated that such patterns can occur in tissue specific patterns. Paraoxonase-1 (Pon1), a gene implicated in arthrosclerosis, was identified as having a dynamic pattern of allelic expression which varies throughout embryonic development. This finding represents the first report of a developmentally regulated pattern of allelic variance. Carboxypeptidase-A4 (Cpa4) was identified as having a tissue-specific imprinted pattern of expression, where the maternal allele was preferentially expressed in all embryonic tissues, with the exception of the brain. The Krüppel-like factor 14 gene (Klf14), a novel imprinted transcript, was found to have ubiquitous maternal expression in all human and murine tissues analyzed. A differentially methylated region, generally associated with imprinted transcripts, was not found in the gene’s CpG island, nor was a differential pattern of histone modifications identified. However, it was determined that maternal methylation regulates the transcript. The data in this thesis contribute to our understanding of the numerous patterns of allelic expression that exist in nature and the diverse mechanisms that regulate them. Ultimately, quantitative analyses of allelic expression patterns and the identification of their underlying genomic DNA sequences will become standard protocol in all biomedical studies. iii ACKNOWLEDGMENTS I would like to thank my supervisor Dr. Stephen Scherer for his confidence in me and for his support, as well as my committee members Dr. Timothy Hughes and Dr. Christopher Pearson for their guidance and encouragement. I would like to thank Dr. Kazuhiko Nakabayashi for introducing me to the field of epigenetics, for the overwhelming amount of guidance that he offered throughout the course of my doctoral degree, for exemplifying scientific creativity and discipline, for answering all my frantic emails, and for his friendship. To Dr. Takahiro Yamada, I thank him for his assistance and collaboration in countless experiments, for his patience in my training, for his wit and humour. To all the post-docs, technicians, and students who assisted me, particularly Dr. Kohji Okamura, Dr. Makiko Meguro-Horike, Dr. Shin-ichi Horike, Dr. Yan Ren, Michelle Lee, Maryam Mashreghi- Mohammadi, Katherine Sansom, and Xiaochu Zhao, I owe a debt of gratitude. I’d like to thank our collaborators who made the findings in this thesis possible. I’d like to thank Dr. Johanna Rommens for her guidance and wisdom. I’d particularly like to thank Dr. Helen Petropoulos for her encouragement, friendship, movie and television critiques, for teaching me all the basics, and for her help troubleshooting several experiments. I thank Adam Smith for his experimental assistance and the critical reading of this thesis. I’d like to thank all past and present members of TCAG and the Scherer lab, particularly the members of the Sequencing and Tissue Culture facilities, as well as the bioinformaticians. I’d like to thank Dr. Ilona Skerjanc and Dr. Robert Hegele for helping me get where I am today. I’d like to thank all the members of the Scherer, Minassian, and Pearson labs for their friendship and help, especially Julie Turnbull, Dr. Iulia Oprea, Julie Tomlinson, Sanjeev Pullenayegum, Dr. Elayne Chan, and Dr. Christian Marshall. To Dr. Lars Feuk and particularly Andrew Carson, with whom I’ve solved over 2000 crossword puzzles, hundreds of wordraces, cricklers, and iSketches, and have spent countless hours discussing the merits of TV shows, I iv owe an endless debt of gratitude for their friendship, support, and assistance. Moving my desk into the lab may have been one of the most difficult, yet responsible decisions I made throughout my PhD, but the ‘conversations’ we had at Sick Kids will always make me smile. To Jennifer Skaug, who epitomizes dedication, discipline, and patience, I can never thank enough. I doubt that I will ever find a co-worker and friend as kind and understanding wherever I may go. I’d like to thank the members of the Association for Baha’i Studies at the University of Toronto, the Baha’i Community of Toronto, Varqa Children’s Magazine, and all the members of my Ruhi study circles for enriching my life. To my friends in Toronto who brightened these past five years: Ted & Lindsay Slavin, Sherine & Nabil Kharooba, Lynn & Jason Arsenault, the Sahba Family, Poopak & Hilda Raad, Robyn & Zayne Raue, and Carol Forster. I thank the countless artists whose music kept me company and entertained me on evenings and weekends ☺ I would like to thank my extended family for their support and prayers along this long trek, particularly my grandparents, my aunt Guity and uncle Faramarz, my cousins Ala, Sina, and Lisa, as well as my in-laws Sam and Barbara, who I consider one of my guardian angels. I would like to thank my siblings Ema, Babak, and Galen and would like to point out that I’m the only real doctor in the family. This thesis would have never been completed without the love, support and encouragement of my parents, Hamid and Mahboobeh, who took care of me when I needed it the most, always believed in me, and let me achieve my goals. Finally, I’d like to dedicate this thesis to my husband and my best friend, David Parker. You’ve brightened every hour of these past five years, including my darkest days. You gave me the strength that I needed to finish and held my hand every step of the way, reminding me why I’m the lucky one. You’ve stood by me and believed in me like nobody ever has. This would have been impossible without you and I look forward to getting where I’m going with you. v TABLE OF CONTENTS THESIS ABSTRACT...................................................................................................................ii ACKNOWLEDGEMENTS............................................................................................................iv TABLE OF CONTENTS..............................................................................................................vi LIST OF TABLES......................................................................................................................ix LIST OF FIGURES......................................................................................................................x LIST OF APPENDICES..............................................................................................................xii CHAPTER I: INTRODUCTION TO UNEQUAL ALLELIC EXPRESSION I.A UNEQUAL ALLELIC EXPRESSION..................................................................................2 I.B UNEQUAL ALLELIC EXPRESSION IN NON-PARENT-OF-ORIGIN PATTERNS....................4 I.B.1 Physiological importance of unequal allelic expression in non-parent of origin patterns............................................................................................................. 4 I.C UNEQUAL ALLELIC EXPRESSION IN PARENT-OF-ORIGIN PATTERNS............................ 5 I.C.1 Imprinting disorders...........................................................................................6 I.C.2 Regulation of imprinting.................................................................................... 9 I.C.2.i DNA methylation................................................................................... 9 I.C.2.ii Histone modifications............................................................................11 I.C.2.iii Insulation............................................................................................12 I.C.2.iv Non-coding RNA................................................................................. 13 I.C.3 Imprinted clusters in human and murine genomes.............................................. 17 I.C.3.i The H19/Igf2 locus............................................................................... 17 I.C.3.ii Imprinted loci on human chromosome 7....................................................18 I.C.3.ii.a GRB10..................................................................................