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(SMCHD1) Regulates Gene Expression Structural Maintenance of Chromosome Hinge Domain Containing 1 (SMCHD1) regulates gene expression by Shabnam Massah B.Sc. (Microbiology and Immunology), Azad University, 2006 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Doctor of Philosophy Program Faculty of Health Sciences Ó Shabnam Massah 2017 SIMON FRASER UNIVERSITY Fall 2017 Approval Name: Shabnam Massah Degree: Doctor of Philosophy (Health Sciences) Title: Structural Maintenance of Chromosome Hinge Domain Containing 1 (SMCHD1) Regulates Gene Expression Examining Committee: Chair: Tania Bubela Professor Gratien Prefontaine Senior Supervisor Associate Professor Tim Beischlag Co-Supervisor Professor Esther Verheyen Supervisor Professor Department of Molecular Biology and Biochemistry Michel Leroux Supervisor Professor Department of Molecular Biology and Biochemistry Colby Zaph Supervisor Professor Department of Biochemistry and Molecular Biology Monash University Nadine Provencal Internal Examiner Assistant Professor Carolyn Brown External Examiner Professor Department of Medical Genetics University of British Columbia Date Defended/Approved: November 17, 2017 ii Abstract Eukaryotic cells evolved by packaging genomic DNA into chromatin where DNA is wrapped around histones. This significantly reduces random transcriptional events by providing a barrier for gene expression. In addition, chemical modifications of histones and cytosine residues on DNA greatly impact regulation of gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is a chromatin modifier. SMCHD1 was originally recognized as essential for X chromosome inactivation and survival in female mice where it plays a critical role in methylation of a subset of CpG islands. Structural studies suggest that SMCHD1 interaction with HP1 binding protein, HBiX1, mediates heterochromatin formation over the X chromosome by linking two chromatin domains enriched for repressive histone marks. In addition, loss of SMCHD1 is lethal in male mice in a mixed background, implying that SMCHD1 regulates genes on non-sex chromosomes. Thus, we identified a need to investigate the role of SMCHD1 in regulating expression of autosomal genes. In addition, I sought to determine SMCHD1 genome occupancy when global DNA methylation was greatly reduced and identify candidate binding partners. I used shRNA technology to knockdown SMCHD1 expression and identified genes that were up and down regulated in human embryonic kidney (HEK293) cells. A number of these genes are expressed in either a stochastic or parent-of-origin monoallelic fashion. Using chromatin immunoprecipitation-sequencing (ChIP), I identified genome-wide occupancy of SMCHD1 and showed that its genomic binding was sensitive to the DNA demethylating reagent, 5-azacytidine. SMCHD1 occupancy correlates with a number of genes belonging to the G protein-coupled receptor superfamily and loss of SMCHD1 in human neuroblastoma SH-SY5Y cells leads to increased levels of cellular cAMP. In addition, loss of SMCHD1 increases KCNQ1 expression, a subunit of the potassium voltage gated channel that plays a critical role in repolarization of the cardiac action potential. Moreover, using tandem tagged affinity purification, I investigated binding partners that potentially interact with SMCHD1 to regulate gene expression. Taken together, SMCHD1 might be involved in variety of diseases including Facioscapulohumeral Muscular Dystrophy (FSHD) and Bosma Arhinia Microphthalmia Syndrome (BAMS). iii Keywords: SMCHD1; CpG methylation; monoallelic; cAMP; KCNQ1; Tap-tag iv To my husband, Payam, my best friend and soul mate and To our son, Aryo, our bundle of joy, love and happiness v Acknowledgements My journey at SFU has been exciting, encouraging and life changing. I would like to express my sincerest gratitude to my senior supervisor, Dr. Gratien Prefontaine for believing in me and providing the immense opportunity to learn and grow as a scientist. I am grateful for his guidance and mentorship throughout these years. Without his support and encouragement, none of this would have been possible. I am thankful to Dr. Tim Beischlag for his endless support, mentorship and guidance over the years. My sincere thanks also goes to Dr. Esther Verheyen, Dr. Michel Leroux and Dr. Colby Zaph for their guidance, valuable feedbacks and helping me to stay on track over the years. I am grateful to Dr. Frank Lee for his insightful comments, continuous support and encouragement. I would like to extend my thanks to my internal examiner, Dr. Nadine Provencal, and my external examiner Dr. Carolyn J. Brown for taking the time to read my thesis and for sitting on my examining committee. I would like to thank all the past and present members of the Prefontaine lab, first and foremost, Rob Hollebakken, Dr. Addie Kolybaba and Dr. Gabrielle Rabu who were my first mentors in the lab and supported me over the years. I also want to express my deep gratitude to my research family and life-long friends, Mandeep Takhar, Nicole Bance, Shazina Khan, Samaneh Khakshour, Hadi Esmaeilsabzali, Mark Labrecque, Julienne Jagdeo, Kevin Tam, Rebecca Nason, Robert Payer, Alex Reers and Lillian Wong. Finally, but most importantly I would like to thank my family. This journey would not have been possible without the support and encouragement of my husband, son, mom, dad, sister and grandma. Thank you for believing in me. Your unconditional love inspires me to work hard and follow my dreams. vi Table of Contents Approval ............................................................................................................................. i Abstract ............................................................................................................................ iii Acknowledgements .......................................................................................................... vi Table of Contents ............................................................................................................ vii List of Tables .................................................................................................................... xi List of Figures .................................................................................................................. xii List of Acronyms .............................................................................................................. xv Chapter 1. General Introduction ................................................................................. 1 1.1. DNA packaging and chromatin structure .................................................................. 1 1.1.1. Chromatin remodeling proteins ................................................................... 3 1.1.2. Chromosome organizers ............................................................................. 5 1.1.2.1 Structural maintenance of chromosome (SMC) complexes and their function ..................................................................................................... 5 1.1.2.1.1 Cohesin .......................................................................................... 7 1.1.2.1.2 Condensin ...................................................................................... 9 1.1.2.1.3 The SMC5/6 complex ................................................................... 10 1.1.2.2 Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) ................................................................................................... 10 1.1.3. Epigenetic modifications and chromatin structure ..................................... 11 1.1.3.1 Histone modifications ............................................................................. 12 1.1.3.2 Histone modification and transcriptional regulation ............................... 13 1.1.3.3 DNA methylation .................................................................................... 14 1.1.3.4 DNA demethylation ................................................................................ 16 1.2. Genes regulated by SMCHD1 ................................................................................ 17 1.2.1. X chromosome inactivation ....................................................................... 17 1.2.1.1 Regulation of X chromosome inactivation ............................................. 17 1.2.1.2 The X inactive specific transcript (Xist) ncRNA ..................................... 21 1.2.1.3 The Tsix ncRNA ..................................................................................... 22 1.2.1.4 Chromatin modifications associated with XCI ........................................ 23 1.2.1.5 The role of DNA methylation in XCI ....................................................... 24 1.2.1.6 The role of regulatory proteins in XCI .................................................... 25 1.2.1.7 The role of SMCHD1 in XCI ................................................................... 26 1.2.2. Genomic imprinting and regulation of imprinted genes ............................. 29 1.2.3. KCNQ1 imprinted locus, its epigenetic regulation and associated diseases .................................................................................................... 31 1.2.3.1 KCNQ1 role in mediating cellular cAMP production by adenylyl cyclases and associated diseases ................................................................. 33 1.2.3.2 Adenosine 3’,5’-cyclic monophosphate (cAMP)
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