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Determining Individual Chromosome Missegregation Rates and The Determining individual chromosome missegregation rates and the responses to aneuploidy in human cells Joseph Thomas Worrall A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy August 2017 Statement of originality I, Joseph Thomas Worrall, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: 21st August 2017 2 Publications during PhD enrolment 1. Phillips M*, Khadeir R* , Worrall JT, Sharpe K, Yuan M, Tookman L, Steele J, Lemoine N, Frezza C, Cutts R, Chelala C, Bomalaski J, Sheaff M, Ghazaly E, Szlosarek P. (2017). Macrophages mediate resistance to ADI-PEG20 in Mesothelioma. Cancer Discovery. In Review. 2. Quétier I, Marshall JJ, Spencer-Dene B, Lachmann S, Casamassima A, Franco C, Escuin S, Worrall JT, Baskaran P, Rajeeve V, Howell M, Copp AJ, Stamp G, Rosewell I, Cutillas P, Gerhardt H, Parker PJ, Cameron AJ. (2016). Knockout of the PKN family of Rho effector kinases reveals a non-redundant role for PKN2 in developmental mesoderm expansion. Cell Reports. 14(3):440-8. 3. Campbell GR, Worrall JT, Mahad DJ. (2014). The central role of mitochondria in axonal degeneration in multiple sclerosis. Multiple Sclerosis Journal. 20(14) 1806-13. Conference abstracts: selected oral presentation 1. Worrall JT and McClelland SE (2016). Determining the missegregation rates of individual human chromosomes. Molecular Biology of the Cell. vol. 27. American Society for Cell Biology annual meeting, San Francisco, USA, December 2016. 2. Worrall JT and McClelland SE (2016). Determining the missegregation rates of individual human chromosomes. Cytogenetics and genome research. vol. 148, 131-131. 21st International Chromosome conference, Foz do Iguaçu, Brazil, July 2016. Runner up: Best Student Oral Presentation Award. Collaborations 1. Single-cell sequencing analysis and RPE-1/DLD1 cells harbouring mutant CENP-A alleles were a generous collaboration with the Fachinetti laboratory, Institut Curie, Paris, France. 2. Figures in chapter 6, and data in chapter 7.1, were generated in collaboration with Dr. Sarah McClelland, Barts Cancer Institute, London, UK. * Equally contributing authors 3 Abstract Background. Genomic instability and aneuploidy, which are ubiquitous hallmarks of cancer cells, encompass both structural and numerical chromosome aberrations. Strikingly, cancer cells often display recurrent patterns of aneuploidy which are thought to be contingent on selection pressures within the tumour microenvironment maintaining advantageous karyotypes. However, it is currently unknown if individual chromosomes are intrinsically vulnerable to missegregation, and therefore whether chromosome bias may also contribute to pathological aneuploidy patterns. Moreover, the earliest responses to chromosome missegregation in non- transformed cells, and how these are overcome in cancer, has remained elusive due to the difficult nature of isolating nascent aneuploid cells. Results. Individual chromosomes displayed recurrent patterns of biased missegregation in response to a variety of cellular stresses across cell lines. Likewise, a small subset of chromosomes accounted for a large fraction of segregation errors following one specific mechanism driving aneuploidy. This was supported by the discovery that chromosomes 1 and 2 are strikingly susceptible to the premature loss of sister chromatid cohesion during prolonged prometaphase arrest. Additionally, I have elucidated the arrangement of individual metaphase human chromosomes, highlighting missegregation vulnerabilities occurring at the metaphase plate periphery following nocodazole wash-out. Finally, I have developed a novel system for isolating nascent aneuploid cells, suggesting the earliest transcriptome responses to chromosome missegregation in non-transformed human cells involve ATM and BCL2-mediated apoptosis. 4 Acknowledgements I am grateful to the Medical Research Council for providing the generous funding for this research and Barts Cancer Institute for the provision of excellent facilities. I would first like to thank Dr. Sarah McClelland for providing unparalleled support, supervision and opportunities throughout this PhD. Sincere thanks is extended to the members of the McClelland lab for frequent sanity-checks, encouragement and experimental teachings. Additionally, I cannot express enough gratitude and appreciation to everyone in the “kindergarten” PhD office for providing the backbone to my most joyous times and memories from BCI – no matter how cramped the desk space. This PhD would have been an insurmountable academic and personal endeavour, had it not have been for the unwavering support of my family and friends, to whom I dedicate this thesis. 5 Table of contents ABSTRACT ....................................................................................................................................... 4 TABLE OF CONTENTS ...................................................................................................................... 6 LIST OF FIGURES ........................................................................................................................... 10 LIST OF TABLES ............................................................................................................................. 12 LIST OF ABBREVIATIONS ............................................................................................................... 13 1. INTRODUCTION ........................................................................................................................ 18 1.1. CHROMOSOMES .......................................................................................................................... 21 1.1.1. Chromosome structure and function ............................................................................... 21 1.1.1.1. Chromatin architecture and remodelling ................................................................................. 22 1.1.1.2. Centromeres ............................................................................................................................. 24 1.1.1.3. Telomeres ................................................................................................................................. 27 1.1.2. The nuclear organisation of chromosomes ...................................................................... 29 1.1.2.1. Radial chromosome positioning ............................................................................................... 29 1.1.2.3. Chromatin domains .................................................................................................................. 32 1.1.2.4. Nuclear membrane-associated chromatin ............................................................................... 32 1.1.3. Mitotic chromosome assembly ........................................................................................ 35 1.1.3.1. The structure and composition of SMC complexes .................................................................. 35 1.1.3.2. Condensins ............................................................................................................................... 36 1.1.3.3. Sister chromatid cohesion ........................................................................................................ 37 1.2. MECHANISMS DRIVING ANEUPLOIDY ............................................................................................... 40 1.2.1. DNA damage .................................................................................................................... 40 1.2.1.1. DNA damage repair .................................................................................................................. 40 1.2.1.2. Cell cycle checkpoints ............................................................................................................... 44 1.2.2. Replication stress ............................................................................................................. 49 1.2.2.1. Replication-transcription collisions .......................................................................................... 49 1.2.2.2. Restarting at collapsed forks .................................................................................................... 50 1.2.2.3. Replication stress in cancer ...................................................................................................... 51 1.2.3. The spindle assembly checkpoint ..................................................................................... 52 1.2.4. Chromosome cohesion abnormalities .............................................................................
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