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Maintenance of Genetic and Physiological Stability in Caenorhabditis Elegans

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Maintenance of Genetic and Physiological Stability in Caenorhabditis Elegans Maintenance of genetic and physiological stability in Caenorhabditis elegans Yannic Chen School of Molecular and Cellular Biology The University of Leeds Submitted in accordance with the requirements for the degree of Doctor of Philosophy January 2020 i The candidate confirms that the work submitted is his own, except where work which has formed part of jointly authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. Part of the experimental work described in Chapter 4 of the thesis is included in the publication below and is directly attributable to the candidate. Pokhrel, B., Chen, Y. & Biro, J. J. (2019) CFP-1 interacts with HDAC1/2 complexes in C. elegans development. FEBS J, 286: 2490-2504. doi:10.1111/febs.14833. The candidate performed the RNAi sensitivity assay of the COMPASS mutants set-2(bn129) and cfp-1(tm6369). Bharat Pokhrel performed most of the work, devised the layout and wrote the manuscript. Jonathan Biro performed heat shock reporter assay. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement The right of Yannic Chen to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. © 2019 The University of Leeds and Yannic Chen ii Acknowledgements I am grateful to the University of Leeds for funding my PhD without which this project would have been impossible. I would like to express my deep gratitude to my supervisor Professor David Westhead and co-supervisor Dr Patricija van Oosten-Hawle for their professional supervision and consistent guidance. My special thanks goes to Dr Ron Chen for initiating and giving me the opportunity to research this PhD project, as well as supervising me for the first half of the PhD program. I am very thankful to my friends Bharat Pokhrel and Dovile Milonaityte for their academic support and their friendship that made my time at Leeds much more enjoyable. My sincere thanks also goes to Dr Laura Jones for her friendly support throughout the second part of the project. I would also like to thank my fellow labmates Rosamund Clifford, Laura Warwick and Hann Ng for their technical support and uplifting the working environment. My sincere appreciation goes to Jeanne Rivera and Dr Edwin Chen for their advice and help. I also want to mention my appreciation to LIDA for their help regarding the Bioinformatics aspect of the project. Last but not least, I am particularly grateful to my father and my mother for their continuous support throughout my life, that allowed me to be where I am today. I give my deep thanks to Wei Li for all her love and support. 我特别感谢我父母不断地支持才能让我有读博的机会 iii Abstract The genetic and physiological stability of an organism is essential to ensure its well-being and survival. This project investigates the effect of epigenetic changes on genetic stability, and the association between different stressors that threaten the physiological stability of C. elegans. Part 1: Gene expression is controlled by epigenetic effects such as DNA methylation and histone modification. The histone modification H3K4me3 is associated with actively transcribed genes and co-localizes with a DNA:RNA hybrid structure known as R-loops which are associated with DNA instability. To investigate the link between H3K4me3 and R-loops, I use Caenorhabditis elegans COMPASS mutants set-2(bn129) and cfp-1(tm6369), that have drastically reduced global H3K4me3 marks. I found that set-2(bn129) has a consistent reduction of R-loop levels compared to wild-type worms, suggesting that SET-2 (or H3K4me3) is vital in sustaining the R-loop levels observed in wild-type worms. Furthermore, seven helicases have been identified to rescue the R-loop levels in set-2(bn129) mutants, four of which are chromatin remodelers, suggesting a link between chromatin remodelling and R- loop aggregation. Part 2: Environmental stress is a common influence that threatens the health of an organism. While different stressors elicit different responses, how these different responses are interconnected is not well understood. To investigate this, I use a bioinformatic approach to compare the response of C. elegans under heat stress and biotic stress inflicted by pathogen infection. Comparison of transcriptomic data from C. elegans infected by different pathogens indicates an overall dissimilar gene expression response. However, a small set of “general pathogen responsive genes” are consistently differentially expressed at a low level under most pathogen infections. Comparing these general pathogen responsive genes with heat shock responsive genes identified a significant overlap of 50 genes. This suggests that the heat shock response and innate immune response partially overlap. iv Table of Contents Acknowledgements ..................................................................................................... ii Abstract ....................................................................................................................... iii List of Figures ............................................................................................................ ix List of Tables ............................................................................................................. xii List of Abbreviations ................................................................................................ xiii Part 1: Investigating the functional relationship between R-loops and the evolutionarily conserved COMPASS complex Chapter 1 Introduction of epigenetics ...................................................................... 2 1.1. Mechanisms of epigenetics ................................................................................... 2 1.1.1. DNA methylation ........................................................................................................................................... 3 1.1.2. Chromatin ........................................................................................................................................................ 4 1.1.3. Histone modification ..................................................................................................................................... 6 1.2. COMPASS complex and H3K4me3 ...................................................................... 10 1.2.1. The COMPASS complex ............................................................................................................................ 10 1.2.2. The H3K4me3 epigenetic mark and its function ................................................................................... 12 1.3. R-loops .................................................................................................................. 14 1.3.1. Formation of R-loops .................................................................................................................................. 15 1.3.2. Preventing R-loop formation ..................................................................................................................... 17 1.3.3. Structure and stability .................................................................................................................................. 18 1.3.4. Resolution of R-loops.................................................................................................................................. 18 1.4. Function and effect of R-loops ............................................................................. 20 1.4.1. Transcription regulation of R-loops ......................................................................................................... 20 1.4.2. R-loop dependent DNA methylation state .............................................................................................. 21 1.4.3. Histone modifications and chromatin compaction ................................................................................ 21 1.4.4. DNA instability ............................................................................................................................................. 24 1.4.5. DNA repair ................................................................................................................................................... 25 1.4.6. Health implications ...................................................................................................................................... 25 1.5. Helicases ............................................................................................................... 26 1.5.1. Types of helicases ......................................................................................................................................... 27 1.5.2. Known helicases that resolve R-loops ...................................................................................................... 31 1.5.3. Other functions of helicases that can affect R-loops ............................................................................. 32 1.6. Aim and objectives ................................................................................................ 35 Chapter 2: Methods for the epigenetic analysis .................................................. 36 2.1.
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