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Chromatin Dynamics in The TRANSCRIPTION AND CHROMATIN DYNAMICS IN THE NOTCH SIGNALLING RESPONSE Zoe Pillidge Churchill College Department of Physiology, Development and Neuroscience University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy September 2018 TRANSCRIPTION AND CHROMATIN DYNAMICS IN THE NOTCH SIGNALLING RESPONSE During normal development, different genes are expressed in different cell types, often directed by cell signalling pathways and the pre-existing chromatin environment. The highly-conserved Notch signalling pathway is involved in many cell fate decisions during development, activating different target genes in different contexts. Upon ligand binding, the Notch receptor itself is cleaved, allowing the intracellular domain to travel to the nucleus and activate gene expression with the transcription factor known as Suppressor of Hairless (Su(H)) in Drosophila melanogaster. It is remarkable how, with such simplicity, the pathway can have such diverse outcomes while retaining precision, speed and robustness in the transcriptional response. The primary goal of this PhD has been to gain a better understanding of this process of rapid transcriptional activation in the context of the chromatin environment. To learn about the dynamics of the Notch transcriptional response, a live imaging approach was used in Drosophila Kc167 cells to visualise the transcription of a Notch-responsive gene in real time. With this technique, it was found that Notch receptor cleavage and trafficking can take place within 15 minutes to activate target gene expression, but that a ligand-receptor interaction between neighbouring cells may take longer. These experiments provide new data about the dynamics of the Notch response which could not be obtained with static time-point experiments. The chromatin accessibility and nucleosome dynamics at Notch-responsive enhancers were also studied using a variety of molecular techniques. These experiments showed that enhancers occupied by Su(H) were highly accessible with a high level of nucleosome turnover, and that Notch signalling promoted a further increase in accessibility. The BRM complex, a SWI/SNF chromatin remodeller implicated in many cancers, was identified as essential for the high chromatin accessibility at these regions and the Notch response. This new insight into the link between a simple signalling pathway and chromatin remodelling could have implications for understanding the complicated process of development and what goes wrong in diseases like cancer. Zoe Pillidge - September 2018 i ACKNOWLEDGEMENTS Firstly, my greatest thanks go to my supervisor, Professor Sarah Bray, without whom this PhD would not have been possible. I am incredibly grateful for her taking me on as a rotation student at short notice and quickly making arrangements for me to learn the experimental techniques in which I was the most interested. Joining the Bray laboratory was one of the best decisions of my life. Sarah has been a fantastic supervisor, providing just the right amount of guidance at all stages of my PhD, and I have learnt a lot from her. I would also like to thank my adviser Rob White, who has been an ongoing source of academic guidance, and a very friendly and supportive person to work alongside. Next, I would like to thank the members of the Bray laboratory who have aided my research immensely over the years. I would like to thank Maria Gomez- Lamarca and Jin Li, particularly for their support when I first joined the Bray laboratory. Jin continued to send advice by email after leaving the country, and Maria has continued to support me throughout the entire PhD, both experimentally and emotionally. I am also deeply grateful for the hard work of our laboratory manager Kat Millen and assistant Agnes Asselin. Together, they have made sure that everything runs smoothly in the Bray laboratory, looking after both the shared space and the people within it. Other member of the Bray laboratory who have either been great friends, offered experimental advice or, in most cases, both include: Matthew Jones, Eva Zacharioudaki, Julia Falo- Sanjuan, Hadi Boukhatmi, Stella Lempidaki, Gustavo Cerda-Moya, Stephen Chan, Silvie Fexova, Torcato Martins, Jonty Townson and Sara Morais-da-Silva. For their contributions to particular experiments, I would like to specifically mention the following people. Fellow PhD student Julia Falo-Sanjuan was heavily involved in setting up the MS2 system in the Bray laboratory, writing the MATLAB code for the analysis. On top of this, I am grateful for her intelligent insights and encouraging discussions, which pushed the project forward. Damiano Porcelli, a former member of the White laboratory, was a great help with setting up the ATAC experiments, sharing his protocol and even some reagents. I deeply appreciate having him as both a laboratory and office iv Zoe Pillidge - September 2018 neighbour for most of my PhD, as he almost always (when he wasn’t drumming too loudly on the desk) brightened my day. The locus tag, as we call it, has been an invaluable tool for studying the live Notch response in vivo, and I am therefore thankful for the hard work by Matthew Jones to implement this system. I would also like to thank Peter Verrijzer for providing antibodies against some BRM complex subunits, Kami Ahmad for sending me flies which express GFP-tagged histone proteins, Neus Visa for sending me plasmids containing the brm sequence, and Dirk Schübeler for sending me plasmids containing V5-tagged histone sequences. I would also briefly like to mention several people who have shaped my life choices over the years, without whom I would not have successfully undertaken a PhD. I am thankful to Rita Monson for pushing me through my undergraduate studies, being the best person at giving pep talks I’ve ever met, and giving me the opportunity to teach after graduating. Without the kindness of Bill Wisden (Imperial College London), I would not have had the amazing opportunity to experience academic research and become an author on a very high impact publication. I am grateful to Hans Hoppe for allowing me to do my PIPS (professional internship) with him and being a generally inspirational character. I am thankful to my friends and family for being a part of my life outside of the PhD. In particular, I deeply appreciate my boyfriend of seven years, Ian Orton, being there for me through it all. I am grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) for selecting me for a PhD and providing my funding. Thank you to Kayla Friedman and Malcolm Morgan (Centre for Sustainable Development, University of Cambridge) for producing the Microsoft Word thesis template used to produce this document. Zoe Pillidge - September 2018 v CONTENTS 1 INTRODUCTION ............................................................. 1 1.1 Transcription and its regulation ................................................. 2 1.1.1 Regulation by enhancers ......................................................................... 2 1.1.2 Transcription factor binding to enhancers ........................................... 5 1.1.3 Transcription activation ......................................................................... 8 1.2 Chromatin structure and its regulation ....................................12 1.2.1 Chromatin structure ............................................................................. 12 1.2.2 Regulation by chromatin modifiers..................................................... 17 1.2.3 Transcription factor interaction with the chromatin......................... 21 1.3 Notch signalling ....................................................................... 23 1.3.1 Notch as a cell signalling pathway ...................................................... 23 1.3.2 Role of Notch signalling in development and disease ........................ 24 1.3.3 Activation mechanism .......................................................................... 25 1.3.4 Nuclear complexes ................................................................................ 27 1.3.5 The switch from repression to activation ............................................ 28 1.3.6 Achieving cell type specificity .............................................................. 29 1.4 Aims and outline of the thesis ................................................... 31 2 MATERIALS AND METHODS ....................................... 33 2.1 Key reagents and methods ....................................................... 33 2.1.1 Molecular cloning .................................................................................. 33 2.1.2 Cell culture conditions .......................................................................... 33 2.1.3 Transfection reagents ........................................................................... 34 2.1.4 Antibiotics and inhibitors ..................................................................... 34 2.1.5 Antibodies .............................................................................................. 34 2.2 Generation of mβ MS2 cell line ............................................... 35 2.2.1 Cloning of constructs ............................................................................ 35 2.2.2 Generation of MCP-GFP-expressing cells ........................................... 38 2.2.3 CRISPR transfection ............................................................................ 39 2.2.4 Removal of the blasticidin resistance cassette ..................................
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