Control of Divergent Noncoding Transcription in Saccharomyces cerevisiae Chun Kit Andrew Wu University College London and The Francis Crick Institute PhD Supervisor: Folkert van Werven A thesis submitted for the degree of Doctor of Philosophy University College London April 2020 Declaration I, Chun Kit Andrew Wu, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The regulation of gene expression underlies all cellular processes and fundamentally enables complexity of eukaryotic organisms. Aberrant expression of noncoding RNAs can compromise normal gene expression. Gene promoters are inherently bidirectional and generate divergent noncoding RNAs along with protein- coding messenger RNAs. Chromatin and RNA turnover pathways limit expression of noncoding RNAs, but how sequence-specific transcription factors regulate divergent noncoding transcription and promoter directionality is not well understood. Here, I investigate how divergent transcription is repressed at highly expressed genes in Saccharomyces cerevisiae. I find that the sequence-specific transcription factor Rap1 limits divergent noncoding transcription at a large fraction of its target genes. Rap1 safeguards normal gene expression by limiting aberrant transcription that overlaps with neighbouring loci. Divergent RNAs initiate at or extremely close to Rap1 binding sites, indicating that Rap1 limits initiation of transcription from divergent core promoters. Stable binding of Rap1 near cryptic promoters is required and sufficient to suppress divergent transcription. Silencing cofactors or transcriptional coactivators associated with Rap1 are not required for repression of noncoding RNAs at promoters. In contrast, a small region within the Rap1 carboxy-terminal domain is required for repression of divergent transcription and affects interaction between Rap1 and the RSC chromatin remodeller. RSC and Rap1 regulate divergent transcription at Rap1-regulated gene promoters in distinct ways. Promoter output shifts from unidirectional to bidirectional transcription in the absence of Rap1, which is partially suppressed after co-depletion of Rap1 and RSC. RSC activity is not strictly required for divergent transcription suggesting that additional regulators also play important roles. I propose that certain sequence- specific transcription factors limit the access of transcription machinery and coactivators to divergent core promoters by steric hindrance, thereby providing directionality towards productive transcription of coding genes. 3 Impact Statement In this thesis, I discovered that the sequence-specific transcription factor Rap1 prevents initiation of divergent noncoding transcription near its binding sites at gene promoters, thereby conferring directionality towards productive transcription of coding genes. This work highlights the molecular basis by which a DNA-binding regulatory protein interprets the information encoded within cis- regulatory element DNA to produce a transcriptional output. These findings have been disseminated through two peer-reviewed articles published in academic journals (Wu et al., 2018b; Wu and Van Werven, 2019). These open access articles are freely available to the scientific community and the wider public, and may to be of interest to scientists investigating fundamental aspects of gene regulation in various systems including humans. Within this thesis, I also demonstrated that CRISPR technology can be used to specifically inhibit expression of divergent long noncoding RNAs by targeting their core promoters. This fundamental information will help to inform design of CRISPR interference (CRISPRi) screens to identify genes involved in diverse cellular processes, and understand how they interact. Noncoding RNAs are associated with a wide range of human diseases, and may offer attractive therapeutic targets. The novel information regarding principles of gene regulation presented in this thesis may help to guide future therapies that exploit CRISPRi to interfere with transcription of coding genes or long noncoding RNAs in a clinical setting. The RNA and transcription start site sequencing data sets generated within this thesis have been deposited and archived in the NCBI Gene Expression Omnibus (GEO) database repository (GEO: GSE110004). These data sets are publicly available and may help to improve future S. cerevisiae genome annotation through community-based resources such as the Saccharomyces Genome Database or Ensembl. 4 Acknowledgement I must start by acknowledging my PhD supervisor, Folkert van Werven. Folkert, thank you for being the best PhD supervisor I could ever have. I have had the opportunity to see first-hand your tireless enthusiasm for science, pragmatic but fearless approach, and astute intuitions that are quite often correct. I’m very grateful for the opportunity to do a PhD in your laboratory where I grew up, learned how to be a scientist, and had the chance to contribute to something meaningful that is much bigger than myself. However, these things cannot compare to the selfless dedication and genuine care you have for all your lab members. I appreciate that you always made the time to discuss my experiments, data, career, and anything else I needed your advice on. I could not have done this PhD without your support, and I will always be grateful for your guidance and mentorship. Thank you for making these past four years a hugely enjoyable experience. I know that your work will shape the future of science and the careers of those who have the privilege of working with you, and I can’t wait to see your amazing discoveries and achievements. Minghao Chia, thank you for being a great collaborator and colleague, but most of all a great friend. I am lucky that I can always count on your amazing expertise and knowledge, wealth of experience, and willingness to help others. I always enjoy laughing at silly things together with you, in science and in life. It has been an amazing four years working together, and I’ve had the chance to see you develop, do amazing work, and even get married! I wish you and Joy all the best as you begin a new chapter of your lives together in Singapore. I’ll be sure to visit you often so we can try some more delicious food together! Janis Tam, what a journey we’ve taken together over the past four years. Thank you for always bringing a laugh and smile to all of us, each and every day in the lab. As with Minghao, it is always fun to share a laugh with you about everything and nothing. It was always reassuring to talk to each other and offer advice or a tasty snack as we took each daunting step together throughout our PhDs. Good luck to you as you complete your thesis and embark on an exciting 5 new journey as well; my best wishes to you and Ryan. Because I know we have very similar tastes, I’m really looking forward to meeting up and discovering new restaurants together. 繼續加油! Radhika Warrier, it has been an absolute pleasure to work together and get to know you. Thank you for always being caring, wise, and sensible with your advice, whether it was scientific or personal. Your encyclopaedic knowledge, quiet confidence, and astute judgement helped to keep all our crazy experiments on track. I wish you and your family good luck in the future, wherever you go and whatever you do. Dora Sideri, thank you for your heroic efforts to maintain the smooth running of the lab, and for making our move from the LRI to the Crick in the middle of our PhDs as seamless as possible. Your calm approach and pragmatic advice always helped us keep our heads on our shoulders when things got hectic. Thanks for all your help, and good luck as you continue to make fantastic contributions. Fabien Moretto, thank you for always taking the time to teach us everything you know. From proper yeast genetics to countless northern blots, your meticulous approach and expertise ensured that we learned to do things the right way. We certainly had quite the journey from the LRI to the Crick! I wish you and your family all the best as you start a new chapter together. Imke Ensinck, it’s been wonderful to get to know you and see your amazing talents continue to develop over the first year of your PhD. Your warmth and sense of humour brightened up each day inside and outside the lab, and I hope we managed to pass some of our knowledge and not too many bad habits on to you. I hope you will continue to take part in London’s amazing theatre scene, and maybe I will see you on a West End production one day! Good luck to you as you continue to do amazing science together with Folkert, I can’t wait to see what you achieve. Alice Rossi, it was fantastic to work with you in our group and get to know you. You are one of the brightest, most enthusiastic, and most Italian people I know. Quite frankly, you’re so smart and motivated, it’s unbelievable. I always 6 enjoyed reminiscing about the Pacific Northwest together, and I look forward to seeing you grow and accomplish amazing things. And thank you for sharing your delicious baked goods and your tiramisu recipe, which I will always keep near and dear to my heart! All the best to you, as you embark on the next phase of your scientific career during your PhD at the Crick. Luc Costello Heaven and Jessie Beck, it was great to have you as valuable members of our group and I wish you the best of luck in your future careers. Thank you to Harshil Patel (Bioinformatics and Biostatistics, The Francis Crick Institute) for your significant contributions towards the design, analysis, and interpretation of our genome-wide data. It has been an absolute pleasure to work with you over these past four years, and you have been the most dedicated, knowledgeable, and genuine collaborator I could have asked for.