A NOVEL BIOINFORMATIC APPROACH FOR COMPREHENSIVE GENOME SCALE ANALYSIS IDENTIFIES KEY REGULATORS OF MACROPHAGE ACTIVATION Samuel Katz Supervisors: Prof. Clare E. Bryant Dr. Iain D.C. Fraser University of Cambridge Fitzwilliam College This dissertation is submitted for the degree of DoCtor of Philosophy. September 2019 Declaration and Statement of Length This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. The dissertation does not exceed the prescribed word limit for the relevant Degree Committee. Samuel Katz September 2019 II A Novel Bioinformatic Approach for Comprehensive Genome Scale Analysis Identifies Key Regulators of Macrophage Activation Samuel Katz Summary The initiation of inflammatory cytokine transcription by bacterial ligands is a central mechanism by which the immune system activates its first line of defense. Macrophage activation by the Toll-like Receptor 4 (TLR4) pathway is initiated with receptor binding of lipopolysaccharides (LPS) and culminates in a large-scale transcriptional response of the inflammatory gene program. Advancements in genome-wide screening technologies have made it possible to interrogate the regulatory landscape of signaling pathways such as those activated by TLR4. Utilizing these high-throughput methods for the comprehensive characterization of pathway components, particularly for regulators that are involved in critical cellular processes such as transcription and translation, however, requires an approach that goes beyond the top scoring and previously characterized hits of genome-scale studies. To address this challenge, I developed the Throughput Ranking by Iterative Analysis of Genomic Enrichment (TRIAGE) method, a bioinformatic analysis model that facilitates the comprehensive identification of likely regulators by iterative sampling of pathway and network databases. I validated the TRIAGE approach by analyzing three previously published genome- wide studies of regulators of early HIV infection and viral transcription. Analysis by TRIAGE showed significantly increased overlap and identified shared novel targets across the three studies. I further developed the TRIAGE analysis method as a globally accessible web-based resource. Applying TRIAGE analysis to three genome-scale studies of LPS treatment in macrophages of mouse and human cell lines, I identified an enrichment for regulators relating to alternative splicing and protein degradation. Using short read and long read RNA-seq of ligand-stimulated macrophages I further characterized the broad transcriptional variation induced by the LPS response and the novel and known transcript variants that define different macrophage activation states. These findings define an approach for comprehensive unbiased discovery of signaling pathway regulators from genome-scale datasets and suggest a model of macrophage activation involving proteasomal removal of negative regulators and remodeling of the macrophage state via a transcriptional shift in splice variant dynamics. III Publications arising from this thesis Sun, J., Katz, S., Dutta, B., Wang, Z., & Fraser, I. D. (2017). Genome-wide siRNA screen of genes regulating the LPS-induced TNF-alpha response in human macrophages. Scientific Data, 4, 170007. doi:10.1038/sdata.2017.7 Li, N., Katz, S., Dutta, B., Benet, Z. L., Sun, J., & Fraser, I. D. (2017). Genome-wide siRNA screen of genes regulating the LPS-induced NF-κB and TNF-α responses in mouse macrophages. Scientific Data, 4, 170008. doi:10.1038/sdata.2017.8 IV Acknowledgements The world so rarely lets us in, let’s praise the luck vista when it does. -Linda Gregerson, Prodigal: New and Selected Poems, 1976 to 2014 At the start of my work that would become this document I came across the above words in a poem describing the findings by Brenner, Horvitz, and Sulston of how the processes of controlled cell death are essential for the development of life. From the day I read them to this moment now as I write this sentence these words have conveyed the luck, joy, and awe I feel about my utter privilege of being able to ask questions of the biological world and the possibility of one day being let in on so elegant an answer. I am filled with gratitude towards the people who have shared and made possible this path that I am on. My family who have shared all the different sides of this journey with me. My sister Pessy who understands better than anyone what the highs and lows of this process mean for me. My sister Chavali with whom I share our own unique joys of what making a new place your home means. My brother-in-law Michael whom I have known for the same amount of time as the work in this thesis, and honestly, both feel like they were always there. My brothers Avi and Gavi who ask me the best questions about my work. Sharry, who prepared my family for what an undertaking this document can be. My father, Ysoscher Katz, Your Heuristic Worldview Happened. And who for years told me that he will only understand the first four words of my thesis (“this thesis is about”) but understands everything that is packed in the space between the words for me. My mother, Esther Katz, who in a world so far away still sees me doing her God’s work. My grandmother, Gita Katz who never misses a chance for a good and real conversation. Two people in my family only got to be on part of this journey with me, my grandfather Leiby Katz who left this world soon after I started this work and my niece Adi Grummer who came into it just as I was completing it. Both of whom are my reminders for how truly awesome the gifts of life, health, and curiosity are. Many friends lifted me up through this journey. Emily Lees, Alison Clasper, and Geoff Eichhorn; thank you for the best of my Cambridge days. My dear friends in New York who always have a pair of eyes to lend me when I forget how to see myself, Malky Brizel Lipshitz, Brooke Sable, and Shmuly Horowitz; thank you for knowing where I come from and seeing where I am going. Marlon Kazmierczak for reminding me of the life that exists outside of this world. Gianmarcco Raddi, who will show up late to read this, but he will show up. Solomon Feuerwerker for sharing with me the joy of seeing what our early dreams became. Anita Gola, V guided me through the hilarity and stress that this process can be and helped meet the finish line with sanity. Nafisa Waziri for the santi of it all. Brian Graves for understanding how it can be like that. Danette Hargraves has been a mentor to me since the days when I was working on my high school equivalency degree and has continued to cheer me on through years of undergraduate education, jobs, applications, and this work. Thank you for your wisdom and guidance at every step, and for the words that got me through at the lowest point in this journey. Dr. Beth Moore gave me my first lab project and showed me the joy of diligent research. It was in her lab that I was first exposed to the fascinating questions of immunology and the wonderful complexity of the macrophage. The NIH-Oxford-Cambridge Scholars program made this work possible. My thanks to the admin team, the advisory board, and the International Biomedical Research Alliance. My gratitude to the members of the Bryant Lab in Cambridge. Lee (Hoppy) Hopkins, who were it not for his Welsh pride I would have mistaken for a long-lost brother from New York. Pani Tourlomousis, Charlotte Macleod, Milton Pereira, and especially Zsofi Digby, we did get it in the end, like I kept saying. The Fraser group at the NIH made every detail of the work in this thesis possible. Jing Sun, Sharat Jacob Vayttaden, Sinu John, Orna Ernst Rabinovich, Clinton Bradfield, Jonathan Liang, and Mike Dorrington. And former members Nicolas Lounsbury, Bin Lin, and Kyle Webb. All of my work in here stands on the shoulders of what they have done and taught me. I am incredibly grateful to Dr. Jian Song who expanded what the TRIAGE project could be and helped me get it to where it is now. Finally, and most importantly, my deepest gratitude goes to my mentors of this work. Clare Bryant gave me the space in her lab to explore in a new way a biological system she knows better than most. Clare modeled and taught me a healthy dose of scientific skepticism that formed so much of how my thoughts were shaped in this process. Iain Fraser gave me time, resources, and support to discover the questions I was passionate about and guided me as I grew in my abilities to pursue it. Living through the logistics of what this project took, both inside and outside of the lab, would not have been possible without his support. Iain never accepted my arguments about not me being innately good at certain kinds of research techniques by reminding me that scientific intuition is learned. Iain taught me that doing something doesn’t mean doing it all by yourself.