REGULATION OF V(D)J RECOMBINATION: MECHANISMS THAT GOVERN LOCUS ACCESSIBILITY AND MITIGATE AGAINST GENOMIC INSTABILITY by Meiling Rose-Anne May A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July 2020 Abstract V(D)J recombination is the process that generates the primary antigen receptor repertoire of the adaptive immune system. This process involves the ordered rearrangement of gene segments within antigen receptor loci and is initiated by the V(D)J recombinase, comprised of the recombination activating gene 1 and 2 proteins (RAG-1 and RAG-2). V(D)J recombination involves the programmed introduction of DNA double stranded breaks in the genome and is therefore tightly regulated to ensure genomic stability. Epigenetic regulation of V(D)J recombination by trimethylation at lysine 4 of histone 3(H3K4me3) is modulated through the plant homeodomain (PHD) of RAG-2. Binding of the RAG-2 PHD to H3K4me3 couples V(D)J recombination to active transcription and H3K4me3 allosterically activates RAG. In this dissertation, we demonstrate that the binding and allosteric activation of H3K4me3 are separable functions of the RAG-2 PHD. Temporal regulation of V(D)J recombination is an additional method of preserving genomic integrity. RAG-2 undergoes periodic destruction at the G1-to-S transition through phosphorylation at T490 and subsequent degradation by the ubiquitin-proteasome pathway. This limits RAG activity to the G1 cell cycle phase, during which nonhomologous end joining (NHEJ) is the predominant form of DNA double stranded break repair. In this dissertation, we employ a mouse model in which RAG-2 protein is constitutively expressed and DNA double stranded breaks accumulate throughout the cell cycle. To characterize genomic rearrangements that result from misregulated RAG activity, we developed a capture-sequencing approach. We demonstrate that this method is capable of identifying endogenous V(D)J rearrangements and novel translocations involving antigen receptor loci. ! ii Ph.D. Dissertation referees for Meiling Rose-Anne May Stephen Desiderio, M.D., Ph.D. Director Emeritus, Institute for Basic Biomedical Sciences Professor (Emeritus), Molecular Biology and Genetics Professor (Emeritus), Medicine Johns Hopkins University School of Medicine (Thesis advisor) Carol Greider, Ph.D. Chair of department, Molecular Biology and Genetics Bloomberg Distinguished Professor, Molecular Biology and Genetics Johns Hopkins School of Medicine (Thesis co-advisor) Ranjan Sen, Ph.D. Professor (Adjunct), Medicine Johns Hopkins University School of Medicine Chief, Laboratory of Cellular and Molecular Biology National Institute on Aging (reader) ! iii Acknowledgements Dr. Stephen Desiderio has been my scientific role model and I aspire to be like him. I chose his lab because I wanted to learn to think about science, design controlled experiments, and perform rigorous science like Steve. I looked to the students that went through his lab and saw how differently and rigorously they approached science compared to students in other laboratories. I knew that this was a direct result of training under Steve. His encouragement, commitment, and guidance throughout my dissertation helped me to accomplish this milestone and gave me the independence to pursue scientific ideas and professional development opportunities. I could not have asked for a more supportive mentor regarding my professional development as Steve let me do a teaching fellowship and an internship at AstraZeneca. He believed in me and my abilities and trusted that even with these time-consuming professional development opportunities that I would be committed to my project and get my work done. Dr. Carol Greider so kindly accepted me into her laboratory as a rotation student and then later as a guest graduate student. She was welcoming and treated me just like one of her own lab members even though I was working on V(D)J recombination and not telomeres. She has always supported me and has been a huge proponent for women in science. I strive to be a leader like Carol, who is a great scientist yet humble and approachable. My dissertation experience would not have been the same if it weren’t for my Desiderio lab mates (John Bettridge, Nick Dordai, Wenyan Lu, and Alyssa Ward) and my Greider lab mates (Kayarash Karimian, Margaret Strong, Callie Shubin, Sam Sholes, Rini Mayangsari, Carla Connelly, Christine Gao). I cannot thank you enough for the endless scientific discussions, ! iv commiseration sessions over failed experiments, and countless laughs. From game nights in the conference room to walks to the hospital to look at poster proofs, I’ve enjoyed every moment spent with you. I’ve learned so much from you scientifically, like protein preparations, southern blots, and scientific resilience, but more importantly I’ve learned how to be a good friend and colleague, how to support each other during the failed experiments and during the triumphs of positive data. I’d like to thank my thesis committee Carol Greider, Kathy Burns, Ranjan Sen, and Sarah Wheelan for their unwavering support of both my science and my professional aspirations. I always left my committee meetings with much clearer direction and wishing I had met with you earlier. You volunteered so much support and offered technical assistance from your lab members. I could not have finished this dissertation without the help and support of the MBG department and the BCMB graduate program. MBG has been such a collaborative environment and through journal clubs and colloquia have helped me to understand topics outside my field and to hone my publication skills. Jess Terzigni has been instrumental in easing my transition between laboratories, in and out of internship, and through the publication process. BCMB, particularly Arhonda Gogos and Sharon Root have been a constant source of support for me throughout all the ups and downs of graduate school. My BCMB classmates are amazing scientists and I’ve learned so much from them both inside and outside of the laboratory. ! v Lastly, I’d like to acknowledge my friends and family (especially Kevin) for being a constant source of support during this journey. Despite not always understanding the science, you have always been there for me to boost my confidence and believe that I can accomplish great things. ! vi Table of Contents Abstract!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!##! Acknowledgements!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!#$! List of Tables!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!#%! List of Figures!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!%! Chapter 1: Binding and allosteric transmission of histone 3 Lys-4 trimethylation to the recombinase RAG-1 are separable functions of the RAG-2 PHD finger!""""""""""""""""""""""""""""""""""!#! Abstract!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!&! Introduction!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!'! The human immune system!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!#! Overview of V(D)J recombination!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!#! The V(D)J recombinase: RAG-1 and RAG-2!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!$! Evolution of RAG-1 and RAG-2!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!%! Domain Structure of RAG-1 and RAG-2!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!&! Mechanism of V(D)J recombination!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!''! 3-Dimensional structure of RAG-1 and RAG-2!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!'#! Regulation of V(D)J recombination!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!'%! Transcription and epigenetic control of the RAG recombinase!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!'&! Approach!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!('! Results!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!&&! Phylogenetic substitution separates H3K4me3 binding from allosteric activation in vitro!""""""""""""""""""""""""""""""!((! A RAG-2 PHD mutant that binds H3K4me3 without supporting V(D)J recombination in vivo!"""""""""""""""""""""""!(#! A back mutation from shark to mouse sequence in the RAG-2 PHD that restores V(D)J recombination activity !""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!(%!