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Structural and Functional Elucidation of PRDM Proteins

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Structural and Functional Elucidation of PRDM Proteins Structural and Functional Elucidation of PRDM Proteins by Danton Ivanochko A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Medical Biophysics University of Toronto © Copyright by Danton Ivanochko, 2021 Structural and Functional Elucidation of PRDM Proteins Danton Ivanochko Doctor of Philosophy Department of Medical Biophysics University of Toronto 2021 Abstract Epigenetic signalling dictates the dynamic patterns of gene expression that are required for life. In humans, epigenetic lysine methylation is produced by chromatin-bound transcription factors that contain a SET (Su(var)3-9-E(z)-Trx-homology) domain or a Rossmann-fold domain. The PRDM (PRDI-BF1-RIZ homology domain containing) proteins are identified by an N-terminal PR/SET domain that shares the canonical SET domain fold, but with just 20-30% amino acid sequence identity. Humans possess 19 PRDM-coding genes with roles in cellular proliferation and differentiation, and dysregulated PRDM gene expression is often associated with diseases. I hypothesize that only a subgroup of PRDM proteins are active lysine methyltransferases and pursue the objective of this thesis to expand the fields of PRDM biology by revealing novel insights regarding the enzymatic and non-enzymatic properties of PRDM proteins. To address this objective, I characterized MRK-740, which is the first and only chemical probe of a PRDM protein. Here I examined the mechanism of inhibition of MRK-740 for its target PRDM9, using biophysical and structural biology techniques. I characterized specificity of MRK-740 within the PRDM family and uncovered the mechanism of inhibition. Remarkably, a structural survey of all chemical probes targeting protein methyltransferases indicated that MRK-740 functions by a previously unobserved mechanism of inhibition. Next, I examined how PRDM proteins regulate epigenetic signaling through a mechanism that is independent of intrinsic methyltransferase activity. Here I examined the PRDM paralogs, PRDM3 (also known as MECOM or MDS1-EVI1) and PRDM16 (also known as MEL1), which may lack intrinsic methyltransferase activity and identified how they directly interact with the Nucleosome Remodeling Deacetylase (NuRD) complex. Finally, I examined 13 of the 19 human PR/SET domains for their ability to bind a chemical analog of the enzyme cofactor, to identify proteins with enzymatic potential. I used nuclear magnetic resonance spectroscopy to detect binding of fluorinated S-adenosyl-L-homocysteine and identified several previously uncharacterized PR/SET domains that likely bind the cofactor. Taken together, these findings provide the impetus, tools and insights for further research into the roles of PRDM proteins in health and disease. ii Acknowledgments First and foremost, I have found myself incredibly fortunate to have Dr. Cheryl Arrowsmith as my thesis advisor for these past four and a half years. To Cheryl, I thank you for the opportunity that you have provided me. Your guidance has helped to expand my research horizons farther than I thought I could see. You provided me the freedom to explore my own ideas and always nudged me back course whenever I got lost in the weeds. I am incredibly grateful to be part of your research ecosystem spanning labs at UHN, the SGC, and with collaborations at Boehringer Ingelheim and MSD. When I first reached out to you for a PhD, I sought to get training in a field that would open doors for me in both academia and industry. Thank you for helping me fulfill this goal and thank you for helping to make me a better scientist. Just like the African proverb that states “It takes a village to raise a child”, it has become apparent to me that my PhD has unquestionably benefited from the large community that has supported me throughout my academic journey. To my thesis supervisory committee, Drs. Gil Prive and Matthieu Schapira, thank you for lending your time to share you expertise with me and helping to guide me towards achieving the best possible PhD thesis that I could achieve. To Matthieu, thank you for going above and beyond your role in the committee and as a collaborator. I am grateful that you included me as a co-author in your review article and thank you for sharing your knowledge of methyltransferases, which has been invaluable to my education and research. My PhD would not have been possible without the mentorship from an extensive network of researchers and scientists. To Shili Duan, thank you for your support and guidance. You are one of the most pleasant and patient people I have ever met and working with you made my day-to- day activities in the lab a joy to take part in. To Dr. Evelyne Lima-Fernandes, thank you for helping me to get started researching the PRDMs and for fostering a fun social culture among the group. To Drs. Levon Halabelian and Scott Houliston, thank you for taking your time to train me in X- ray crystallography and NMR spectroscopy. These are two of the most interesting techniques I have ever learned, and I am incredible grateful for your explanations, guidance and insights. To Drs. Masoud Vedadi and Dalia Barsyte-Lovejoy, thank you for providing me opportunities to work within your groups and for the helpful advice that has guided me towards being a better scientist. To Drs. Rachel Harding, Abdellah Allali-Hassani, Guillermo Senisterra, and Mani Ravi, your advice, guidance, and patience formed an indispensable component of my training and research. To Dr. Jark Boettcher, thank your for taking me under your wing during my time in Vienna. You instantly made me feel like I was a part of the team and I am very grateful that you opened the door to the inner workings of pharmaceutical development for me. I had an excellent experience and look forward to our next meeting! To my friends and family, I thank you for your cheer. To my parents, I am only here today because you have always nurtured my goals and supported my endeavours. To all my friends, I want you to know that this would have been a drag without you. Whether around the foosball table, on the rugby pitch, or just kicking back on a patio, you all have been a stable source of energy that has kept me moving forward. To Marcus, Mike, Max, and Julian, thank you for standing with me on my wedding day. Three of you are friends that I can call my brothers, and one of you is a brother I am glad to call my friend. Finally, to my beautiful and brilliant wife, Victoria, thank you for being the smiling face beside me on this adventure. You have been my stability in times of uncertainty and my inspiration on a cloudy day. You showed me how to find my motivation and since then, the two of us have been moving forward together. I can’t wait to add the third! « iv Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents .............................................................................................................................v List of Figures .................................................................................................................................x List of Tables .............................................................................................................................. xiii Chapter 1 ..........................................................................................................................................1 General Introduction ...................................................................................................................1 1.1 Background ..........................................................................................................................1 1.1.1 The genome and beyond. .........................................................................................1 1.1.2 On epigenetics – “above” the gene. .........................................................................1 1.1.3 On epigenetic lysine methylation.............................................................................2 1.1.4 Writers, readers and erasers of lysine methylation. .................................................5 1.1.5 On SET-fold as KMTs. ............................................................................................5 1.1.6 On drugging SET-fold domains. ..............................................................................6 1.2 The PRDM proteins. ............................................................................................................7 1.2.1 The PRDM family....................................................................................................7 1.2.2 Discovery of the PRDM family. ..............................................................................9 1.2.3 Biological functions of PRDMs in health and disease.............................................9 1.2.4 PRDMs: enzymes or pseudoenzymes? ..................................................................11 1.2.5 Comparing PR/SET domains with canonical SETs. ..............................................13 1.2.6 Structural dynamics in PR/SET enzymes. .............................................................15 1.3 Structural and functional elucidation of PRDM proteins ..................................................17 1.3.1 Rationale for investigating PRDMs. ......................................................................17 1.3.2 Research objective. ................................................................................................19
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