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Dissecting Bmi1 Protein-Protein Interactions Through Chemical Biology

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DISSECTING BMI1 PROTEIN-PROTEIN INTERACTIONS THROUGH CHEMICAL BIOLOGY by Felicia Gray A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2015 Doctoral Committee: Assistant Professor Tomasz Cierpicki, Co-Chair Assistant Professor Jolanta E. Grembecka, Co-Chair Associate Professor Elizabeth Lawlor Professor Anna K. Mapp Associate Professor Raymond C. Trievel © Felicia Gray All rights reserved, 2015 Dedication For Dorothy Kithinji ii Acknowledgements I first need to acknowledge Dr. Tomasz Cierpicki and Dr. Jolanta Grembecka for their mentorship, patience and support over these past four years. I am grateful for their unending enthusiasm for science and for providing a lab environment in which I could be successful. I thank my committee members, Dr. Anna Mapp, Dr. Ray Trievel and Dr. Beth Lawlor for providing support and guidance throughout this process. I have always looked forward to my committee meetings and appreciate their time and insight. I would like to thank the members of the Cierpicki and Grembecka labs, past and present, for creating a collegial and engaging environment in which to learn and work. I have enjoyed working with Shihan, Bhavna, Hyoje, Shirish, Weijiang and Qingjie in tackling interdisciplinary problems. I am especially grateful for Dmitry, Dave, George and Jon for proving both scientific support and opportunities to relax outside of lab. I thank many friends in the Chemical Biology program: Alison, Hong, Matt W., Matt P., JP, Carol Ann, Elin, Beth, Chinmay and Carrie. I especially thank Ningkun for guiding me as a naïve rotation student and still being my friend afterwards! My graduate experience would not have been the same without the friendship of Paul and Victoria, essentially from the beginning, and I appreciate learning from their personal and scientific insights many, many times over the course of this processes. iii I am grateful to have worked with the wonderful women in FEMMES for four years as we have worked towards our shared ambition to increase diversity in STEM fields and to just plain excite young girls about science. This was a very meaningful group for me to have been a part of and I am continuously inspired and enlightened by the spirit of the group. I am excited to see where the program goes with the next generation. I am thankful to Noah who has tremendous patience and who challenges me to be my best person. I thank him especially for reading this thesis and providing his critical insight. And finally, I am forever grateful to my family for providing a solid foundation to follow my passion and supporting my every move in my less-than-linear path to this point. I would not be where I am without Bev’s unending faith in me and her insistence that I live up to it! Molly inspires me every day and I am so honored to be her big sister. I thank my Dad and Bebe for their love, enthusiastic interest in my work and for providing me a second home in the Midwest. My mother never gives herself enough credit and I’m sure I cannot do it here but it is through her eternal support and wise council that she has set me up for success and her regular notes of encouragement provided confidence and inspiration. iv Table of Contents Dedication ...................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii List of Figures ............................................................................................................................... ix List of Tables ................................................................................................................................ xi List of Equations ......................................................................................................................... xii List of Abbreviations ................................................................................................................. xiii Abstract ........................................................................................................................................ xv Chapter 1: Introduction ............................................................................................................... 1 A. Motivation .............................................................................................................................. 1 B. Transcriptional regulation through chromatin modification ........................................... 2 C. Polycomb proteins negatively regulate gene transcription ................................................ 3 C.1. Canonical polycomb gene silencing mechanisms ....................................................... 3 C.2. Non-canonical polycomb gene silencing mechanisms ................................................ 5 C.3. Architecture of the canonical PRC1 complex ............................................................. 5 D. Polycomb function in biology ............................................................................................... 7 D.1. Phenotypes of polycomb deletion suggest complex functional roles .......................... 7 D.2. Delineating polycomb function through identification of gene targets ...................... 8 E. BMI1 is an oncogene implicated in many cancers ............................................................. 9 E.1. Clinical implications of BMI1 overexpression ............................................................ 9 E.2. Mechanistic insight into BMI1 as an oncogene ........................................................ 10 v E.3. Genetic inhibition of BMI1 in cancer models supports small molecule inhibitor development for this target ............................................................................................... 14 F. Targeting protein-protein interactions with small molecule inhibitors ......................... 16 F.1. Challenges for small molecule inhibition of protein-protein interactions ................ 16 F.2. Methods of identifying protein-protein interaction inhibitors................................... 17 F.3. Examples of PPI inhibitors for chromatin modifying proteins ................................. 18 G. Thesis summary .................................................................................................................. 20 H. References ............................................................................................................................ 20 Chapter 2: Structural Characterization of BMI1 Protein-Protein Interaction Domain ..... 37 A. Abstract ................................................................................................................................ 37 B. Background .......................................................................................................................... 37 C. Results ................................................................................................................................... 38 C.1. The second domain of BMI1 interacts directly with PHC2 ...................................... 38 C.2. Optimization of BMI1 second domain construct for structural studies .................... 39 C.3. Characterization of BMI1-binding domain in PHC2 ................................................ 42 C.4. Structure determination of BMI1-PHC2 complex ..................................................... 44 C.5. Design of mutations in BMI1 to disrupt BMI1-PHC2 interaction ............................ 49 C.6. BMI1 ULD can form higher order oligomers ........................................................... 50 C.7. Multiple BMI1 protein-protein interactions regulate cellular proliferation ............ 54 D. Discussion and Conclusion ................................................................................................. 56 D.1. Need for using multiple methodologies to determine structure of BMI1-PHC2 complex ............................................................................................................................. 56 D.2. Structural studies of BMI1-PHC2 reveal basis for polycomb ULD specificity ........ 57 D.3. Implications of BMI1 protein-protein interactions on PRC1 architecture ............... 60 Acknowledgements .................................................................................................................. 61 E. Experimental methods ........................................................................................................ 62 F. References ............................................................................................................................ 68 Chapter 3: High-throughput Screening to Identify Inhibitors of the BMI1-PHC2 Protein- Protein Interaction ...................................................................................................................... 72 A. Abstract ................................................................................................................................ 72 B. Background .......................................................................................................................... 73 B.1. Rationale for developing small molecule inhibitors of BMI1-PHC2 protein-protein interaction ........................................................................................................................
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    SUP. FIGURE S1 DAPI NFAT Merge DMSO Ac­5SGlcNAc Figure S1. Inhibition of OGT does not prevent nuclear translocation of NFAT. Jurkat cells !"#$%&'()*+(!!,-.'/012"#..(3'4056'7(+('"+(#"(3'7,"8'9:';<'5=29>/%=45='?+'@<>A'B?+'CD' 8+!E''F(%%!'7(+('"8(-'*%#"(3'?-'#-",2F@GHF@ID2=?#"(3'=?J(+!%,*!'B?+'G:'K,-!E''5B"(+'B,)#",?-L' =(%%!'7(+('#-#%&M(3'$&'=?-B?=#%'K,=+?!=?*&E SUP. FIGURE S2 Labeled Unlabeled Labeled Unlabeled 1h 18h ­Az ­PEG 1h 19h ­Az ­PEG !"#$%"#&'( ­ + ­ + + + !"#$%"#&'( ­ + ­ + + + 110 EWSR1 160 *34+&5 80 &,/ 160 )*+-2$ 160 110 )*+,& 80 60 &,/ 160 110 80 0"1"- RUNX1 110 80 60 60 50 160 SP1 160 110 ELF1 110 80 160 NUP98 110 80 )*+&-. &,/ Figure S2. PEG mass tags allow visualization of O­GlcNAc stoichiometry. 5K PEG mass tags were affixed to O­GlcNAc groups on proteins from control or activated T cells via enzy­ matic labeling with azide and copper­free click chemistry. Proteins were then analyzed for shifts in electrophoretic mobility by immunoblot. For the unlabeled control samples, either the azide (­Az) or PEG (­PEG) reagent was omitted during the labeling procedure. Note that HCFC1 appears as multiple bands because the protein is expressed as a single polypeptide that under­ goes proteolytic processing. UBAP2L appears as two bands in the unlabeled control samples due to alternative splicing. Table S1. Details of 133 higher confidence and 81 lower confidence O-GlcNAc glycoproteins1. Confidence Uniprot ID Symbol Specificity Chi Higher P55265 ADAR 100% 9.11E-04 Higher Q09666 AHNAK 92% 2.36E-16 Higher Q8IWZ3 ANKHD1 100% 1.19E-03 Higher
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