Characterizing Novel Interactions of Transcriptional Repressor Proteins BCL6 & BCL6B by Geoffrey Graham Lundell-Smith A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Geoffrey Lundell-Smith, 2017 Characterizing Novel Interactions of Transcriptional Repression Proteins BCL6 and BCL6B Geoffrey Graham Lundell-Smith Masters of Science Department of Biochemistry University of Toronto 2016 Abstract B-cell Lymphoma 6 (BCL6) and its close homolog BCL6B encode proteins that are members of the BTB-Zinc Finger family of transcription factors. BCL6 plays an important role in regulating the differentiation and proliferation of B-cells during the adaptive immune response, and is also involved in T cell development and inflammation. BCL6 acts by repressing genes involved in DNA damage response during the affinity maturation of immunoglobulins, and the mis- expression of BCL6 can lead to diffuse large B-cell lymphoma. Although BCL6B shares high sequence similarity with BCL6, the functions of BCL6B are not well-characterized. I used BioID, an in vivo proximity-dependent labeling method, to identify novel BCL6 and BCL6B protein interactors and validated a number of these interactions with co-purification experiments. I also examined the evolutionary relationship between BCL6 and BCL6B and identified conserved residues in an important interaction interface that mediates corepressor binding and gene repression. ii Acknowledgments Thank you to my supervisor, Gil Privé for his mentorship, guidance, and advice, and for giving me the opportunity to work in his lab. Thanks to my committee members, Dr. John Rubinstein and Dr. Jeff Lee for their ideas, thoughts, and feedback during my Masters. Thank you to Dr. Etienne Coyaud for performing the mass spec experiments and providing help with data analysis, advice, and troubleshooting. An incredible amount of thanks to Neil Pomroy without whom much of this work would have been so much more difficult, if not nigh-impossible. Thanks also to Dr. Doug Kuntz, Dr. Wesley Errington, and Dr. Yong Wei, your guidance and advice has been invaluable. A big thank you to my fellow grad students in the lab, Andrew, Alan, Darren, Xiong, Yaseen, and the new guy, for the advice, support, thoughtful discussion, and allowing me free reign over the lab’s music playlist. I’d also like to thank the staff of the Department of Biochemistry, especially Carrie Harber, who works administrative wonders. Special thanks to my friends, you guys helped me through all the ups and downs, and all of the craziness. T follicular helper cells, right? Most importantly, to my mother and father, my sister Kierstin and my brother Connor, your support has meant the world to me. Thank you so much for being there. iii Table of Contents Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Abbreviations .................................................................................................................... vii List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Appendices ........................................................................................................................ xii 1 Introduction ................................................................................................................................ 1 1.1 BTB Domains ..................................................................................................................... 1 1.2 BTB-Zinc finger proteins .................................................................................................... 1 1.3 General Mechanism of Transcriptional Repression ............................................................ 4 1.4 BCL6 ................................................................................................................................... 6 1.4.1 Introduction ............................................................................................................. 6 1.4.2 BCL6 Biological Roles ........................................................................................... 7 1.4.3 BCL6 and the Germinal Centre Reaction ............................................................... 8 1.4.4 BCL6 as Master Regulator of the Germinal Centre Reaction .............................. 10 1.4.5 BCL6 Regulation and Lymphomagenesis ............................................................ 11 1.5 BCL6B is a paralog of BCL6 ............................................................................................ 12 1.6 The Structure of the BCL6 BTB domain .......................................................................... 14 1.7 The BCL6 Interactome ..................................................................................................... 20 1.8 Protein-Protein Interactions .............................................................................................. 22 1.9 BioID ................................................................................................................................. 24 1.10 Thesis Objectives .............................................................................................................. 27 2 Materials and Methods ............................................................................................................. 28 iv 2.1 Cloning and Generation of Constructs .............................................................................. 28 2.2 SDS-PAGE and Western Blots ......................................................................................... 28 2.3 Mammalian Tissue Culture ............................................................................................... 29 2.4 Analysis of BioID Results ................................................................................................ 30 2.5 Transient Transfection and Co-Immunoprecipitation ....................................................... 30 2.6 Expression and Purification of BCL6BBTB ....................................................................... 31 2.7 Circular Dichroism of BCL6B BTB in Sarcosyl .............................................................. 32 2.8 Microscale Thermophoresis .............................................................................................. 32 2.9 Crystallization of BCL6BBTB ............................................................................................ 33 2.10 Phylogenetic Analysis and Sequence Conservation ......................................................... 33 3 Results ...................................................................................................................................... 35 3.1 BioID identifies known and novel interactors of BCL6 and BCL6B ............................... 35 3.2 Validation of Identified Interactions ................................................................................. 45 3.3 Purification of the BCL6B BTB domain .......................................................................... 46 3.3.1 Constructs and Purification Strategy .................................................................... 46 3.3.2 Evaluation of stability, solubility, and folding of BCL6BBTB in the presence of Sarcosyl ................................................................................................................. 49 3.3.3 Crystallization Trials ............................................................................................. 51 3.4 Biophysical Characterization of the interaction between BCL6B and SMRT ................. 52 3.5 Bioinformatic Analyses .................................................................................................... 55 3.5.1 Phylogenetic analysis of BCL6/B ......................................................................... 55 3.5.2 Conservation of Corepressor BBDs ...................................................................... 60 3.5.3 Conservation of the BCL6 and BCL6B lateral grooves ....................................... 60 3.5.4 Co-evolving Residues in the BTB Domain .......................................................... 64 4 Discussion ................................................................................................................................ 67 4.1 Experimental Rationale ..................................................................................................... 67 v 4.1.1 Choice of Technique ............................................................................................. 67 4.1.2 Identification of Statistically Significant Interactors ............................................ 70 4.1.3 Validation of Target Interactions: ......................................................................... 70 4.2 Interactions Identified by BioID ....................................................................................... 71 4.2.1 The BCL6 BTB domain interacts with TLE1 and TLE3 ...................................... 75 4.2.2