Characterization of E2E Ubiquitin-Conjugating Enzymes and Ubiquitin-Specific Protease 7 (USP7) in Histone H2A Ubiquitination Hossein Davarinejad A thesis submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of Master of Science Graduate Program in Biology York University Toronto, Ontario December 2017 © Hossein Davarinejad, 2017 Abstract Ubiquitin(Ub)-conjugating E2 enzymes play essential roles in ubiquitination of proteins. The UbE2E sub-family members UbE2E1, UbE2E2, and UbE2E3 have N-terminal extensions to the conserved E2 core which contain Ubiquitin-Specific Protease 7 (USP7) binding sequences (P/A/ExxS). Here, we continued our investigations to established that USP7 can interact with E2Es in vitro and in vivo. Our new data indicated that the N-terminal extensions of E2E2 or E2E3 can directly associate with USP7 TRAF domain. We demonstrated that E2E2 or E2E3 are stabilized by USP7 in cells. We also showed that E2Es interact with Ring1B:BMI1, the core components of the Polycomb Repressive Complex 1 (PRC1) and established E2E1 as an in vivo E2 for monoubiquitination of histone H2A on lysine(K) 119. We demonstrated that E2Es can modulate the levels of H2A monoubiquitination in cells and that USP7 may exert an effect on K119-UbH2A levels through regulating E2Es. ii Dedications For my dearest, and departed grandmother. Your memories, love, and grace are eternal. I also dedicate this to Agnieszka, GhoorGhoor, and Charlie for their invaluable support. And for science; may our collective and ever developing understanding of the universe and life lead us to make the world a better and a more peaceful place, for all life is precious. iii Acknowledgements I am grateful to my two dear supervisors, Dr. Vivian Saridakis and Dr. Yi Sheng, for the opportunity to start and complete these projects, a great work and learning experience, and their guidance. Thank you, Dr. Peter Cheung, for your time and efforts as my advisor. A special thanks to Raghav, Heather, Rahima, Ira, Anna, Olga, Niharika, Varvara, Sahar, and Roland in my two labs for all the help, teachings, and great memories. I am forever indebted to my father Masoud Davarinejad for his continuous financial support throughout my studies and beyond. Thank you, governments of Ontario and Canada, for providing student loans and grants. Thank you, the York University administration and CUPE 3903, for the employment opportunities during my degree. Thank you Canadian Institute of Health Research (CIHR) for funding my projects. Thank you Jelena & Giga and Jyotsna for your kindness, support, and help for my defense. Thank you, lab members in Bayfield [Sam, Jyotsna, Ana, Rawa], Audette [Agnesa, Ayat, Cristina], Peng [Mohamed, Stephanie, Jelena, Heyam, Shahin, Alina], Donaldson [Jamie, Sarah, Kate], Zoidl [Paige, Cherie, Ryan], Rosonina [Veroni, John, Akhi], Cheung [Marlee, Keyur], Kubiseski [Dayana, Queenie], McDermott [Saviz, Stephanie, Dandan, Christina, Ali], and Yusaf [Paul, Sina] groups and all the supervisors and personnel on the 3rd floor of the Life Sciences Building (LSB) including the custodian Maria, for a wonderful and enjoyable time while completing my degree. Thank you executives, directors, administrators, staff and members of the York University Graduate Students’ Association (YUGSA), Association of Graduate Students in Biological Sciences (AGSBS), the Canadian Federation of Students (CFS); Department of Biology, Faculty of Graduate Studies, and the Office of the President at York University for extending my education beyond biology into advocacy, governance, politics, administration, and student movements. iv Table of Contents Abstract ................................................................................................................................... ii Dedications ............................................................................................................................ iii Acknowledgements ................................................................................................................ iv Table of Contents .................................................................................................................... v List of Tables............................................................................................................................ x List of Figures ..........................................................................................................................xi List of Abbreviations .............................................................................................................. xiii Chapter 1: Ubiquitin Systems ............................................................................................. 1 1.1 Ubiquitin: A Ubiquitous Small Signalling Protein ............................................................ 1 1.2 Ubiquitin and Ubiquitin-Like Post-Translational Modification ....................................... 2 1.3 Diversity of Ubiquitin Signaling ....................................................................................... 5 1.4 The Ubiquitination Pathway & Machinery ..................................................................... 6 1.5 E1 Ubiquitin-Activating Enzymes .................................................................................... 7 1.6 E3 Ubiquitin-Ligases ........................................................................................................ 8 1.7 E2 Ubiquitin-Conjugating Enzymes ............................................................................... 10 1.7.1 E2 Classification: A Tail of Tails ............................................................................................................ 12 1.7.2 Mechanism of E2 Conjugation by UBA1 .............................................................................................. 14 1.7.3 E2 Functional Roles and Specificities ................................................................................................... 15 v 1.7.4 E2E Sub-Family of Ubiquitin-Conjugating Enzymes ............................................................................. 17 1.8 Deubiquitinating Enzymes (DUBs) ................................................................................ 20 1.8.1 DUB Classification ................................................................................................................................ 20 1.8.2 DUBs Regulate Ubiquitin Pathways ..................................................................................................... 23 1.8.3 Ubiquitin-Specific Protease 7 (USP7): One DUB, Many Roles .............................................................. 25 1.8.3.1 USP7 Structure and Mechanism of Function ............................................................................. 26 1.8.3.2 USP7’s TRAF Domain has a Consensus Binding Site ................................................................... 29 1.9 Ubiquitin & The Histone Code ...................................................................................... 31 1.9.1 Mono-Ubiquitination of H2A by Polycomb Group Proteins ................................................................ 32 1.10 Ub-Ligases and Ub-Conjugases as Targets of USP7 ...................................................... 34 1.10.1 USP7 is a Regulator of Ring1B ......................................................................................................... 34 1.10.2 USP7 is a Regulator of E2E1 ............................................................................................................ 35 1.10.3 USP7 Is a Potential Regulator of E2E2 and E2E3 ............................................................................. 35 1.10.3.1 E2E2 and E2E3 Contain TRAF Binding Motif ............................................................................... 35 1.10.3.2 Full-Length E2E2 and E2E3 Proteins Bind USP7 TRAF ................................................................ 35 1.10.3.3 USP7 Binds E2E2 and E2E3 in vivo .............................................................................................. 36 1.10.3.4 Molecular Analysis of the USP7:E2E2 or E2E3 Interactions ....................................................... 36 1.11 Rationale ....................................................................................................................... 37 Chapter 2: Materials & Methods ...................................................................................... 39 2.1 Bacteria | Culture, Gene Expression, and Protein Purification .................................... 39 2.1.1 DNA Plasmid Constructs and Amplifications ....................................................................................... 39 2.1.2 Preparation of Chemically-Competent Bacteria .................................................................................. 40 2.1.3 Gene Expression (Transformations)..................................................................................................... 40 2.1.4 Inducible Protein Expression ............................................................................................................... 41 vi 2.1.5 Harvest and Lysis ................................................................................................................................. 41 2.1.6 Histidine-Nickel Protein Purifications (His-Tag) ................................................................................... 41 2.1.7 GST-Glutathione Protein