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Systematic Functional Proteomic Investigation of Mammalian Histone Methylation-Related Protein Complexes

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Systematic Functional Proteomic Investigation of Mammalian Histone Methylation-Related Protein Complexes Systematic Functional Proteomic Investigation of Mammalian Histone Methylation-Related Protein Complexes by Jonathan B. Olsen A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Jonathan B. Olsen 2014 Investigation of Histone Methylation-Related Protein Complexes Jonathan B. Olsen Doctor of Philosophy Molecular Genetics University of Toronto 2014 Abstract Epigenetic regulation of chromatin structure, which involves site-specific methylation of histone proteins, is critical for regulating gene expression during development. While the enzymatic components of histone methylation-related protein complexes have been discovered, little is generally known regarding the associated tissue-specific co-factors that regulate complex activity in vertebrate/mammalian embryos. To better understand the composition and control of histone methylation-related complexes in mammals, I used an affinity purification and mass spectrometry (AP-MS) approach to identity stably interacting proteins of histone methylation- related proteins in human embryonic kidney (HEK) 293 cells. My AP-MS approach included replicate isolation and characterization of 33 histone methylation- and/or transcription-related proteins, resulting in an interaction network encompassing 573 protein-protein interactions (PPIs). Among the findings were a novel set of RNA polymerase II (RNAPII) binding proteins, known as the “RPRD” proteins, which recognize phosphorylated RNAPII, and several putative new components of the evolutionarily conserved gene silencing machinery. Most notably, I identified and characterized ZNF644, a protein that is putatively causally linked to high-grade myopia, as a novel interacting partner for the paralogous histone methyltransferases (HMTs) G9a and GLP and as a new co-regulator of histone H3 at lysine 9 dimethylation (H3K9me2). Using a zebrafish embryo model system, I characterized the roles of zebrafish g9a and two orthologues of ii ZNF644, termed znf644a and znf644b, in the forming retina and midbrain. I found that even modest disruption of g9a activity caused widespread apoptosis in progenitor and differentiating cell populations in the retina. I also found that znf644a and znf644b have common retina-specific roles in suppressing progenitor gene expression (i.e., vsx2 and ccnd1) by regulating promoter H3K9me2. However, despite the overlapping roles in progenitor gene suppression, the two znf644 paralogues controlled distinct aspects of retinal differentiation; specifically, I observed that disruption of znf644b caused retina-specific developmental delays, resulting in the delayed differentiation of RPCs, whereas disruption of znf644a caused the mislocalized expression of H3K9me2 marked genes (i.e., znf644a and znf644b) during differentiation, resulting in the impaired viability of differentiated neurons. Importantly, I used complementation assays as well as “genetic cooperativity” assays to demonstrate that, despite the disparate phenotypes of g9a, znf644a and znf644b morphant retinas, the regulatory roles of znf644a and znf644b are critically dependent on their ability to functionally and physically interact with g9a. In addition, I provided evidence that the functions of znf644a and znf644b are encompassed by mammalian ZNF644 and that the retinal roles of g9a, znf644a and znf644b are recapitulated in progenitor cells of the developing midbrain. Collectively, my thesis provides both a high-confidence global molecular interaction map that furthers understanding of the physical and functional associations underlying the mammalian histone methylation machinery and highlights the unexpected role of a previously unappreciated co-factor, ZNF644 as a co-regulator of H3K9me2-mediated gene silencing and a multifaceted regulator of neural differentiation. iii Acknowledgments The work in this doctoral dissertation was supported in part by grants from the Canadian Foundation for Innovation, Ontario Research Fund, and Canadian Institute for Health Research to Dr. Andrew Emili. I was supported in part by a doctoral Ontario Graduate Scholarship (2010- 2012). First and foremost, I would like to thank and acknowledge my PhD supervisor, Andrew Emili, for extending his support and guidance over the past 6 years. Not only did Andrew grant me the flexibility to tread into unfamiliar waters with my projects, but also served at a personal level as a great mentor and motivator. I also express my appreciation to my supervisory committee, Drs. Jeff Wrana and Dev Sidhu, for their critical insights to help keep me on track through my PhD. I also thank the many talented collaborators with whom I have had the privilege of working for their support and for taking interest in my thesis work. In particular, a special thanks to Drs. Loksum Wong and Vincent Tropepe for all things zebrafish-related; their expertise and insights were critical for applying meaningful in vivo insights to my proteomics data. I also extend my gratitude to Dr. Jack Greenblatt for his contagious enthusiasm for chromatin biology and for always being willingness to discuss all things related to transcription. I extend my sincere gratitude all the members of the Emili Lab for ongoing collegiality and support. Last, but certainly not least, I thank my family for their patient, loving support over the past few years. iv Table of Contents Acknowledgments .............................................................................................................................. iv Table of Contents .................................................................................................................................. v List of Tables ......................................................................................................................................... ix List of Figures ......................................................................................................................................... x List of Appendices ............................................................................................................................. xiii List of Abbreviations ........................................................................................................................ xiv Chapter 1 Introduction ....................................................................................................................... 1 1 Introduction .................................................................................................................................... 2 1.1 Chromatin and its modification ...................................................................................................... 2 1.1.1 Histone methylation and the related protein machinery ............................................................... 6 1.1.2 The impact of histone methylation on transcription ........................................................................ 9 1.1.3 Histone methylation, stem cells and cellular differentiation ..................................................... 13 1.1.4 Histone methylation complexes and their importance in transcriptional regulation .... 14 1.2 The paralogous H3K9 HMTs G9a and GLP form a heterodimer required for euchromatic gene silencing ....................................................................................................................... 20 1.2.1 Long-term gene silencing mediated by G9a/GLP ........................................................................... 23 1.3 The differentiation of retinal progenitor cells during development ............................... 27 1.3.1 The origin of highly proliferative progenitor cell of the retina ................................................. 27 1.3.2 Regulation of RPC proliferation and cell fate decisions ............................................................... 28 1.3.3 The role of G9a and H3K9me2 in RPCs ............................................................................................... 29 1.4 Aims and objectives ........................................................................................................................... 30 Chapter 2 A physical interaction map for histone methylation-related proteins ........ 32 2 A physical interaction map for histone methylation-related proteins ..................... 33 2.1 Abstract ................................................................................................................................................. 33 2.2 Introduction ......................................................................................................................................... 34 2.3 Methods ................................................................................................................................................. 37 2.3.1 Plasmid construction ................................................................................................................................... 37 2.3.2 Cell culture and lentivirus production ................................................................................................. 37 v 2.3.3 Affinity purification and mass spectrometry .................................................................................... 38 2.3.4 Immunoprecipitation and western blotting ...................................................................................... 39 2.3.5 Lentiviral-based shRNA-mediated mRNA knockdown ...............................................................
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