University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2020-07-29 Characterizing Inhibitor of Growth (ING) family evolution and ING1 structure and function Bertschmann, Jessica Bertschmann, J. (2020). Characterizing Inhibitor of Growth (ING) family evolution and ING1 structure and function (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/112355 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Characterizing Inhibitor of Growth (ING) family evolution and ING1 structure and function by Jessica Bertschmann A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOCHEMISTRY AND MOLECULAR BIOLOGY CALGARY, ALBERTA JULY, 2020 © Jessica Bertschmann 2020 1 Abstract The INhibitior of Growth (ING) family of tumor suppressors have emerged as a versatile family of phospholipid effectors, histone mark sensors, and growth regulators. An updated phylogenetic analysis of this protein family using sequences from 42 eukaryotic species reveals that ING4 is likely most similar to the ancestral ING protein, not ING3 as previously reported. Previous studies have shown that the major ING1 isoforms, ING1a and ING1b serve distinct cellular functions by differentially regulating apoptosis and senescence in primary cells. The ING1a isoform encodes a sequence unique in the human proteome. To identify ING1a homologs in other species we searched all available databases and found that sequences corresponding to ING1a were only found in great apes and old-world monkeys. However, only select primates had start codons capable of encoding full-length ING1a. Moreover, when we expressed ING1a with and without it’s unique N-terminal sequence, the unique sequence promoted localization to the mitochondria. Given the natural induction of this isoform as cells age in culture, expression of ING1a may serve to help limit the replicative lifespan of cells from long-lived primates, in part through its activity in the mitochondria. 2 Acknowledgements I would like to first express my gratitude for my supervisor, Dr. Karl Riabowol, for allowing me to join his lab. He has been incredibly supportive of both my academic and professional development. He has always encouraged me to ask questions and to pursue my curiosities, and I am a much better scientist for it. I would also like to thank the current and former members of the Riabowol lab: Arthur Dantas, Nancy Adam, Buthaina Shueili, Yang Yang, Karen Blote, Hari Thoppil, Whitney Alpaugh, Charles Ricordel, and Subhash Thalappilly for their help with experiments and for making my time in the lab much more enjoyable. I would like to extend a special thank you to Dr. Hamed Hojjat for his invaluable support and guidance. He offered many helpful ideas and patiently helped me troubleshoot many experiments. I would like to thank my supervisory committee members, Dr. Jennifer Cobb and Dr. Jason de Koning, for their valuable suggestions throughout my project. I would also like to thank all my friends within the Charbonneau research group and the BMB department. I wouldn’t have made through this degree without all the scientific discussion and laughs over coffee. 3 Table of Contents Abstract .............................................................................................................................. 1 Acknowledgements ........................................................................................................... 2 Table of Contents .............................................................................................................. 3 List of Tables ..................................................................................................................... 4 List of Figures and Illustrations ...................................................................................... 5 List of Symbols, Abbreviations and Nomenclature ....................................................... 6 Chapter 1 Introduction ................................................................................................... 10 1.1 The ING1 family of tumor suppressor genes ..................................................... 10 1.1.1 Structural features of ING proteins ............................................................ 11 1.1.2 Biological roles of ING proteins ................................................................... 16 1.1.2.1 Chromatin modification ....................................................................... 16 1.1.2.2 Cellular senescence .............................................................................. 18 1.1.2.3 DNA damage response ......................................................................... 19 1.1.2.4 rRNA transcriptional regulation ......................................................... 21 1.1.2.5 Cell stress response .............................................................................. 22 1.1.2.6 Cell migration, metastasis, and angiogenesis suppression ................. 22 1.1.2.7 Cell differentiation and stem cell maintenance .................................. 23 1.1.3 ING proteins in model organisms ................................................................ 24 1.1.4 Evolution of ING proteins ............................................................................ 28 1.2 Cellular Senescence ............................................................................................... 29 1.2.1 Aging and cellular senescence ..................................................................... 29 1.2.2 The senescent phenotype .............................................................................. 31 1.2.3 Senescence signalling pathways ................................................................... 33 1.2.4 ING1a induces senescence ............................................................................ 34 1.3 Mitochondria Biology ......................................................................................... 37 1.3.1 Mitochondrial structure ............................................................................... 38 1.3.2 Energy production ........................................................................................ 39 1.3.3 Apoptosis ....................................................................................................... 40 1.3.4 Mitochondria-associated senescence ........................................................... 41 1.4 Hypothesis and objectives .................................................................................... 42 Chapter 2 ING Family Evolution .................................................................................. 44 2.1 Introduction ........................................................................................................... 44 2.2 Methods .................................................................................................................. 44 2.2.1 Selection of organisms for analysis ............................................................. 44 2.2.2 Protein sequence retrieval ........................................................................... 46 2.2.3 Protein sequence alignments and phylogenetic analysis ........................... 48 2.2.5 Protein sequence alignments ....................................................................... 48 2.2.5 Genomic DNA sequence retrieval ............................................................... 49 2.2.6 Genomic DNA sequence alignments ........................................................... 50 2.3 Results .................................................................................................................... 51 2.3.1 Distribution of ING proteins in eukaryotes ............................................... 51 4 2.3.4 Analyses of plant and fungi ING sequences ............................................... 59 2.3.4 Emergence of the novel ING1a isoform ...................................................... 62 2.4 Discussion ......................................................................................................... 64 Chapter 3 ING1 Structure and Function ...................................................................... 66 3.1 Introduction ........................................................................................................... 66 3.2 Methods .................................................................................................................. 66 3.2.1 Cells and cell culture .................................................................................... 66 3.2.2 Freezing and thawing cells ........................................................................... 67 3.2.3 Sub-culturing of cells .................................................................................... 68 3.2.4 Cell nucleofection .......................................................................................... 68 3.2.5 DNA constructs and mutagenesis ...............................................................