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Functional Genomic Characterization of Transcription Factors in Fission Yeast University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2016 Functional Genomic Characterization of Transcription Factors in Fission Yeast Vachon, Lianne Vachon, L. (2016). Functional Genomic Characterization of Transcription Factors in Fission Yeast (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26245 http://hdl.handle.net/11023/3218 doctoral 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 Functional Genomic Characterization of Transcription Factors in Fission Yeast by Lianne Vachon A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA AUGUST, 2016 © Lianne Vachon 2016 Abstract Mapping an organism’s transcriptional regulatory network (TRN), which consists of all interactions between its transcription factors (TF) and target genes, is a necessary step in developing a complete understanding of how that organism normally develops, and how disease states can arise. However, the number of TFs, target genes, and potential regulatory interactions make this a difficult task, even in simple eukaryotes. In this study, the TRN of Schizosaccharomyces pombe was studied. To do so, we systematically deleted over 80% of fission yeast TFs, and characterized the effects of TFΔ on cell growth, length, and gene expression. Deletion of most TFs did not appear to impact the cell, suggesting that many may be inactive in rich medium. To circumvent this issue, we used two approaches. First, hypersensitivity of TFΔ strains to various drug compounds was determined to identify conditions that might induce TF activity. A four-way microarray expression profiling scheme was then used to identify the target genes and function of uncharacterized TF Toe1. This revealed that Toe1 regulated several genes implicated in the pyrimidine salvage pathway. Secondly, we systematically overexpressed S. pombe TF genes under control of the nmt1 promoter. Over 70% of the overexpressed TF genes resulted in altered cell length or fitness, indicating that their target genes might be inappropriately expressed. Expression microarrays and ChIP-chip were thus used to identify the putative target genes for three uncharacterized fungal TFs, SPBC1773.16, SPBC16G5.17, and SPAC25B8.11, revealing potential roles for each of these TFs in regulating the utilization of alternative nitrogen sources. Finally, screens of our TFΔ array in flocculation inducing and rich mediums revealed six novel transcriptional activators (Foe1, Prr1, Prt1, SPBC530.08, Fep1, and Grt1) and two ii transcriptional repressors (Scr1 and SPBC56F2.05) of flocculation. Microarray expression profiling was used to identify potential target genes for six of these TFs. Additionally, ChIP-chip was used to identify direct Foe1 targets, revealing that this TF directly binds and regulates the expression of several genes encoding flocculins and cell wall remodeling/biosynthesis proteins. Collectively, these results should contribute to a better understanding of transcriptional regulation in S. pombe as a whole. iii Acknowledgements I would like to thank everyone who supported and encouraged me while I was pursuing my PhD. Without all of your help, this wouldn’t have been possible. In particular, I’d like to thank my PhD supervisor Dr. Gordon Chua, and my committee members Dr. Dave Hansen and Dr. Gregory Moorhead, for their insight and guidance along the way. I would also like to thank all of the members of the Chua lab, not only for helping train me when I came into the lab and discussing challenging problems, but also for making the lab a fun place to be. Finally, I would like to thank my family, especially my brother Eric, my father Mario, and my mom and stepdad Julie and Dwight, for your continuous support and encouragement throughout this process. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... xi List of Figures and Illustrations ........................................................................................ xii List of Symbols, Abbreviations and Nomenclature ......................................................... xiv CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Regulation of gene expression ...................................................................................1 1.1.1 Regulated stages of gene expression .................................................................1 1.2 Regulation of transcription ........................................................................................3 1.2.1 The basal transcriptional machinery ..................................................................3 1.2.2 Sequence specific transcription factors .............................................................4 1.2.2.1 DNA-binding domains and the classification of TFs ...............................4 1.2.2.2 TF regulatory domains and mechanisms of activation and repression ..................................................................................................6 1.2.3 Eukaryotic cis-regulatory elements ...................................................................9 1.2.3.1 Core promoter region ..............................................................................9 1.2.3.2 Proximal promoter elements ..................................................................10 1.2.3.3 Other regulatory sequences in higher eukaryotes .................................11 1.2.4 Cis-regulatory elements ...................................................................................12 1.3 Transcriptional-regulatory networks ........................................................................12 1.3.1 Recurrent network motifs in transcriptional regulatory networks ...................13 1.3.2 Semi independent modules ..............................................................................15 1.3.3 Overall network topology ................................................................................16 1.3.4 Reconstruction of the S. pombe TRN ..............................................................16 1.4 The model organism Schizosaccharomyces pombe .................................................17 1.4.1 Sequencing of the S. pombe genome ..............................................................17 1.4.2 Advantages of S. pombe as a model organism ................................................18 1.5 Identification of TF target genes ..............................................................................19 1.5.1 Systematic TF deletion and overexpression ....................................................20 1.5.2 Identifying global changes in gene expression to elucidate TF target genes ..21 1.5.3 Identifying TF sequence specificities and mapping TF binding locations ......23 1.6 Flocculation .............................................................................................................24 1.6.1 Proteins mediating cell-cell adhesion ..............................................................25 1.6.2 Flocculation mechanisms ................................................................................26 1.6.3 Environmental conditions and signalling pathways that induce flocculation ......................................................................................................27 1.6.4 Transcriptional regulation of flocculation .......................................................28 1.7 Objective of this study .............................................................................................29 1.7.1 Specific aims ...................................................................................................29 CHAPTER TWO: MATERIALS AND METHODS ........................................................31 2.1 Media .......................................................................................................................31 2.2 Construction of transcription factor deletion library ...............................................32 v 2.2.1 PCR amplification and stitching ......................................................................32 2.2.2 Lithium acetate transformation ........................................................................33 2.2.3 Yeast colony screens .......................................................................................34 2.2.4 Determination of cell length and generation time ...........................................34 2.2.5 Flocculation assays ..........................................................................................35
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