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A STUDY of the COMPOSITION and FUNCTION of TELOMERIC CHROMATIN in Drosophila Melanogaster

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A STUDY of the COMPOSITION and FUNCTION of TELOMERIC CHROMATIN in Drosophila Melanogaster A STUDY OF THE COMPOSITION AND FUNCTION OF TELOMERIC CHROMATIN IN Drosophila melanogaster. by James Gregory Brian Doheny B.A., Simon Fraser University, 1987 B. Sc. The University of British Columbia, 1990 M. Sc. The University of British Columbia, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA January 2008 © James Gregory Brian Doheny, 2008 ABSTRACT The telomeres of most organisms are characterized by a protein-capping complex that protects chromosome ends, a series of repetitive subtelomeric sequences known as Telomere-Associated Sequences (TAS), and a behavioral phenomenon known as Telomere Position Effect (TPE). TPE is a phenomenon whereby normally active genes become repressed and silenced if relocated near to telomeres, and is thought to be a property of the proteins that constitute telomeric heterochromatin. Genetic dissection was used to exploit this phenomenon in order to identify components of telomeric heterochromatin in Drosophila melanogaster. Using genetic dissection, followed by a chromatin analysis technique known as Chromatin ImmunoPrecipitation (ChIP) I was able to identify three proteins, HDAC1, SU(VAR)3-9, and HP1c, as integral components of telomeric heterochromatin in Drosophila. HDAC1 and SU(VAR)3-9 are both believed to be involved in the gene-silencing process, and thus, their presence at telomeres could explain the phenomenon of TPE. Furthermore, I found that these proteins were specifically associated with the TAS region on the centromere-proximal side of the HeT- A transposable elements that maintain telomere length in Drosophila. As a result of this, I proposed a model, which I call the ‘pairing-sliding model of telomere length control in Drosophila,’ which proposes that temporary incorrect pairing of Drosophila telomeres results in the deacetylation and subsequent methylation of the nucleosomes associated with the HeT-A and TART elements by TAS-associated HDAC1 and SU(VAR)3-9, resulting in these elements being transcriptionally silent. Thus, I propose that the TAS region, and the HDAC1 and SU(VAR)3-9 associated with it play a role in the negative regulation of telomere length in Drosophila. ii TABLE OF CONTENTS ABSTRACT…………………………………………..……………………………………ii TABLE OF CONTENTS…………………………..………………………………...……iii LIST OF TABLES…………………………….…………………………………………..vi LIST OF FIGURES………………………………….………………………..………….vii LIST OF ABBREVIATIONS……………………….……………………………………..x ACKNOWLEDGEMENTS……………………….…………………………………..….xii DEDICATION……………………………………….………………………………..…xiii CO-AUTHORSHIP STATEMENT……………..………..………………………..…….xiv CHAPTER ONE........................................................................................................................................... 1 INTRODUCTORY CHAPTER .................................................................................................................. 1 PUTTING THE STUDY OF THE TELOMERES OF DROSOPHILA MELANOGASTER INTO AN APPROPRIATE CONTEXT....................................................................................................................... 1 I Linear chromosomes and telomere maintenance................................................................................. 2 II Characteristic features of yeast telomeres. ......................................................................................... 4 III Characteristic features of mammalian telomeres. ............................................................................. 8 IV Current knowledge about Drosophila telomeres............................................................................. 11 IVA Telomere length maintenance in Drosophila. ....................................................................... 12 IVB Evidence for t-loop formation at Drosophila telomeres as part of a telomere repair mechanism.......................................................................................................................................... 14 IVC Genetic mutations that influence telomere length maintenance. ......................................... 15 IVD The Drosophila telomere capping complex........................................................................... 19 IVE TPE in Drosophila.................................................................................................................. 20 V Overview of thesis and objectives.................................................................................................... 25 VI REFERENCES ............................................................................................................................... 27 CHAPTER TWO........................................................................................................................................ 33 A GENETIC SCREEN TO FIND MUTATIONS THAT SUPPRESS TELOMERE POSITION EFFECT IN DROSOPHILA. .................................................................................................................... 33 I INTRODUCTION............................................................................................................................. 34 II MATERIALS AND METHODS...................................................................................................... 39 IIA Assay used to test suppression of TPE.................................................................................... 39 IIB Assay used to test suppression of PEV.................................................................................... 44 IIC Generation of w- stocks for testing.......................................................................................... 44 IID Crosses used to score suppression of TPE and PEV included an internal wild-type standard. 45 IIE Eye pigment assays.................................................................................................................. 46 IIF Fly crosses............................................................................................................................... 49 IIG Protein homology searches. .................................................................................................... 50 IIH Lethal complementation analysis. .......................................................................................... 50 III RESULTS ....................................................................................................................................... 53 IIIA Suppression of TPE by previously identified Su(var) mutations.......................................... 59 IIIB Previously identified Su(TPE) mutations.............................................................................. 67 IIIC A candidate gene screening approach to finding additional Su(TPE) loci.......................... 70 IV DISCUSSION................................................................................................................................. 93 iii IVA TPE as a separate phenomenon from PEV........................................................................... 93 IVB Summary and analysis of TPE-suppressing loci................................................................... 94 V REFERENCES .............................................................................................................................. 105 CHAPTER THREE.................................................................................................................................. 110 AN EXAMINATION OF THE CHROMATIN COMPOSITION OF THE DROSOPHILA 2L TELOMERE............................................................................................................................................. 110 I INTRODUCTION........................................................................................................................... 111 II MATERIALS AND METHODS ................................................................................................... 116 IIA Generation of 39C-X/Su(var)3-9GFP and 39C-5/Su(var)3-9GFP flies. ............................. 116 IIB ChIP Assays........................................................................................................................... 118 IIC Quantitative PCR (qPCR) of Reporter Gene, Actin 42A, and various regions of the 2L telomere. ........................................................................................................................................... 120 III RESULTS ..................................................................................................................................... 126 IIIA The HDAC1, SU(VAR)3-9, and HP1c proteins are present and enriched at the reporter gene when it is located in the telomere............................................................................................ 126 IIIB HDAC1, SU(VAR)3-9, HP1c are present at the native 2L telomere, but are preferentially associated with the TAS region........................................................................................................ 134 IIIC Distribution of histone modifications at the telomeric reporter gene................................. 143 IIID Distribution of histone modifications across the native Drosophila 2L telomere.............. 147 IV DISCUSSION............................................................................................................................... 154 IVA TPE and the chromatin composition of TAS regions. .......................................................
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