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The Role of the Mis18α-Β Complex and Its Interactions with HJURP and CENP-A in Human Centromeric Chromatin Establishment

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The role of the Mis18α-β complex and its interactions with HJURP and CENP-A in human centromeric chromatin establishment Isaac Kaufman Nardi, VA B.S. Biology, Virginia Tech, 2010 M.S. Biochemistry and Molecular Genetics, University of Virginia, 2012 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Biochemistry and Molecular Genetics University of Virginia February, 2016 1 Table of Contents ABSTRACT ............................................................................................................... 4 CHAPTER 1: GENERAL INTRODUCTION ............................................................ 5 General Perspective and Significance .................................................................... 5 The Epigenetic Propagation of the Centromere ...................................................... 6 Centromeric Chromatin ...................................................................... 6 The CCAN: A Platform for Kinetochore Formation ............................................. 10 CENP-A Structural Characteristics ...................................................................... 16 The CENP-A Deposition Pathway ....................................................................... 18 Temporal Regulation of CENP-A Deposition ........................................ 19 Centromere Priming Components: CENP-C ......................................... 22 Centromere Priming Components: HJURP ........................................... 23 Centromere Priming Components: Mis18 ............................................. 27 DNA Replication Licensing .................................................................................. 32 CHAPTER 2: LICENSING OF CENTOMERIC CHROMATIN ASSEMBLY THROUGH THE MIS18-MIS18 HETEROTETRAMER .................................... 36 ABSTRACT ....................................................................................................... 37 INTRODUCTION ............................................................................................. 38 RESULTS ......................................................................................................... 41 Mis18 proteins form a conserved tetramer ............................................ 40 Mis18 proteins interact through conserved coiled-coils ......................... 41 Mis18 localization requires formation of the heterotetramer via the coiled-coil domain ................................................................................ 42 Mis18 recruits HJURP through a direct interaction with the coiled- coil domain ........................................................................................... 44 The Mis18 coiled-coils recruit HJURP .................................................. 45 HJURP binding disrupts the Mis18 heterotetramer ............................... 46 Mis18α is stable and does not turnover at the centromere during G1 .... 48 HJURP facilitates removal of Mis18 from centromeres ......................... 48 CENP-A levels at centromeres are controlled by Mis18 ........................ 49 DISCUSSION ...................................................................................................... 51 MATERIALS & METHODS ............................................................................... 57 REFERENCES ..................................................................................................... 61 FIGURE LEGENDS ............................................................................................ 65 PRIMARY FIGURES .......................................................................................... 70 SUPPLEMENTAL FIGURES .............................................................................. 77 SUPPLEMENTAL FIGURE LEGNEDS .............................................................. 83 SUPPLEMENTAL MATERIALS & METHODS ................................................ 87 CHAPTER 3: MIS18 AS A COMPONENT OF THE CENP-A DEPOSITION MACHINERY ......................................................................................................... 90 ABSTRACT ....................................................................................................... 91 INTRODUCTION ............................................................................................. 92 RESULTS ......................................................................................................... 95 Mis18 complex directly binds to CENP-A CATD ................................... 95 Mis18recognizes histidine 104 within CATD of CENP-A ....................... 96 2 Mis18 YIPPEE domain required for CENP-A retention at chromatin .... 98 DISCUSSION .................................................................................................... 101 MATERIALS & METHODS ............................................................................. 104 FIGURE LEGENDS .......................................................................................... 109 PRIMARY FIGURES ........................................................................................ 112 CHAPTER 4: THE CENP T/W/S/X EXPERIMENTS: THE LONG LOST PROJECT/ HJURP AND ITS INVOLVMENT IN DNA DAMAGE ........................................ 117 ABSTRACT ..................................................................................................... 118 INTRODUCTION ........................................................................................... 119 RESULTS ....................................................................................................... 124 S/X ...................................................................................................... 124 T/W ..................................................................................................... 127 HJURP & DNA Damage ..................................................................... 128 DISCUSSION .................................................................................................... 132 FIGURE LEGENDS .......................................................................................... 135 MATERIALS&METHODS ............................................................................... 137 PRIMARY FIGURES ........................................................................................ 139 REFERENCES ................................................................................................... 149 APPENDIX ........................................................................................................ 158 ABBREVIATIONS ............................................................................................ 161 3 Abstract The centromere, in higher eukaryotes, is an epigenetically specified locus that is the site kinetochore formation on each chromosome during mitosis. The epigenetic mark that defines a centromere as of this writing is the histone H3 variant Centromere Protein-A (CENP-A). The establishment and maintenance of CENP-A at centromeres is absolutely critical for proper chromosome segregation and ploidy in cells. Several key factors have been identified as crucial machinery involved in CENP-A deposition. Understanding how a specific subset of these factors maintains CENP-A specifically at centromeric chromatin is the theme of this work. The second chapter of this work demonstrates the CENP-A chaperone HJURP is directly targeted to centromeres by the Mis18 complex. Furthermore, binding of Mis18 by HJURP mislocalizes the complex from centromeres giving cells control over where CENP-A deposition occurs and how much CENP-A gets consigned at centromeres. The third chapter characterizes a direct interaction between Mis18 and CENP-A that has never been characterized before. This work, though in its infancy, alludes to an important role of Mis18 as being an integral part of the CENP-A deposition machinery into centromeric chromatin. The fourth chapter describes the CENP-T/W/S/X complex and its ability to form “nucleosome-like” structures that may be involved in further differentiating the centromeres from outside chromatin. I also go over a potential role of HJURP in the DNA damage response which was touched on several years ago but not fully followed up ever since HJURPs role in CENP-A deposition has been discovered. 4 CHAPTER I: GENERAL INTRODUCTION General Perspective and Significance Accurate chromosome segregation is absolutely critical for proper cell ploidy, viability, and survival. To ensure this process happens properly every cell cycle the centromere, an epigenetically specified locus in higher eukaryotes, is absolutely required to be positioned and properly maintained on every chromosome to allow for the proteinacous kinetochore apparatus to form during mitosis. Chromosome mis-segregation and the resulting aneuploidy is a hallmark of a majority of cancers and is shown to be heavily involved in a variety of birth defects. Therefore, understanding how proper and improper chromosome segregation occur and how the centromere is involved in this process is of the utmost importance in not only understanding the process but also for the identification of novel targets for future chemotherapeutics. The work presented here is significant to the centromere field and cancer research because it furthers our understanding of the essential steps of CENP-A assembly into chromatin through a never before seen, and hugely critical interaction, between HJURP and Mis18. 5 The Epigenetic
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