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title | AN IN VITRO CENP-A ASSEMBLY ASSAY REVEALS A ROLE FOR CENP-C
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title | IN CENP-A DEPOSITION
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text | A DISSERTION
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text | SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY
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text | AND THE COMMITTEE ON GRADUATE STUDIES
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text | OF STANFORD UNIVERSITY
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text | IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
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text | FOR THE DEGREE OF
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text | DOCTOR OF PHILOSOPHY
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|
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text | Carissa Bove Meyer
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text | October 2011
| © 2012 by Carissa Bove Meyer. All Rights Reserved.
| Re-distributed by Stanford University under license with the author.
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|
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text | This work is licensed under a Creative Commons Attribution-
| Noncommercial 3.0 United States License.
| http://creativecommons.org/licenses/by-nc/3.0/us/
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|
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text | This dissertation is online at: http://purl.stanford.edu/bh410qk3771
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meta | ii
text | I certify that I have read this dissertation and that, in my opinion, it is fully adequate
| in scope and quality as a dissertation for the degree of Doctor of Philosophy.
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text | Aaron Straight, Primary Adviser
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|
|
text | I certify that I have read this dissertation and that, in my opinion, it is fully adequate
| in scope and quality as a dissertation for the degree of Doctor of Philosophy.
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text | Suzanne Pfeffer
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|
|
text | I certify that I have read this dissertation and that, in my opinion, it is fully adequate
| in scope and quality as a dissertation for the degree of Doctor of Philosophy.
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text | Julie Theriot
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|
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|
text | Approved for the Stanford University Committee on Graduate Studies.
| Patricia J. Gumport, Vice Provost Graduate Education
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|
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text | This signature page was generated electronically upon submission of this dissertation in
| electronic format. An original signed hard copy of the signature page is on file in
| University Archives.
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|
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meta | iii
title | Abstract
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text | Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic
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text | spindle through a chromosomal microtubule-binding site called the kinetochore.
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text | Kinetochores assemble on a specialized chromosomal locus termed the centromere,
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text | characterized by the replacement of histone H3 in centromeric nucleosomes with the
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text | essential histone H3 variant centromere protein A (CENP-A). CENP-A nucleosomes
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text | are believed to epigenetically specify centromere identity, thus understanding how
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text | CENP-A chromatin is assembled and maintained is central to understanding
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text | chromosome segregation mechanisms. CENP-A nucleosome assembly requires the
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text | Mis18 complex and the CENP-A chaperone HJURP. HJURP binds to pre-
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text | nucleosomal CENP-A and facilitates the deposition of new CENP-A nucleosomes into
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text | centromeric chromatin. The mechanistic roles of the Mis18 complex in CENP-A
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text | assembly and maintenance are not well understood. The Mis18 complex and HJURP
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text | localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled.
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text | The molecular interactions that control their targeting are unknown. Several
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text | constitutive centromere proteins that remain associated with CENP-A chromatin
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text | throughout the cell cycle have also been implicated in CENP-A assembly, including
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text | CENP-C. Their functions in CENP-A assembly are unknown.
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text | The first chapter of this thesis work describes the development of an in vitro
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text | system for centromeric chromatin assembly in Xenopus laevis egg extracts. We show
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text | that CENP-A assembly in extract recapitulates the cell cycle dependence and HJURP
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text | requirement of CENP-A assembly in somatic cells. We then use this in vitro system to
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text | identify a novel role for CENP-C in recruiting CENP-A assembly factors to the
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meta | iv
text | centromere to promote CENP-A assembly, described in Chapter 2. We show that
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text | CENP-C is required to target the Mis18 complex protein M18BP1 to Xenopus sperm
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text | centromeres in metaphase. In the absence of CENP-C, M18BP1 and HJURP targeting
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text | to centromeres is disrupted and new CENP-A assembly into centromeric chromatin is
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text | inhibited. We find that CENP-C interacts directly with M18BP1 through conserved
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text | domains in the CENP-C protein. Thus, CENP-C provides a link between existing
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text | CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.
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text | Overall, this work extends our mechanistic understanding of how the pre-
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text | existing centromere directs the local assembly of new CENP-A nucleosomes to ensure
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text | stable propagation of the centromere. Furthermore, the ability to assemble centromeric
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text | chromatin in vitro will provide a valuable tool for dissecting the biochemical and cell
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text | cycle regulatory mechanisms that control new CENP-A assembly and ensure faithful
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text | centromere propagation.
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meta | v
title | Acknowledgements
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text | First thanks go to the members of the Straight lab, a fantastic group of
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text | scientific colleagues and friends. I want to especially thank Ben Moree, who taught me
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text | how to make extracts and who did far more than his fair share of frog injections over
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text | the past two years. I am grateful to have had him as a collaborator on this project; our
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text | spirited scientific discussions were fun and made this work stronger. I also thank Ben
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text | for re-introducing me to the joys of meat-eating, in particular his pulled pork and
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text | anything with bacon. I am profoundly grateful to Colin Fuller for always being
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text | available when I needed an “assist” with computer or experimental issues, and for
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text | many stimulating discussions about the merits of various gluten-free flours. Thanks to
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text | my mentees and collaborators Bradley French and Andy Nguyen; I am happy to leave
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text | this project in Bradley’s capable hands. I thank Annika, my fellow frog lady, for her
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text | scientific mentorship and her friendship. Topher has provided much valuable and
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text | practical experimental advice and professional guidance. Whitney, Teddy, and
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text | Amanda have always been enthusiastic about and supportive of my work. Special
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text | thanks to former Straight lab member Kristina Godek, who taught me how to purify
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text | proteins and who was very generous with her time when I first joined the lab. Finally,
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text | I am extremely grateful to Aaron Straight for serving as my advisor. His enthusiasm
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text | for experimental science is inspiring, and his optimism and faith in our abilities to
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text | make experiments work got me through the frustrating parts of this project. I thank
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text | him for pushing me to think about the “big picture” when planning experiments and
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text | interpreting data, and for teaching me how to effectively communicate our science to a
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text | larger audience.
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meta | vi
text | I am grateful to my thesis committee members, Suzanne Pfeffer and Julie
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text | Theriot, for their scientific guidance and encouragement over the past four years.
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text | Thanks to James Nelson for serving as my oral defense committee chair and to Raj
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text | Rohatgi for serving on my oral defense committee.
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text | Thanks to all of my Biochemistry department colleagues; I have benefited
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text | greatly from the connectedness of this department, both scientifically and socially. I
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text | am also grateful for the many friends I have made outside the department. Special
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text | thanks to my fellow Biosciences graduate students Karen Colbert and Erica Machlin
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text | for their support and for our many lunch and dinner dates over the past four years.
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text | Thanks also to Annika and her husband Jens for many evenings of delicious food and
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text | drink and stimulating conversations about food, politics, and funny American idioms.
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text | I am grateful to the National Science Foundation, the Department of Defense’s
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text | National Defense Science and Engineering Graduate Fellowship program, and to the
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text | Stanford Graduate Fellowship program for supporting me financially.
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text | Many thanks to my grandparents, parents, and brother for their love and
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text | encouragement. Even though they don’t understand quite what I do on a daily basis,
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text | they have been enthusiastic every step of the way. Finally, I am profoundly grateful to
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text | my husband, Scott, for his support and encouragement over the past four years.
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text | Though living apart has been extremely difficult, I know that he is proud of what I
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text | have accomplished. I thank him for making me laugh at the end of tough days in lab,
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text | for keeping me mentally balanced, and for always believing in me.
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meta | vii
text | This thesis is dedicated to the memory of my grandfather Harry Meyer, who passed
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text | away a few months ago. He was extremely supportive of my academic endeavors
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text | throughout my life.
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meta | viii
title | Table of Contents
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text | Preliminary Pages
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text | Title Page i
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text | Copyright Notice Page ii
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text | Signature Page iii
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text | Abstract iv
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text | Acknowledgements vi
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text | Table of Contents ix
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text | List of Tables x
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text | List of Figures xi
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text | Text
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text | Introduction 1
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text | Chapter 1 Development of an in vitro system for 61
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text | centromeric chromatin assembly in
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text | Xenopus laevis egg extracts
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text | Chapter 2 CENP-C recruits M18BP1 to centromeres 105
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text | to promote CENP-A chromatin assembly
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text | Concluding Remarks 176
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text | References 187
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meta | ix
title | List of Tables
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text | Introduction
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text | Table 1 CENP-A nomenclature in different species 60
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text | Chapter 1
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text | Supplemental Table 1 Plasmid constructs used in this study 103
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text | Supplemental Table 2 Antibodies used in this study 104
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text | Chapter 2
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text | Supplemental Table 1 Plasmid constructs used in this study 173
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text | Supplemental Table 2 Antibodies used in this study 175
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meta | x
title | List of Figures
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text | Introduction
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text | Figure 1 Chromatin and nucleosome structure 58
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text | Figure 2 Domain architecture of CENP-A and CENP-C 59
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|
|
text | Chapter 1
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text | Figure 1 CENP-A assembly in Xenopus extracts requires 96
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text | xHJURP addition and mitotic exit.
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text | Figure 2 Myc-CENP-A is stably incorporated into sperm 97
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text | chromatin, and CENP-A assembly in Xenopus
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text | extracts is independent of DNA replication.
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text | Figure 3 Exogenously added xHJURP promotes assembly of 98
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text | exogenous myc-CENP-A and endogenous CENP-A.
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text | Figure 4 xHJURP, myc-CENP-A, and the sperm chromatin 99
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text | template do not need to pass through mitosis for
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text | efficient myc-CENP-A assembly.
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text | Figure S1 Characterization of xHJURP-mediated CENP-A 100
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text | assembly assay in Xenopus egg extract.
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text | Figure S2 Myc-CENP-A assembly continues throughout 101
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text | interphase in Xenopus extracts.
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text | Figure S3 xHJURP, but not hHJURP, promotes CENP-A 102
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text | assembly in Xenopus extract.
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meta | xi
text | Chapter 2
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text | Figure 1 Characterization of X. laevis M18BP1. 158
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text | Figure 2 Depletion of M18BP1 inhibits xHJURP-mediated 159
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text | CENP-A assembly in extract.
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text | Figure 3 CENP-C regulates M18BP1 assembly at centromeres 160
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text | in Xenopus egg extract.
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text | Figure 4 CENP-C and M18BP1 promote the recruitment 161
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text | of HJURP to centromeres.
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text | Figure 5 CENP-C depletion inhibits M18BP1 targeting to 162
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text | metaphase centromeres and xHJURP dependent
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text | CENP-A assembly.
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text | Figure 6 CENP-C associates with M18BP1-1 in metaphase 163
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text | extract.
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text | Figure 7 CENP-C directly interacts with M18BP1 through 164
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text | two conserved domains in CENP-C.
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text | Figure S1 Additional characterization of xM18BP1. 165
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text | Figure S2 Both isoforms of M18BP1 rescue CENP-A assembly 166
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text | in M18BP1-depleted extract.
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text | Figure S3 Both M18BP1 isoforms localize to interphase 167
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text | centromeres in CENP-C-depleted extract.
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text | Figure S4 M18BP1 accumulates at interphase centromeres 168
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text | prior to HJURP.
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text | Figure S5 hCENP-C directly interacts with hM18BP1, and the 169
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meta | xii
text | M18BP1 binding domain of CENP-C is conserved.
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text | Figure S6 Several point mutations within the CENP-C motif 170
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text | abolish CENP-C targeting to centromeres but do
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text | not affect CENP-C binding to M18BP1.
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text | Figure S7 A C-terminally truncated fragment of M18BP1-2 171
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text | co-precipitates with CENP-C and targets to
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text | metaphase centromeres.
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text | Figure S8 CENP-C depletion inhibits CENP-N assembly 17
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text | at centromeres
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meta | xiii
title | Introduction
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|
meta | 1
text | Accurate chromosome segregation depends on the mitotic kinetochore.
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text | Cell division is a fundamental process during reproduction, development, and
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text | throughout the lifetime of an organism. One important aspect of cell division is
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text | propagation of the genetic material: each daughter cell inherits an exact copy of the
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text | parental genome. This involves two steps: first, DNA is faithfully replicated during S
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text | phase of the cell cycle, and second, replicated (‘sister’) chromosomes are equally
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text | partitioned between daughter cells during mitosis. Mistakes in chromosome
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text | segregation lead to the loss or gain of genetic material (aneuploidy), which is
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text | detrimental on both the cellular and organismal level. Aneuploidy is the leading
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text | known cause of pregnancy loss, and for those pregnancies that survive to term, the
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text | primary genetic cause of developmental disabilities and mental retardation (Hassold
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text | and Hunt, 2001). Furthermore, aneuploidy is a common characteristic of human
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text | cancers and is correlated with poor prognosis (Weaver and Cleveland, 2006).
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text | Experimentally, aneuploidy has been shown to promote tumorigenesis (Weaver and
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text | Cleveland, 2009; Weaver et al., 2007) or cause embryonic lethality (Williams et al.,
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text | 2008) in some mouse models. Consequently, cells have evolved stringent mechanisms
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text | for regulating chromosome segregation during mitosis and meiosis.
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text | Chromosome segregation is mediated by a proteinacious structure called the
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text | kinetochore, which assembles at a single site on each chromosome during mitosis. The
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text | kinetochore serves as the primary attachment site for microtubules of the mitotic
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text | spindle and couples spindle forces to chromosome movement during anaphase. The
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text | kinetochore also mediates the spindle assembly checkpoint, which delays anaphase
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text | onset until all sister chromosomes are attached to opposite poles of the spindle, thus
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meta | 2
text | ensuring that each daughter cell receives a precise complement of the genome
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text | (reviewed in (Cheeseman and Desai, 2008)). The kinetochore assembles on a
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text | specialized chromatin domain called the centromere.
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|
text | The centromere is a specialized chromatin domain that serves as the site of
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text | mitotic kinetochore assembly.
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text | The centromere was first defined cytologically, as the primary constriction
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text | point of paired mitotic chromosomes. On a molecular level, a collection of ~20
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text | proteins called the constitutive centromere associated network (CCAN) remain
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text | associated with this locus throughout the cell cycle and serve as the platform for
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text | mitotic kinetochore assembly. Mutation or loss of proteins of the CCAN results in
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text | kinetochore formation defects and chromosome missegregation (Amano et al., 2009;
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text | Cheeseman et al., 2008; Foltz et al., 2006; Hori et al., 2008; McClelland et al., 2007).
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text | Thus, understanding how the centromere is formed is central to understanding
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text | chromosome segregation mechanisms.
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text | In this introduction, I will first introduce the general structure and organization
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text | of chromatin, then I will discuss what makes centromeric chromatin distinctive. On a
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text | molecular level, a hallmark of centomeric chromatin is the replacement of histone H3
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text | with the centromere-specific histone H3 variant centromere protein A (CENP-A) in
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text | centromeric nucleosomes (Palmer et al., 1991; Palmer et al., 1987; Sullivan et al.,
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text | 1994). I will present evidence suggesting that CENP-A nucleosomes epigenetically
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text | specify centromere identity, then I will end with a discussion on the molecular
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text | mechanisms of centromere propagation.
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title | Eukaryotic DNA is packaged into chromatin.
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text | To fit about two meters of DNA into a nucleus that is merely ten to twenty
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text | microns in diameter, eukaryotic cells ‘package’ their DNA into chromatin fibers
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text | (Annunziato, 2008). The basic unit of chromatin is a protein-DNA complex called the
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text | nucleosome, which can be visualized as the ‘bead’ component of the ‘beads on a string’
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text | structure adopted by chromatin extracted from cells under conditions that cause it to
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text | unravel ((Olins and Olins, 1974; Olins and Olins, 2003), Figure 1A). The protein
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text | components of nucleosomes are histones, a family of small, positively charged
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text | proteins with unstructured N-terminal tails followed by a conserved histone fold
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text | domain (HFD). The ‘canonical’ histones, which comprise the bulk of histones found
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text | within chromatin, are termed H2A, H2B, H3, and H4; in solution, these proteins exist
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text | as H2A-H2B dimers and H3-H4 tetramers. To form a nucleosome, two copies of each
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text | histone protein form a compact histone octamer that is wrapped 1.65 times by a left-
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text | handed DNA superhelix 146 base pairs in length (Luger et al., 1997) (Figure 1b). The
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text | histone HFDs mediate histone-histone interactions and histone-DNA interactions that
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text | drive formation of the histone octamer and the nucleosome, and the histone N-terminal
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text | tails protrude from the nucleosome core particle. The histone tails are highly basic and
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text | are substrates for a variety of post-translational modifications that affect chromatin
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text | structure and function, including methylation, acetylation, and phosphorylation
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text | (reviewed in (Kouzarides, 2007)).
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text | Within cells, nucleosomes form at regularly spaced intervals, with an average
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text | of 20 base pairs of linker DNA (‘string’, Figure 1A) between nucleosomes
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meta | 4
text | (Annunziato, 2008). The packaging of DNA into arrays of nucleosomes compacts the
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text | DNA approximately seven-fold. However, during mitosis, when chromosomes are in
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text | their most compacted state, the DNA is approximately 10,000-fold more compact than
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text | in its linear form (Annunziato, 2008). Thus, to achieve additional compaction,
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text | nucleosome arrays are folded into higher-order structures, which are poorly
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text | understood at a structural level ((Annunziato, 2008; Felsenfeld and Groudine, 2003)).
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text | In addition to facilitating compaction of the DNA, the formation of
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text | nucleosomes allows for the specialization of specific chromosomal regions. For
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text | example, nucleosome modifications can mark chromosomal loci as transcriptionally
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text | active, transcriptionally silent, or in need of DNA repair. Additionally, specific regions
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text | of chromatin, such as the centromere, can direct the assembly of protein complexes
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text | (the kinetochore) that regulate whole chromosome behaviors (chromosome
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text | segregation). In general, chromatin is specialized in two ways, through post-
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text | translational modification (PTMs) of the histone tails and through replacement of
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text | conventional histones with histone variants. Common PTMs include methylation,
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text | acetylation, and phosphorylation (reviewed in (Kouzarides, 2007)), and histone
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text | variants for H2A, H2B, and H3 have been discovered (reviewed in (Bernstein and
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text | Hake, 2006)). Both PTMs and histone variants can affect intra- and inter-nucleosomal
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text | contacts, thus influencing nucleosome and higher-order chromatin structures
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text | (reviewed in (Bernstein and Hake, 2006; Kouzarides, 2007). Additionally, PTMs can
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text | recruit effector proteins that modulate chromatin structure and function (reviewed in
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text | (Bernstein and Hake, 2006; Kouzarides, 2007)). Histone variants, through sequence
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text | differences with their conventional histone counterparts, can present unique sites for
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text | post-translational modification and/or effector binding. Characterizing the biological
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text | functions of histone variants and their distribution within chromatin is an area of
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text | active research (reviewed in (Bernstein and Hake, 2006)).
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|
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text | Centromeric chromatin contains CENP-A nucleosomes.
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text | Centromeric chromatin is specialized by the replacement of histone H3 with
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text | the centromere-specific H3 variant centromere protein A (CENP-A) within
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text | centromeric nucleosomes (Palmer et al., 1991; Palmer et al., 1987; Sullivan et al.,
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text | 1994). The N-terminal tails of H3 and CENP-A are divergent, but the histone fold
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text | domains share ~60% sequence identity at the amino acid level (Figure 2A). Shortly
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text | after the crystal structure of the H3 nucleosome was solved, Yoda et al. showed that
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text | CENP-A could replace H3 in octameric nucleosomes reconstituted in vitro, suggesting
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text | that the overall structure of centromeric and canonical nucleosomes was likely to be
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text | similar (Yoda et al., 2000). The crystal structure of the CENP-A nucleosome was
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text | recently solved, confirming this prediction (Figure 1C). The CENP-A nucleosome is a
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text | histone octamer made up of two copies each of the histones H2A, H2B, CENP-A, and
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text | H4, wrapped by DNA in a left-handed manner. Furthermore, SAXS measurements
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text | showed that the global structures of CENP-A and H3 nucleosomes are quite similar
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text | (Tachiwana et al., 2011). One notable difference is that only the central 121 base pairs
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text | of DNA are visible in the CENP-A nucleosome crystal structure (compared to 146
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text | base pairs in the H3 nucleosome structure), indicating that 13 base pairs on either end
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text | are unstructured (Tachiwana et al., 2011). This observation is consistent with
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text | biophysical studies showing that the DNA at the entry and exit sites of the CENP-A
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text | nucleosome is more flexible than in the H3 nucleosome (Conde e Silva et al., 2007;
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text | Kingston et al., 2011). Another notable difference is that CENP-A contains two extra
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text | amino acids in the loop 1 region within the histone fold domain, which protrude from
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text | the nucleosome core particle and are solvent accessible (Tachiwana et al., 2011). The
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text | authors speculated that this might provide a CENP-A-specific protein-protein
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text | interaction surface that could contribute to the exclusive localization of CENP-A at
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text | centromeres and/or centromere-specific localization of other centromere and
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text | kinetochore proteins.
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text | Several alternative models for the structure of the centromeric nucleosome
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text | have been proposed, that differ from the crystal structure with respect to the protein
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text | composition of the nucleosome and the directionality of the DNA wrapped around the
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text | nucleosome core particle. However, it is important to note that many biochemical
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text | experiments using CENP-A nucleosomes reconstituted from human or budding yeast
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text | proteins also suggest that the histone protein complex is octameric and that it is
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text | wrapped by a left-handed DNA superhelix (Camahort et al., 2009; Conde e Silva et al.,
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text | 2007; Kingston et al., 2011; Sekulic et al., 2010; Tachiwana et al., 2011; Xiao et al.,
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text | 2011).
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text | The most controversial proposal is that the protein core of centromeric
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text | nucleosomes is a histone tetramer, comprised of one copy of each of histones H2A,
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text | H2B, CENP-A, and H4 – the so-called ‘hemisome’ model. Specifically, primary
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text | amine crosslinking of interphase chromatin from Drosophila S2 cells followed by
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text | immunoprecipitation with anti-CID (Drosophila CENP-A, Table 1) antibodies yielded
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text | a complex whose size was consistent with a tetramer containing one copy of each
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text | histone protein. In support of this model, atomic force microscopy (AFM)
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text | measurements of immunoprecipitated chromatin showed that the height of CID
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text | nucleosomes was roughly half that of nucleosomes within bulk chromatin (Dalal et al.,
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text | 2007). Similar AFM observations were recently made using chromatin prepared from
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text | asynchronous HeLa cells, leading the authors to propose that hemisomes are present
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text | within human centromeres as well (Dimitriadis et al., 2010). However, several groups
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text | have shown that when epitope-tagged CENP-A is expressed in cells and
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text | immunoprecipitated from extensively digested chromatin, it is associated with
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text | endogenous CENPA. This suggests that multiple CENP-A proteins, e.g. both epitope-
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text | tagged and endogenous, are present within the same nucleosome (Erhardt et al., 2008;
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text | Shelby et al., 1997; Xiao et al., 2011). At present, it is not clear whether these
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text | conflicting results reflect differences in experimental methodology, or whether the
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text | hemisome and the octasome represent real differences in centromeric nucleosome
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text | structure between species and/or cell cycle states. One limitation to the in vivo
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text | crosslinking approach used in Drosophila is that CID lacks lysine residues within the
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text | region predicted to hold the two tetrameric “halves” of the nucleosome together
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text | (which are present in H3) (Black and Bassett, 2008). This raises the possibility that
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text | hemisomes were recovered during immunoprecipitations because of inefficient
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text | crosslinking, which is consistent with the observation that CENP-A nucleosomes are
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text | more easily disassembled than H3 nucleosomes in vitro (Conde e Silva et al., 2007).
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text | But, immunoprecipitation data showing a ‘homotypic’ association between epitope-
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text | tagged and endogenous CENP-A must also be interpreted cautiously, as digested
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text | chromatin likely contained a mixed population of mononucleosomes and short
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text | nucleosome arrays (e.g. di- and tri-nucleosomes). Thus, it is possible that epitope-
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text | tagged CENP-A and endogenous CENP-A were present in adjacent nucleosomes
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text | rather than within the same nucleosome.
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text | Another controversial aspect of the hemisome model is that centromeric
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text | nucleosomes are wrapped by DNA in a right-handed manner (Furuyama, 2006). In
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text | vitro reconstitution of CID (Drosophila CENP-A) nucleosomes using the histone
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text | chaperone protein Rbap48 introduced positive supercoils into a plasmid, whereas
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text | reconstitution of H3 nucleosomes under the same conditions introduced negative
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text | supercoils. In addition, the effect of centromeric nucleosome assembly on plasmid
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text | topology in vivo was investigated using budding yeast. Similar to their in vitro results,
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text | the authors found that assembly of Cse4 (budding yeast CENP-A, Table 1)
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text | nucleosomes introduced positive supercoils into a plasmid. Given that conventional
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text | nucleosomes are wrapped by DNA in a left-handed manner, the authors asserted that
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text | right-handed DNA wrapping is structurally compatible with a tetrameric nucleosome
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text | but not with an octameric nucleosome (Furuyama, 2006). It is important to note that
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text | the authors used an unconventional nucleosome reconstitution method for their in vitro
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text | experiments, which can influence the structure of the reconstituted product (Furuyama
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text | and Henikoff, 2009). Furthermore, the mutations they used to manipulate Cse4
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text | nucleosome assembly in budding yeast experiments also disrupted assembly of other
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text | centromere proteins; thus, it is possible that plasmid topology differences were caused
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text | by the loss of Cse4-associated proteins rather than Cse4 itself (Hegemann et al., 1988;
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text | McGrew et al., 1986; Ortiz et al., 1999; Pearson et al., 2003). Thus, further
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text | experiments are needed to determine whether the ‘hemisome’ model is broadly
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text | relevant in vivo.
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text | In budding yeast, it has been proposed that the nonhistone protein Scm3
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text | replaces H2A and H2B at the centromere to form a unique hexameric nucleosome,
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text | with two copies each of Scm3, Cse4, and H4 (Mizuguchi et al., 2007; Xiao et al.,
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text | 2011). Scm3 is required for Cse4 localization to the centromere (Camahort et al.,
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text | 2007; Mizuguchi et al., 2007; Stoler et al., 2007), and its role in Cse4 nucleosome
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text | assembly will be discussed later (see “Scm3/HJURP: CENP-A chaperone”). The
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text | ‘hexameric nucleosome’ model stemmed from the observation that Scm3 localizes to
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text | centromeric DNA sequences throughout the cell cycle, whereas H3 and H2B are
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text | absent (Mizuguchi et al., 2007; Xiao et al., 2011). Recently, it was shown that
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text | centromeric DNA sequences are refractory to reconstitution of octameric Cse4
blank |
text | nucleosomes in vitro but support the assembly of Scm3-Cse4-H4-DNA complexes,
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text | presumed to contain two copies of each protein based on the electrophoretic mobility
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text | shift of the complex. Resistance to octameric nucleosome reconstitution was mediated
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text | by a specific centromere sequence element (CDEII, discussed below), as replacement
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text | of this element with an equivalent length of non-centromeric sequence significantly
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text | increased the efficiency of octameric nucleosome reconstitution. In conflict with the
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text | hexameric nucleosome model, one group detected an association between Cse4 and
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text | H2B but not between Cse4 and Scm3 within solubilized chromatin (Camahort et al.,
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text | 2009). Furthermore, overexpression of Cse4 rescued the lethality of an Scm3 deletion,
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text | indicating that an Scm3-Cse4-H4 nucleosome is not essential (Camahort et al., 2009).
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text | Interestingly, Xiao et al. reported that reconstitution of octameric Cse4 nucleosomes
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text | on centromeric DNA was more efficient at higher temperatures (e.g. 23°C or 37°C
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text | versus 4°C) (Xiao et al., 2011). Thus, it is possible that the structure of the budding
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text | yeast Cse4 nucleosome is flexible in vivo. Camahort et al. observed low levels of Cse4
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text | at non-centromeric loci (Camahort et al., 2009), which could be in the form of
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text | octameric nucleosomes. If so, it will be interesting to determine whether and how
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text | nucleosome structure influences kinetochore formation and removal of mis-
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text | incorporated Cse4 from non-centromeric loci.
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text | Further investigation of the structural differences between CENP-A and H3
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text | nucleosomes and how these differences influence chromatin structure and protein-
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text | nucleosome interactions will provide insight into how the centromere uniquely
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text | nucleates kinetochore formation. CENP-A nucleosomes have been shown to present
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text | novel sites for effector binding (Carroll et al., 2010) and may alter the physical
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text | properties of centromeric chromatin (Black et al., 2004; Sekulic et al., 2010), and both
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text | modes of chromatin regulation are likely to contribute to centromere function.
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|
|
title | Centromere DNA sequences
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text | The simplest and best characterized centromere is that of the budding yeast
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text | Saccharomyces cerevisiae. Budding yeast assemble a single Cse4-containing
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text | nucleosome at a specific 125 base pair ‘CEN’ DNA sequence (Furuyama and Biggins,
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text | 2007). CEN DNA is comprised of three sequence elements, termed CDEI, CDEII, and
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text | CDEIII. CDEI and CDEIII are conserved across all chromosomes and interact with
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text | sequence-specific DNA binding proteins that promote Cse4 nucleosome assembly and
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text | kinetochore formation. CDEII, which physically interacts with Cse4, is variable
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text | between chromosomes, but is of similar length (~80 base pairs) and AT base pair
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text | content (>90%) in all chromosomes (Cleveland et al., 2003; Fitzgerald-Hayes et al.,
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text | 1982). CEN DNA is necessary and sufficient for centromere formation (Gaudet and
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text | Fitzgerald-Hayes, 1987; Hegemann et al., 1988; Kingsbury and Koshland, 1991;
blank |
text | McGrew et al., 1986; Niedenthal et al., 1991; Sorger et al., 1994).
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text | Unlike the so-called ‘point centromeres’ of budding yeast, centromeres in
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text | fission yeast and most higher eukaryotes are comprised of many CENP-A
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text | nucleosomes spanning a long stretch of repetitive DNA, referred to as ‘regional’
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text | centromeres. Interestingly, both the length and the base pair composition of
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text | centromeric DNA sequences are extremely divergent between species. The simplest
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text | regional centromere, that of the fission yeast Saccharomyces pombe, spans 40-100 kb
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text | of DNA. Each centromere locus contains a 4-7 kb non-repetitive central sequence
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text | (cnt) flanked by centromere-specific innermost repeat sequences (imr), which together
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text | form the site for Cnp1 (fission yeast CENP-A, Table 1) nucleosome assembly. This
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text | central domain is flanked by long tandem arrays of outer repeat sequences (otr), which
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text | are heterochromatic (discussed further below) (reviewed in (Carroll and Straight,
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text | 2006)). In contrast, human centromeres are assembled on arrays of a tandemly
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text | repeated 171 base pair sequence element called α-satellite, ranging from 0.5 to 1.5
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text | Mbp in length (reviewed in (Carroll and Straight, 2006)). An additional feature of
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text | human centromeric DNA is a 17 base pair sequence element called the CENP-B box,
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text | which is found within many α-satellite repeats. The CENP-B box is bound in a
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text | sequence-dependent manner by the centromere protein CENP-B (Masumoto et al.,
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text | 1989). Regional centromeres occur at a single site on each chromosome that is
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