The ATAD2/ANCCA homolog Yta7 cooperates with Scm3HJURP to deposit Cse4CENP-A at the centromere in yeast

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP. The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.

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Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e12-12-0893 ◽

2013 ◽

Vol 24 (12) ◽

pp. 2034-2044 ◽

Cited By ~ 36

Author(s):

Lars Boeckmann ◽

Yoshimitsu Takahashi ◽

Wei-Chun Au ◽

Prashant K. Mishra ◽

John S. Choy ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Cell Biology ◽

Fluorescent Protein ◽

Functional Characterization ◽

Histone H3 ◽

In Vitro Assays ◽

Histone H3 Variant

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

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Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

Essays in Biochemistry ◽

10.1042/ebc20190080 ◽

2020 ◽

Vol 64 (2) ◽

pp. 251-261

Author(s):

Jessica E. Fellmeth ◽

Kim S. McKim

Keyword(s):

Chromosome Segregation ◽

New Technologies ◽

Early Prophase ◽

Unique Role ◽

Kinetochore Assembly ◽

M Phase ◽

In Vivo Analysis ◽

Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0426 ◽

2007 ◽

Vol 18 (1) ◽

pp. 129-141 ◽

Cited By ~ 53

Author(s):

Yasunari Takami ◽

Tatsuya Ono ◽

Tatsuo Fukagawa ◽

Kei-ichi Shibahara ◽

Tatsuo Nakayama

Keyword(s):

Dna Replication ◽

Essential Role ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

10.1101/176206 ◽

2017 ◽

Cited By ~ 1

Author(s):

Kentaro Ohkuni ◽

Reuben Levy-Myers ◽

Jack Warren ◽

Wei-Chun Au ◽

Yoshimitsu Takahashi ◽

...

Keyword(s):

Genome Stability ◽

Histone H3 ◽

Physiological Role ◽

Centromeric Chromatin ◽

E3 Ligases ◽

Physiological Conditions ◽

Evolutionarily Conserved ◽

Histone H3 Variant

AbstractStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

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CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

10.1101/836262 ◽

2019 ◽

Author(s):

N Cortes-Silva ◽

J Ulmer ◽

T Kiuchi ◽

E Hsieh ◽

G Cornilleau ◽

...

Keyword(s):

Chromosome Segregation ◽

Gene Editing ◽

Mitotic Progression ◽

Critical Function ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Essential Function ◽

Necessary And Sufficient ◽

Assembly Pathways

AbstractAccurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

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CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

The Journal of Cell Biology ◽

10.1083/jcb.201106078 ◽

2011 ◽

Vol 195 (4) ◽

pp. 563-572 ◽

Cited By ~ 100

Author(s):

Valerie C. Coffman ◽

Pengcheng Wu ◽

Mark R. Parthun ◽

Jian-Qiu Wu

Keyword(s):

Saccharomyces Cerevisiae ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Budding Yeast ◽

Histone H3 ◽

Kinetochore Assembly ◽

Microtubule Attachment ◽

Attachment Sites ◽

Histone H3 Variant ◽

Architectural Models

The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ∼40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1–DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.

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Epigenetic inheritance of centromere identity in a heterologous system

10.1101/560391 ◽

2019 ◽

Cited By ~ 2

Author(s):

Virginie Roure ◽

Bethan Medina-Pritchard ◽

Eduard Anselm ◽

A. Arockia Jeyaprakash ◽

Patrick Heun

Keyword(s):

Chromosome Segregation ◽

Chromosomal Region ◽

Histone H3 ◽

Epigenetic Inheritance ◽

Centromeric Chromatin ◽

Centromere Proteins ◽

Heterologous System ◽

Histone H3 Variant ◽

Self Association

SUMMARYThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

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CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

The Journal of Cell Biology ◽

10.1083/jcb.201106079 ◽

2011 ◽

Vol 194 (6) ◽

pp. 855-871 ◽

Cited By ~ 134

Author(s):

Ben Moree ◽

Corey B. Meyer ◽

Colin J. Fuller ◽

Aaron F. Straight

Keyword(s):

Chromosome Segregation ◽

Protein A ◽

Histone H3 ◽

Chromatin Assembly ◽

Nucleosome Assembly ◽

Chromosomal Locus ◽

C Protein ◽

Centromere Protein A ◽

Histone H3 Variant ◽

Complex Protein

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

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The role of heterochromatin in centromere function

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2004.1611 ◽

2005 ◽

Vol 360 (1455) ◽

pp. 569-579 ◽

Cited By ~ 100

Author(s):

Alison L Pidoux ◽

Robin C Allshire

Keyword(s):

Fission Yeast ◽

Histone H3 ◽

Centromeric Heterochromatin ◽

Chromatin Domain ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Centromere Function ◽

Kinetochore Assembly ◽

Histone H3 Variant

Chromatin at centromeres is distinct from the chromatin in which the remainder of the genome is assembled. Two features consistently distinguish centromeres: the presence of the histone H3 variant CENP-A and, in most organisms, the presence of heterochromatin. In fission yeast, domains of silent ‘heterochromatin’ flank the CENP-A chromatin domain that forms a platform upon which the kinetochore is assembled. Thus, fission yeast centromeres resemble their metazoan counterparts where the kinetochore is embedded in centromeric heterochromatin. The centromeric outer repeat chromatin is underacetylated on histones H3 and H4, and methylated on lysine 9 of histone H3, which provides a binding site for the chromodomain protein Swi6 (orthologue of Heterochromatin Protein 1, HP1). The remarkable demonstration that the assembly of repressive heterochromatin is dependent on the RNA interference machinery provokes many questions about the mechanisms of this process that may be tractable in fission yeast. Heterochromatin ensures that a high density of cohesin is recruited to centromeric regions, but it could have additional roles in centromere architecture and the prevention of merotely, and it might also act as a trigger for kinetochore assembly. In addition, we discuss an epigenetic model for ensuring that CENP-A is targeted and replenished at the kinetochore domain.

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The N-terminal Tail of C. elegans CENP-A Interacts with KNL-2 and is Essential for Centromeric Chromatin Assembly

10.1101/2020.12.28.424576 ◽

2020 ◽

Author(s):

Christian de Groot ◽

Jack Houston ◽

Bethany Davis ◽

Adina Gerson-Gurwitz ◽

Joost Monen ◽

...

Keyword(s):

Histone H3 ◽

Direct Interaction ◽

Chromatin Assembly ◽

Nucleosome Assembly ◽

Centromeric Chromatin ◽

C Elegans ◽

Histone Fold ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Evolutionary Variation

ABSTRACTCentromeres are epigenetically defined by the presence of the centromere-specific histone H3 variant CENP-A. A specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, which rely on a CENP-A-dependent centromere. Here, we show that the extended N-terminal tail of C. elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly. Removal of this region of the CENP-A N-Tail prevents loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-Tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-Tail containing the predicted structured region binds to KNL-2, a conserved SANTA and Myb domain-containing protein (referred to as M18BP1 in vertebrates), that is specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode C. briggsae, despite divergence of the N-Tail and KNL-2 primary sequences. Thus, the extended N-Tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates an interaction of the specialized histone fold of CENP-A with KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.

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