MHF1–2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination

The histone-fold proteins Mhf1/CENP-S and Mhf2/CENP-X perform two important functions in vertebrate cells. First, they are components of the constitutive centromere-associated network, aiding kinetochore assembly and function. Second, they work with the FANCM DNA translocase to promote DNA repair. However, it has been unclear whether there is crosstalk between these roles. We show that Mhf1 and Mhf2 in fission yeast, as in vertebrates, serve a dual function, aiding DNA repair/recombination and localizing to centromeres to promote chromosome segregation. Importantly, these functions are distinct, with the former being dependent on their interaction with the FANCM orthologue Fml1 and the latter not. Together with Fml1, they play a second role in aiding chromosome segregation by processing sister chromatid junctions. However, a failure of this activity does not manifest dramatically increased levels of chromosome missegregation due to the Mus81–Eme1 endonuclease, which acts as a failsafe to resolve DNA junctions before the end of mitosis.

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Emerging roles for centromere-associated proteins in DNA repair and genetic recombination

Biochemical Society Transactions ◽

10.1042/bst20130200 ◽

2013 ◽

Vol 41 (6) ◽

pp. 1726-1730 ◽

Cited By ~ 9

Author(s):

Fekret Osman ◽

Matthew C. Whitby

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Genetic Recombination ◽

Current Knowledge ◽

Complementation Group ◽

Histone Fold ◽

Centromere Proteins ◽

Associated Proteins ◽

And Function ◽

Dna Translocase

Centromere proteins CENP-S and CENP-X are members of the constitutive centromere-associated network, which is a conserved group of proteins that are needed for the assembly and function of kinetochores at centromeres. Intriguingly CENP-S and CENP-X have alter egos going by the names of MHF1 (FANCM-associated histone-fold protein 1) and MHF2 respectively. In this guise they function with a DNA translocase called FANCM (Fanconi’s anemia complementation group M) to promote DNA repair and homologous recombination. In the present review we discuss current knowledge of the biological roles of CENP-S and CENP-X and how their dual existence may be a common feature of CCAN (constitutive centromere-associated network) proteins.

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How Does SUMO Participate in Spindle Organization?

Cells ◽

10.3390/cells8080801 ◽

2019 ◽

Vol 8 (8) ◽

pp. 801

Author(s):

Abrieu ◽

Liakopoulos

Keyword(s):

Dna Repair ◽

Protein Assemblies ◽

Cellular Mechanisms ◽

Future Directions ◽

Kinetochore Assembly ◽

Associated Proteins ◽

Long Time ◽

And Function ◽

Dependent Processes

The ubiquitin-like protein SUMO is a regulator involved in most cellular mechanisms. Recent studies have discovered new modes of function for this protein. Of particular interest is the ability of SUMO to organize proteins in larger assemblies, as well as the role of SUMO-dependent ubiquitylation in their disassembly. These mechanisms have been largely described in the context of DNA repair, transcriptional regulation, or signaling, while much less is known on how SUMO facilitates organization of microtubule-dependent processes during mitosis. Remarkably however, SUMO has been known for a long time to modify kinetochore proteins, while more recently, extensive proteomic screens have identified a large number of microtubule- and spindle-associated proteins that are SUMOylated. The aim of this review is to focus on the possible role of SUMOylation in organization of the spindle and kinetochore complexes. We summarize mitotic and microtubule/spindle-associated proteins that have been identified as SUMO conjugates and present examples regarding their regulation by SUMO. Moreover, we discuss the possible contribution of SUMOylation in organization of larger protein assemblies on the spindle, as well as the role of SUMO-targeted ubiquitylation in control of kinetochore assembly and function. Finally, we propose future directions regarding the study of SUMOylation in regulation of spindle organization and examine the potential of SUMO and SUMO-mediated degradation as target for antimitotic-based therapies.

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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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CBIO-16. SUPPRESSION OF HISTONE H3.3 MITOTIC PHOSPHORYLATION DRIVES DEVELOPMENT OF H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.076 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii18-ii19

Author(s):

Charles Day ◽

Alyssa Langfald ◽

Florina Grigore ◽

Leslie Sepaniac ◽

Jason Stumpff ◽

...

Keyword(s):

Chromosome Segregation ◽

Treatment Options ◽

Chromosome Instability ◽

Pericentromeric Heterochromatin ◽

Low Grade ◽

High Grade ◽

Chromosome Missegregation ◽

Mitotic Phosphorylation ◽

Chk1 Kinase

Abstract Pediatric midline gliomas – including DIPG – are lethal brain tumors in children, with poor prognosis and limited treatment options that provide only short-term benefits. The majority have a lysine-to-methionine substitution at residue 27 (H3K27M) in genes expressing histone H3 – predominantly in the H3.3 variant. This causes a global reduction in H3 Lys27 tri-methylation (H3K27Me3), comprehensive epigenetic reprogramming, and is a key driver in gliomagenesis. We show that the H3.3K27M mutation also induces chromosome segregation defects, which in high-grade tumors, results in extensive copy number alterations (CNAs). Ser31 is one of five amino acid substitutions differentiating H3.3 from canonical H3.1. Mitotic phosphorylation of H3.3 Ser31 by Chk1 kinase is restricted to pericentromeric heterochromatin, where it plays a role in chromosome segregation. We show that the K27M mutation affects neighboring Ser31 phosphorylation and pericentromeric heterochromatin organization. We demonstrate that (i) H3.3 K27M protein is defective for Ser31 phosphorylation by Chk1 kinase in vitro; (ii) DIPG cell lines have significantly decreased mitotic Ser31 phosphorylation, and are chromosomally unstable; and (iii) CRISPR-reversion of H3.3K27M to Lys27 restores phospho-Ser31 (and Lys27 tri-methylation) and significantly decreases chromosome instability. Expression of H3.3K27M or non-phosphorylatable H3.3S31A mutants in WT cells results in chromosome missegregation; this is suppressed by co-expression of phospho-mimetic H3.3K27M/S31E. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. Finally, in a novel mouse model of glioma, mean survival of mice with tumors induced with H3.3K27M and H3.3S31A was 81 and 68 days: 100% of H3.3S31A mice developed high-grade tumors. H3.3 WT controls developed only low-grade tumors and all survived 100 days. H3.3S31A is WT for Lys27 tri-methylation and thus, loss of Ser31 phosphorylation alone is oncogenic.

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Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

The Journal of Cell Biology ◽

10.1083/jcb.200706015 ◽

2007 ◽

Vol 179 (2) ◽

pp. 255-267 ◽

Cited By ~ 147

Author(s):

Karthik Jeganathan ◽

Liviu Malureanu ◽

Darren J. Baker ◽

Susan C. Abraham ◽

Jan M. van Deursen

Keyword(s):

Cell Death ◽

Chromosome Segregation ◽

Physiological Role ◽

Mitotic Checkpoint ◽

Critical Threshold ◽

Wild Type ◽

Checkpoint Protein ◽

Chromosome Missegregation ◽

Mitotic Checkpoint Protein

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

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The structure of the yeast Ctf3 complex

eLife ◽

10.7554/elife.48215 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Stephen M Hinshaw ◽

Andrew N Dates ◽

Stephen C Harrison

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

High Resolution ◽

Atomic Model ◽

Kinetochore Assembly ◽

Cryo Electron Microscopy ◽

Molecular Interpretation ◽

Spindle Microtubules ◽

And Function ◽

Assembly Site

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

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Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.201909136 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 2

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Mitotic Chromosome ◽

Budding Yeast ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

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Heterochromatin: Guardian of the Genome

Annual Review of Cell and Developmental Biology ◽

10.1146/annurev-cellbio-100617-062653 ◽

2018 ◽

Vol 34 (1) ◽

pp. 265-288 ◽

Cited By ~ 100

Author(s):

Aniek Janssen ◽

Serafin U. Colmenares ◽

Gary H. Karpen

Keyword(s):

Chromosome Segregation ◽

Genome Stability ◽

Constitutive Heterochromatin ◽

Repetitive Sequences ◽

Genome Integrity ◽

Chromatin Domain ◽

Cellular Processes ◽

Eukaryotic Nucleus ◽

And Function ◽

Heterochromatin Structure

Constitutive heterochromatin is a major component of the eukaryotic nucleus and is essential for the maintenance of genome stability. Highly concentrated at pericentromeric and telomeric domains, heterochromatin is riddled with repetitive sequences and has evolved specific ways to compartmentalize, silence, and repair repeats. The delicate balance between heterochromatin epigenetic maintenance and cellular processes such as mitosis and DNA repair and replication reveals a highly dynamic and plastic chromatin domain that can be perturbed by multiple mechanisms, with far-reaching consequences for genome integrity. Indeed, heterochromatin dysfunction provokes genetic turmoil by inducing aberrant repeat repair, chromosome segregation errors, transposon activation, and replication stress and is strongly implicated in aging and tumorigenesis. Here, we summarize the general principles of heterochromatin structure and function, discuss the importance of its maintenance for genome integrity, and propose that more comprehensive analyses of heterochromatin roles in tumorigenesis will be integral to future innovations in cancer treatment.

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Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

The Journal of Cell Biology ◽

10.1083/jcb.201210091 ◽

2012 ◽

Vol 200 (1) ◽

pp. 21-30 ◽

Cited By ~ 49

Author(s):

Fabienne Lampert ◽

Christine Mieck ◽

Gregory M. Alushin ◽

Eva Nogales ◽

Stefan Westermann

Keyword(s):

Chromosome Segregation ◽

Protein Complexes ◽

Structural Elements ◽

Large Protein ◽

Kinetochore Assembly ◽

Sister Chromatids ◽

Dam1 Complex ◽

Shed Light ◽

Kinetochore Function

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

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