Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

Download Full-text

The structure of the Ctf19c/CCAN from budding yeast

eLife ◽

10.7554/elife.44239 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 36

Author(s):

Stephen M Hinshaw ◽

Stephen C Harrison

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

Budding Yeast ◽

Histone H3 ◽

Kinetochore Assembly ◽

Microtubule Attachment ◽

Cryo Electron Microscopy ◽

Histone H3 Variant

Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917814117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5386-5393 ◽

Cited By ~ 2

Author(s):

Sara Shahnejat-Bushehri ◽

Ann E. Ehrenhofer-Murray

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Histone H3 ◽

Nucleosome Assembly ◽

Direct Role ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Assembly Factor

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.

Download Full-text

CDK-regulated dimerization of M18BP1 on a Mis18 hexamer is necessary for CENP-A loading

eLife ◽

10.7554/elife.23352 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 23

Author(s):

Dongqing Pan ◽

Kerstin Klare ◽

Arsen Petrovic ◽

Annika Take ◽

Kai Walstein ◽

...

Keyword(s):

Cell Cycle ◽

Histone H3 ◽

G1 Phase ◽

Cyclin Dependent Kinase ◽

2A Peptide ◽

Macromolecular Complexes ◽

Histone H3 Variant ◽

Genetic Features ◽

Spindle Microtubules ◽

Chromosomal Loci

Centromeres are unique chromosomal loci that promote the assembly of kinetochores, macromolecular complexes that bind spindle microtubules during mitosis. In most organisms, centromeres lack defined genetic features. Rather, they are specified epigenetically by a centromere-specific histone H3 variant, CENP-A. The Mis18 complex, comprising the Mis18α:Mis18β subcomplex and M18BP1, is crucial for CENP-A homeostasis. It recruits the CENP-A-specific chaperone HJURP to centromeres and primes it for CENP-A loading. We report here that a specific arrangement of Yippee domains in a human Mis18α:Mis18β 4:2 hexamer binds two copies of M18BP1 through M18BP1’s 140 N-terminal residues. Phosphorylation by Cyclin-dependent kinase 1 (CDK1) at two conserved sites in this region destabilizes binding to Mis18α:Mis18β, limiting complex formation to the G1 phase of the cell cycle. Using an improved viral 2A peptide co-expression strategy, we demonstrate that CDK1 controls Mis18 complex recruitment to centromeres by regulating oligomerization of M18BP1 through the Mis18α:Mis18β scaffold.

Download Full-text

Centromeric chromatin gets loaded

The Journal of Cell Biology ◽

10.1083/jcb.200702020 ◽

2007 ◽

Vol 176 (6) ◽

pp. 735-736 ◽

Cited By ~ 3

Author(s):

Christopher W. Carroll ◽

Aaron F. Straight

Keyword(s):

Chromosome Segregation ◽

Molecular Mechanisms ◽

Protein A ◽

Histone H3 ◽

Chromatin Assembly ◽

Centromeric Chromatin ◽

Kinetochore Assembly ◽

Centromere Protein A ◽

Specific Loading ◽

Histone H3 Variant

Centromeric nucleosomes contain a histone H3 variant called centromere protein A (CENP-A) that is required for kinetochore assembly and chromosome segregation. Two new studies, Jansen et al. (see p. 795 of this issue) and Maddox et al. (see p. 757 of this issue), address when CENP-A is deposited at centromeres during the cell division cycle and identify an evolutionally conserved protein required for CENP-A deposition. Together, these studies advance our understanding of centromeric chromatin assembly and provide a framework for investigating the molecular mechanisms that underlie the centromere-specific loading of CENP-A.

Download Full-text

CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization

10.1101/355487 ◽

2018 ◽

Cited By ~ 1

Author(s):

Bradley T. French ◽

Aaron F. Straight

Keyword(s):

Cell Cycle ◽

Xenopus Laevis ◽

Chromosome Segregation ◽

Nuclear Localization ◽

Mitotic Spindle ◽

Histone H3 ◽

Nucleosome Assembly ◽

Histone H3 Variant ◽

Cdk Phosphorylation

AbstractChromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18α, Mis18β, M18BP1). Here we show that Xenopus M18BP1 localizes to centromeres during metaphase - prior to CENP-A assembly - by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase.

Download Full-text

De Novo Kinetochore Assembly Requires the Centromeric Histone H3 Variant

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0771 ◽

2005 ◽

Vol 16 (12) ◽

pp. 5649-5660 ◽

Cited By ~ 54

Author(s):

Kimberly A. Collins ◽

Andrea R. Castillo ◽

Sean Y. Tatsutani ◽

Sue Biggins

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

De Novo ◽

Histone H3 ◽

Centromeric Chromatin ◽

Centromeric Dna ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Chromosome Attachment ◽

Dna Specificity

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.

Download Full-text

An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

10.1101/381897 ◽

2018 ◽

Author(s):

Jackie Lang ◽

Adrienne Barber ◽

Sue Biggins

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

De Novo ◽

Budding Yeast ◽

Centromeric Dna ◽

Kinetochore Assembly ◽

Ndc80 Complex ◽

Spindle Microtubules ◽

Underlying Mechanisms ◽

Mitotic Phosphorylation

ABSTRACTChromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

Download Full-text

An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

eLife ◽

10.7554/elife.37819 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 25

Author(s):

Jackie Lang ◽

Adrienne Barber ◽

Sue Biggins

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

De Novo ◽

Budding Yeast ◽

Centromeric Dna ◽

Kinetochore Assembly ◽

Ndc80 Complex ◽

Spindle Microtubules ◽

Underlying Mechanisms ◽

Mitotic Phosphorylation

Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

Download Full-text

CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state

The Journal of Cell Biology ◽

10.1083/jcb.201301006 ◽

2013 ◽

Vol 201 (1) ◽

pp. 23-32 ◽

Cited By ~ 56

Author(s):

Karen E. Gascoigne ◽

Iain M. Cheeseman

Keyword(s):

Chromosome Segregation ◽

Mitotic Exit ◽

Cyclin Dependent Kinase ◽

Temporal Regulation ◽

Regulatory Processes ◽

Kinetochore Assembly ◽

Nuclear Exclusion ◽

Ndc80 Complex ◽

Mitotic Phosphorylation

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex. Prior work has identified more than 100 different kinetochore components in human cells. However, little is known about the regulatory processes that specify their assembly upon mitotic entry and disassembly at mitotic exit. In this paper, we used a live-cell imaging–based assay to quantify kinetochore disassembly kinetics and systematically analyze the role of potential regulatory mechanisms in controlling kinetochore assembly state. We find that kinetochore assembly and disassembly was driven primarily by mitotic phosphorylation downstream of cyclin-dependent kinase (CDK). In addition, we demonstrate that nuclear exclusion of the Ndc80 complex helped restrict kinetochore formation to mitosis. Combining constitutive CDK-dependent phosphorylation of CENP-T and forced nuclear localization of the Ndc80 complex partially prevented kinetochore disassembly at mitotic exit and led to chromosome segregation defects in subsequent divisions. In total, we find that the coordinated temporal regulation of outer kinetochore assembly is essential for accurate cell division.

Download Full-text

The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽

10.1093/genetics/165.2.489 ◽

2003 ◽

Vol 165 (2) ◽

pp. 489-503 ◽

Cited By ~ 1

Author(s):

Karen E Ross ◽

Orna Cohen-Fix

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Chromosome Loss ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Growth Defects ◽

Genes Encoding ◽

Specificity Factor

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

Download Full-text