scholarly journals Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A

2001 ◽  
Vol 114 (19) ◽  
pp. 3529-3542 ◽  
Author(s):  
Aaron A. Van Hooser ◽  
Ilia I. Ouspenski ◽  
Heather C. Gregson ◽  
Daniel A. Starr ◽  
Tim J. Yen ◽  
...  
Keyword(s):  
Dna Binding Protein ◽  
Centromeric Heterochromatin ◽  
Additional Mechanism ◽  
Centromeric Chromatin ◽  
Microtubule Associated Proteins ◽  
Kinetochore Assembly ◽  
Double Minute ◽  
Histone H3 Variant ◽  
Associated Proteins ◽  
Double Minute Chromosomes

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

scholarly journals Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin

2007 ◽  
Vol 176 (6) ◽  
pp. 757-763 ◽  
Author(s):  
Paul S. Maddox ◽  
Francie Hyndman ◽  
Joost Monen ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Functional Genomics ◽  
Protein A ◽  
Common Mechanism ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Close Proximity ◽  
Single Protein ◽  
Histone H3 Variant ◽  
New Protein

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

scholarly journals Centromere repositioning induced by inner kinetochore impairment generates a meiosis barrier

2018 ◽  
Author(s):  
Min Lu ◽  
Xiangwei He
Keyword(s):  
Meiotic Chromosome ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Eukaryotic Species ◽  
Histone H3 Variant ◽  
Single Deletion ◽  
Centromere Inactivation ◽  
Evolutionary New Centromeres ◽  
Two Populations

AbstractCentromeres dictate the sites for kinetochore assembly on chromosomes, while their own position on each chromosome is determined epigenetically by a specific histone H3 variant CENP-A. For all eukaryotic species, the chromosomal position of each centromere is distinct and inherited with high fidelity, although the mechanisms underlying the epigenetic stability and its functional significance remain largely unknown. Here in the fission yeast Schizosaccharomyces pombe, we show that mutations in inner kinetochore components influence centromeric chromatin organization to various levels. In extreme cases, a single deletion of wip1, mhf1 and mhf2 (the conserved CENP-T-W-S-X complex subunits) or double deletions of cnp3 (a homologue of mammalian CENP-C) and fta6 (a pombe specific component) induce centromere repositioning - inactivation of the original centromere and formation of a neocentromere - in one of the three chromosomes at random. Neocentromeres tend to locate in pericentromeric heterochromatin regions, although heterochromatin is not required for centromere inactivation. Cells carrying a neocentromere are competent in mitosis and in meiosis of homozygotes. However, when these cells are crossed to cells carrying the original centromere, the progeny suffers severe lethality due to defects in meiotic chromosome segregation. These results recapitulate a meiosis barrier that could initiate genetic divergence between two populations with mismatched centromeres, documenting a potential role of the Evolutionary New Centromeres (ENCs) in speciation.Significance StatementIn eukaryotes, centromeres are chromosomal regions where kinetochores are assembled and the positions of centromeres are accurately inherited. While the centromere and kinetochore assembly are extensively studied, the mechanisms that each centromere maintain its identity on chromosomes are still not well understood. In this study, we demonstrated that the inner kinetochore is required for the normal centromere identity as single depletion of the inner kinetochore CENP-T-W-S-X complex or double deletions of cnp3/CENP-C and fta6 induce centromere repositioning. We further showed cells carrying a neocentromere are reproductively isolated from the wildtype population carrying the original centromere. Taken together, these results suggest that induced centromere repositioning mimics the evolutionary new centromeres and is sufficient to cause reproductive isolation.

scholarly journals The role of heterochromatin in centromere function

2005 ◽  
Vol 360 (1455) ◽  
pp. 569-579 ◽  
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.

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

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.

scholarly journals Assembly principles and stoichiometry of a complete human kinetochore module

2020 ◽  
Author(s):  
Kai Walstein ◽  
Arsen Petrovic ◽  
Dongqing Pan ◽  
Birte Hagemeier ◽  
Dorothee Vogt ◽  
...  
Keyword(s):  
Cell Division ◽  
Full Length ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Binding Interface ◽  
Histone H3 Variant ◽  
Multiple Copies ◽  
Assembly Principles ◽  
Chromosomal Loci

Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes, and permits further assembly of all other kinetochore subunits in vitro with relative ratios that closely match those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module, and may have important implications for trans-generational inheritance of centromeric chromatin.

scholarly journals Centromeric chromatin gets loaded

2007 ◽  
Vol 176 (6) ◽  
pp. 735-736 ◽  
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.

scholarly journals Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex

2019 ◽  
Author(s):  
Engin Demirdizen ◽  
Matthias Spiller-Becker ◽  
Arion Förtsch ◽  
Alexander Wilhelm ◽  
Samuel Corless ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cultured Cells ◽  
Mass Spectrometry Analysis ◽  
Nurd Complex ◽  
Centromeric Chromatin ◽  
Spectrometry Analysis ◽  
Centromere Function ◽  
Histone H3 Variant ◽  
Associated Proteins ◽  
Specific Association

Abstract Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

scholarly journals Sim4

2003 ◽  
Vol 161 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Alison L. Pidoux ◽  
William Richardson ◽  
Robin C. Allshire
Keyword(s):  
Chromatin Structure ◽  
Coiled Coil ◽  
Central Core ◽  
Centromeric Heterochromatin ◽  
Kinetochore Assembly ◽  
Powerful Approach ◽  
Histone H3 Variant ◽  
Specific Proteins ◽  
Domain Functional ◽  
Functional Kinetochore

Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

scholarly journals De Novo Kinetochore Assembly Requires the Centromeric Histone H3 Variant

2005 ◽  
Vol 16 (12) ◽  
pp. 5649-5660 ◽  
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.

scholarly journals A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

2014 ◽  
Vol 206 (4) ◽  
pp. 509-524 ◽  
Author(s):  
Peter Hornung ◽  
Paulina Troc ◽  
Francesca Malvezzi ◽  
Michael Maier ◽  
Zuzana Demianova ◽  
...  
Keyword(s):  
Protein C ◽  
Protein Complexes ◽  
Point Mutations ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
N Terminus ◽  
Associated Proteins ◽  
Rate Limiting ◽  
Step Mechanism ◽  
Kinetochore Function

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1CENP-U and Okp1CENP-Q form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.

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