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 The ratio between centromeric proteins CENP-A and CENP-C maintains homeostasis of human centromeres

2019 ◽  
Author(s):  
Daniël P. Melters ◽  
Tatini Rakshit ◽  
Minh Bui ◽  
Sergei A. Grigoryev ◽  
David Sturgill ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
De Novo ◽  
Histone H3 ◽  
Chromosomal Locus ◽  
Centromeric Chromatin ◽  
Dna Accessibility ◽  
Nucleosome Dynamics ◽  
Histone H3 Variant ◽  
Correct Ratio

AbstractThe centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

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 Centromeric DNA destabilizes H3 nucleosomes to promote CENP-A deposition during the cell cycle

2017 ◽  
Author(s):  
Manu Shukla ◽  
Tong Pin ◽  
Sharon A. White ◽  
Puneet P. Singh ◽  
Angus M. Reid ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Chromosomal Location ◽  
Nucleosome Occupancy ◽  
Histone H3 ◽  
S Phase ◽  
Intrinsic Properties ◽  
Centromeric Dna ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Specific Sequences

SummaryActive centromeres are defined by the presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and kinetochores are maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A itself promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences. Intrinsic properties of centromeric DNA may favour the assembly of CENP-A rather than H3 nucleosomes. Here we investigate histone dynamics on centromeric DNA. We show that during S-phase histone H3 is deposited as a placeholder at fission yeast centromeres and is subsequently evicted in G2 when we detect deposition of the majority of new CENP-ACnp1. We also find that centromeric DNA has an innate property of driving high rates of turnover of H3 containing nucleosomes resulting in low nucleosome occupancy. When placed at an ectopic chromosomal location in the absence of any CENP-ACnp1 assembly, centromeric DNA retains its ability to impose S-phase deposition and G2 eviction of H3, suggesting that features within this DNA program H3 dynamics. As RNAPII occupancy on this centromere DNA coincides with H3 eviction in G2, we propose a model in which RNAPII-coupled chromatin remodelling promotes replacement of H3 with CENP-ACnp1 nucleosomes.

scholarly journals Epigenetic inheritance of centromere identity in a heterologous system

2019 ◽  
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.

scholarly journals Trans-generational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

2020 ◽  
Author(s):  
Reinier F. Prosée ◽  
Joanna M. Wenda ◽  
Caroline Gabus ◽  
Kamila Delaney ◽  
Francoise Schwager ◽  
...  
Keyword(s):  
De Novo ◽  
Germ Line ◽  
Protein A ◽  
Histone H3 ◽  
Centromeric Chromatin ◽  
C Elegans ◽  
Centromere Function ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Meiosis I

AbstractCentromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is established de novo on chromatin during diplotene of meiosis I. Here we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but dispensable for centromere maintenance during embryogenesis. Worms homozygous for a CENP-A tail deletion maintain a functional centromere during development, but give rise to inviable offspring because they fail to re-establish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2, and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

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

2020 ◽  
Vol 117 (10) ◽  
pp. 5386-5393 ◽  
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.

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 Hap2-Ino80 facilitated transcription promotes de novo establishment of CENP-A chromatin

2019 ◽  
Author(s):  
Puneet P. Singh ◽  
Manu Shukla ◽  
Sharon A. White ◽  
Pin Tong ◽  
Tatsiana Auchynnikava ◽  
...  
Keyword(s):  
Fission Yeast ◽  
De Novo ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Centromere Function ◽  
Kinetochore Assembly ◽  
Auxiliary Subunit ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant ◽  
Chromatin Integrity

SUMMARYCentromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily-conserved histone H3 variant, which directs kinetochore assembly and hence, centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity selected solubilised fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilises H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.

scholarly journals Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

PLoS Biology ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. e3000968
Author(s):  
Reinier F. Prosée ◽  
Joanna M. Wenda ◽  
Isa Özdemir ◽  
Caroline Gabus ◽  
Kamila Delaney ◽  
...  
Keyword(s):  
De Novo ◽  
Germ Line ◽  
Protein A ◽  
Histone H3 ◽  
Transgenerational Inheritance ◽  
Centromeric Chromatin ◽  
C Elegans ◽  
Centromere Function ◽  
Centromere Protein A ◽  
Histone H3 Variant

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

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

2021 ◽  
Vol 118 (48) ◽  
pp. e2104459118
Author(s):  
Qianhua Dong ◽  
Xue-lei Liu ◽  
Xiao-hui Wang ◽  
Yu Zhao ◽  
Yu-hang Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Competitive Exclusion ◽  
Histone H3 ◽  
Cyclin Dependent Kinase ◽  
Null Mutant ◽  
Kinetochore Assembly ◽  
N Terminus ◽  
Ndc80 Complex ◽  
Histone H3 Variant

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.

Sign in / Sign up

Export Citation Format

Share Document