The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID

Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.

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

10.1101/215624 ◽

2017 ◽

Cited By ~ 1

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.

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Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres

eLife ◽

10.7554/elife.02203 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 59

Author(s):

Jan Wisniewski ◽

Bassam Hajj ◽

Jiji Chen ◽

Gaku Mizuguchi ◽

Hua Xiao ◽

...

Keyword(s):

Cell Cycle ◽

Elevated Temperatures ◽

De Novo ◽

Histone H3 ◽

S Phase ◽

Nuclear Accumulation ◽

Histone H3 Variant ◽

Functionally Impaired ◽

Cell Cycle Dynamics ◽

Cell Lethality

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.

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Ser68 phosphoregulation is essential for CENP-A deposition, centromere function and viability in mice

10.1101/2021.11.23.469796 ◽

2021 ◽

Author(s):

Yuting Liu ◽

Kehui Wang ◽

Li Huang ◽

Jicheng Zhao ◽

Xinpeng Chen ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Histone H3 ◽

Dependent Manner ◽

Centromere Function ◽

Histone H3 Variant ◽

A Cell ◽

Cycle Dependent ◽

Spatiotemporal Control

Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.

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How Not To Be in the Wrong Place at the Wrong Time: An Education Primer for Use with “Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation”

Genetics ◽

10.1534/genetics.120.303493 ◽

2020 ◽

Vol 216 (2) ◽

pp. 333-342

Author(s):

Yee Mon Thu

Keyword(s):

Molecular Mechanisms ◽

Genetic Diseases ◽

Histone H3 ◽

Model Organism ◽

Post Translational Modification ◽

Genetic Changes ◽

Equal Distribution ◽

Link Type ◽

Histone H3 Variant ◽

Big Picture

Recent work by Kentaro Ohkuni and colleagues exemplifies how a series of molecular mechanisms contribute to a cellular outcome—equal distribution of chromosomes. Failure to maintain structural and numerical integrity of chromosomes is one contributing factor in genetic diseases such as cancer. Specifically, the authors investigated molecular events surrounding centromeric histone H3 variant Cse4 deposition—a process important for chromosome segregation, using Saccharomyces cerevisiae as a model organism. This study illustrates an example of a post-translational modification—sumoylation—regulating a cellular process and the concept of genetic interactions (e.g., synthetic dosage lethality). Furthermore, the study highlights the importance of using diverse experimental approaches in answering a few key research questions. The authors used molecular biology techniques (e.g., qPCR), biochemical experiments (e.g., Ni-NTA/8His protein purification), as well as genetic approaches to understand the regulation of Cse4. At a big-picture level, the study reveals how genetic changes can lead to subsequent molecular and cellular changes.

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Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Open Biology ◽

10.1098/rsob.150236 ◽

2016 ◽

Vol 6 (2) ◽

pp. 150236 ◽

Cited By ~ 24

Author(s):

Yahui Liu ◽

Arsen Petrovic ◽

Pascaline Rombaut ◽

Shyamal Mosalaganti ◽

Jenny Keller ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Chromosome Segregation ◽

Functional Organization ◽

Histone H3 Variant ◽

Centromeric Protein ◽

Spindle Microtubules ◽

Specialized Region ◽

Structural Methods ◽

Mitosis And Meiosis ◽

Shed Light

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.

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CAL1 is the Drosophila CENP-A assembly factor

The Journal of Cell Biology ◽

10.1083/jcb.201305036 ◽

2014 ◽

Vol 204 (3) ◽

pp. 313-329 ◽

Cited By ~ 87

Author(s):

Chin-Chi Chen ◽

Mekonnen Lemma Dechassa ◽

Emily Bettini ◽

Mary B. Ledoux ◽

Christian Belisario ◽

...

Keyword(s):

Histone H3 ◽

Specific Protein ◽

Terminal Domain ◽

Left Handed ◽

C Terminus ◽

Assembly Factors ◽

Histone H3 Variant ◽

Assembly Factor ◽

Drosophila Cells ◽

Assembly Activity

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. Here, we show that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the “missing” CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms.

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