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Centromere protein A (CENP-A) nucleosomes containing the centromere-specific histone H3 variant CENP-A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing the CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and the RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Bub1 is required for the kinetochore/centromere localization of two essential mitotic kinases Plk1 and Aurora B. Surprisingly, stable depletion of Bub1 by ∼95% in human cells marginally affects whole chromosome segregation fidelity. We show that CENP-U, which is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex, is required to prevent chromosome mis-segregation in Bub1-depleted cells. Mechanistically, Bub1 and CENP-U redundantly recruit Plk1 to kinetochores to stabilize kinetochore-microtubule attachments, thereby ensuring accurate chromosome segregation. Furthermore, unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B. Consistently, depletion of Bub1 or CENP-U sensitizes cells to the inhibition of Plk1 but not Aurora B kinase activity. Taken together, our findings provide mechanistic insight into the regulation of kinetochore function, which may have implications for targeted treatment of cancer cells with mutations perturbing kinetochore recruitment of Plk1 by Bub1 or the CENP-O complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The centromere is a critical genomic region that enables faithful chromosome segregation during mitosis, and must be distinguishable from other genomic regions to facilitate establishment of the kinetochore. The centromere-specific histone H3-variant CENP-A forms a special nucleosome that functions as a marker for centromere specification. In addition to the CENP-A nucleosomes, there are additional H3 nucleosomes that have been identified in centromeres, both of which are predicted to exhibit specific features. It is likely that the composite organization of CENP-A and H3 nucleosomes contributes to the formation of centromere-specific chromatin, termed 'centrochromatin'. Recent studies suggest that centrochromatin has specific histone modifications that mediate centromere specification and kinetochore assembly. We use chicken non-repetitive centromeres as a model of centromeric activities to characterize functional features of centrochromatin. This review discusses our recent progress, and that of various other research groups, in elucidating the functional roles of histone modifications in centrochromatin.

Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A-H4-HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48-Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A-H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.

H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone variant has two isoforms, H2A.Z.1 and H2A.Z.2, each of which is coded by an individual gene. H2A.Z is involved in multiple epigenetic regulations, and in humans, it also has relevance to carcinogenesis. In this study, we used the H2A.Z DKO cells, in which both H2A.Z isoform genes could be inducibly knocked out, for the functional analysis of H2A.Z by a genetic complementation assay, as the first example of its kind in vertebrates. Ectopically expressed wild-type H2A.Z and two N-terminal mutants, a nonacetylable H2A.Z mutant and a chimera in which the N-terminal tail of H2A.Z.1 was replaced with that of the canonical H2A, complemented the mitotic defects of H2A.Z DKO cells similarly, suggesting that both acetylation and distinctive sequence of the N-terminal tail of H2A.Z are not required for mitotic progression. In contrast, each one of these three forms of H2A.Z complemented the transcriptional defects of H2A.Z DKO cells differently. These results suggest that the N-terminal tail of vertebrate H2A.Z makes distinctively different contributions to these epigenetic events. Our results also imply that this genetic complementation system is a novel and useful tool for the functional analysis of H2A.Z., (© 2016 The Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The kinetochore connects chromosomes to microtubules during mitosis and therefore plays an essential role in faithful chromosome segregation. It is built at the centromeric region of the chromosome and is comprised of many protein complexes. CENP-S, -T, -W, and -X are kinetochore components with histone-folds. These proteins play important roles in establishment of kinetochore chromatin. Similar to canonical histones, these kinetochore histone-fold proteins form heteromeric complexes (CENP-S/CENP-X complex and CENP-T/CENP-W complex) and bind DNA in sequence independent manner. In addition, they form a CENP-T-W-S-X heterotetrameric complex and bind DNA in a manner that is different from both CENP-S-X and CENP-T-W. To understand how kinetochores form and function it is necessary to characterize the components in detail. Here, we describe our approaches in purification and characterization of the kinetochore histone-fold complexes.

The centromere-specific histone H3 variant, CENP-A, is overexpressed in particular aggressive cancer cells, where it can be mislocalized ectopically in the form of heterotypic nucleosomes containing H3.3. In the present study, we report the crystal structure of the heterotypic CENP-A/H3.3 particle and reveal its "hybrid structure", in which the physical characteristics of CENP-A and H3.3 are conserved independently within the same particle. The CENP-A/H3.3 nucleosome forms an unexpectedly stable structure as compared to the CENP-A nucleosome, and allows the binding of the essential centromeric protein, CENP-C, which is ectopically mislocalized in the chromosomes of CENP-A overexpressing cells.

Purpose: The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs)., Methods and Materials: To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation., Results: FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells., Conclusions: We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A. Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. Here, we used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. We found that H4K20 monomethylation (H4K20me1) is enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. We conclude that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Recent studies revealed that both disseminated tumor cells and noncancerous cells contributed to cancer progression cooperatively in the bone marrow. Here, RNA-seq analysis of bone marrow from gastric cancer patients was performed to identify prognostic markers for gastric cancer., Experimental Design: Bone marrow samples from eight gastric cancer patients (stages I and IV: n = 4 each) were used for RNA-seq analysis. Results were validated through quantitative real-time PCR (qRT-PCR) analysis of HIST1H3D expression in 175 bone marrow, 92 peripheral blood, and 115 primary tumor samples from gastric cancer patients. miR-760 expression was assayed using qRT-PCR in 105 bone marrow and 96 primary tumor samples. Luciferase reporter assays were performed to confirm whether histone mRNAs were direct targets of miR-760. miR-760 expression was also evaluated in noncancerous cells from gastric cancer patients., Results: RNA-seq analysis of bone marrow samples from gastric cancer patients revealed higher expression of multiple histone mRNAs in stage IV patients. HIST1H3D expression in the bone marrow, peripheral blood, and primary tumor of stage IV patients was higher than that in stage I patients (P = 0.0284, 0.0243, and 0.0006, respectively). In contrast, miR-760 was downregulated in the bone marrow and primary tumor of stage IV patients compared with stage I patients (P = 0.0094 and 0.0018, respectively). Histone mRNA and miR-760 interacted directly. Furthermore, miR-760 was downregulated in noncancerous mucosa in stage IV gastric cancer patients., Conclusion: Histone mRNA was upregulated, whereas miR-760 was downregulated in the bone marrow and primary tumor of advanced gastric cancer patients, suggesting that the histone mRNA/miR-760 axis had a crucial role in the development of gastric cancer., (©2013 AACR.)

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes.

Histone variants play important roles in the epigenetic regulation of genome function. The histone variant H2A.Z is evolutionarily conserved from yeast to vertebrates, and it has been reported to have multiple effects upon gene expression and insulation, and chromosome segregation. Recently two genes encoding H2A.Z were identified in the vertebrate genome. However, it is not yet clear whether the proteins transcribed from these genes are functionally distinct. To address this issue, we knocked out each gene individually in chicken DT40 cells. We found that two distinct proteins, H2A.Z-1 and H2A.Z-2, were produced from these genes, and that these proteins could be separated on a long SDS-PAGE gel. The two isoforms were deposited to a similar extent by the SRCAP chromatin-remodeling complex, suggesting redundancy to their function. However, cells lacking either one of the two isoforms exhibited distinct alterations in cell growth and gene expression, suggesting that the two isoforms have differential effects upon nucleosome stability and chromatin structure. These findings provide insight into the molecular basis of the multiple functions of the H2A.Z gene products.

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A-H2B, we identified protein phosphatase (PP) 2C gamma subtype (PP2Cgamma/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A-H2B. The disruption of PP2Cgamma in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cgamma-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.