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1.
Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.  相似文献   

2.
The human CENP-A centromeric nucleosome-associated complex   总被引:11,自引:0,他引:11  
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3.
The centromere is an essential chromatin domain required for kinetochore recruitment and chromosome segregation in eukaryotes. To perform this role, centro-chromatin adopts a unique structure that provides access to kinetochore proteins and maintains stability under tension during mitosis. This is achieved by the presence of nucleosomes containing the H3 variant CENP-A, which also acts as the epigenetic mark defining the centromere. In this review, we discuss the role of CENP-A on the structure and dynamics of centromeric chromatin. We further discuss the impact of the CENP-A binding proteins CENP-C, CENP-N, and CENP-B on modulating centro-chromatin structure. Based on these findings we provide an overview of the higher order structure of the centromere.  相似文献   

4.
The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity.  相似文献   

5.
The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.  相似文献   

6.
CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.  相似文献   

7.
8.
The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.  相似文献   

9.
10.
CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells.  相似文献   

11.
Perpelescu M  Fukagawa T 《Chromosoma》2011,120(5):425-446
Equal distribution of DNA in mitosis requires the assembly of a large proteinaceous ensemble onto the centromeric DNA, called the kinetochore. With few exceptions, kinetochore specification is independent of the DNA sequence and is determined epigenetically by deposition at the centromeric chromatin of special nucleosomes containing an H3-related histone, CENP-A. Onto centromeric CENP-A chromatin is assembled the so-called constitutive centromere-associated network (CCAN) of 16 proteins distributed in several functional groups as follows: CENP-C, CENP-H/CENP-I/CENP-K/, CENP-L/CENP-M/CENP-N, CENP-O/CENP-P/CENP-Q/CENP-R/CENP-U(50), CENP-T/CENP-W, and CENP-S/CENP-X. One role of the CCAN is to recruit outer kinetochore components further, such as KNL1, the Mis12 complex, and the Ndc80 complex (KMN network) to which attach the spindle microtubules with their structural and regulatory proteins. Among the CENPs in CCAN, CENP-C and CENP-T are required in parallel for operational kinetochore specification and spindle attachment. This review presents discussion of the latest structural and functional data on CENP-A and CENPs from the CCAN as well as their interaction with the KMN network.  相似文献   

12.
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.  相似文献   

13.
Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro–translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.  相似文献   

14.
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.  相似文献   

15.
16.
In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in?vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.  相似文献   

17.
Centromeric loci of chromosomes are defined by nucleosomes containing the histone H3 variant CENP-A, which bind their DNA termini more permissively than their canonical counterpart, a feature that is critical for the mitotic fidelity. A recent cryo-EM study demonstrated that the DNA termini of CENP-A nucleosomes, reconstituted with the Widom 601 DNA sequence, are asymmetrically flexible, meaning one terminus is more clearly resolved than the other. However, an earlier work claimed that both ends could be resolved in the presence of two stabilizing single chain variable fragment (scFv) antibodies per nucleosome, and thus are likely permanently bound to the histone octamer. This suggests that the binding of scFv antibodies to the histone octamer surface would be associated with CENP-A nucleosome conformational changes, including stable binding of the DNA termini. Here, we present computational evidence that allows to explain at atomistic level the structural rearrangements of CENP-A nucleosomes resulting from the antibody binding. The antibodies, while they only bind the octamer façades, are capable of altering the dynamics of the nucleosomal core, and indirectly also the surrounding DNA. This effect has more drastic implications for the structure and the dynamics of the CENP-A nucleosome in comparison to its canonical counterpart. Furthermore, we find evidence that the antibodies bind the left and the right octamer façades at different affinities, another manifestation of the DNA sequence. We speculate that the cells could use induction of similar allosteric effects to control centromere function.  相似文献   

18.
Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle–restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle–restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.  相似文献   

19.
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.  相似文献   

20.
Centromeres are the chromosomal loci that direct the formation of the kinetochores. These macromolecular assemblies mediate the interaction between chromosomes and spindle microtubules and thereby power chromosome movement during cell division. They are also the sites of extensive regulation of the chromosome segregation process. Except in the case of budding yeast, centromere identity does not rely on DNA sequence but on the presence of a special nucleosome that contains a histone H3 variant known as CenH3 or CENP-A (Centromere Protein A). It has been therefore proposed that CENP-A is the epigenetic mark of the centromere. Upon DNA replication the mark is diluted two-fold and must be replenished to maintain centromere identity. What distinguishes CENP-A nucleosomes from those containing histone H3, how CENP-A nucleosomes are incorporated specifically into centromeric chromatin, and how this incorporation is coordinated with other cell cycle events are key issues that have been the focus of intensive research over the last decade. Here we review some of the highlights of this research.  相似文献   

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