The chromatin remodeler RSC prevents ectopic CENP-A propagation into pericentromeric heterochromatin at the chromatin boundary

Centromeres of most eukaryotes consist of two distinct chromatin domains: a kinetochore domain, identified by the histone H3 variant, CENP-A, and a heterochromatic domain. How these two domains are separated is unclear. Here, we show that, in Schizosaccharomyces pombe, mutation of the chromatin remodeler RSC induced CENP-A^Cnp1 misloading at pericentromeric heterochromatin, resulting in the mis-assembly of kinetochore proteins and a defect in chromosome segregation. We find that RSC functions at the kinetochore boundary to prevent CENP-A^Cnp1 from spreading into neighbouring heterochromatin, where deacetylated histones provide an ideal environment for the spread of CENP-A^Cnp1. In addition, we show that RSC decompacts the chromatin structure at this boundary, and propose that this RSC-directed chromatin decompaction prevents mis-propagation of CENP-A^Cnp1 into pericentromeric heterochromatin. Our study provides an insight into how the distribution of distinct chromatin domains is established and maintained.     

The role of heterochromatin in centromere function

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.     

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.     

Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.     

Centromere/kinetochore localization of human centromere protein A (CENP-A) exogenously expressed as a fusion to green fluorescent protein

Three human centromere proteins, CENP-A, CENP-B and CENP-C, are a set of autoantigens specifically recognized by anticentromere antibodies often produced by patients with scleroderma. Microscopic observation has indicated that CENP-A and CENP-C localize to the inner plate of metaphase kinetochore, while CENP-B localizes to the centromere heterochromatin beneath the kinetochore. The antigenic structure, called "prekinetochore", is also present in interphase nuclei, but little is known about its molecular organization and the relative position of these antigens. Here, to visualize prekinetochore in living cells, we first obtained a stable human cell line, MDA-AF8-A2, in which human CENP-A is exogenously expressed as a fusion to a green fluorescent protein of Aequorea victoria. Simultaneous staining with anti-CENP-B and anti-CENP-C antibodies showed that the recombinant CENP-A colocalized with the endogenous CENP-C and constituted small discrete dots attaching to larger amorphous mass of CENP-B heterochromatin. When the cell growth was arrested in G1/ S phase with hydroxyurea, CENP-B heterochromatin was sometimes highly extended, while the relative location between GFP-fused CENP-A and the endogenous CENP-C was not affected. These results indicated that the fluorescent CENP-A faithfully localizes to the centromere/kinetochore throughout the cell cycle. We then obtained several mammalian cell lines where the same GFP-fused human CENP-A construct was stably expressed and their centromere/kinetochore is fluorescent throughout the cell cycle. These cell lines will further be used for visualizing the prekinetochore locus in interphase nuclei as well as analyzing kinetochore dynamics in the living cells.     

CENP-B: a major human centromere protein located beneath the kinetochore

The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.     

Building the centromere: from foundation proteins to 3D organization

At each mitosis, accurate segregation of every chromosome is ensured by the assembly of a kinetochore at each centromeric locus. Six foundation kinetochore proteins that assemble hierarchically and co-dependently have been identified in vertebrates. CENP-A, Mis12, CENP-C, CENP-H and CENP-I localize to a core domain of centromeric chromatin. The sixth protein, CENP-B, although not essential in higher eukaryotes, has homologues in fission yeast that bind pericentric DNA and are essential for heterochromatin formation. Foundation kinetochore proteins have various roles and mutual interactions, and their associations with centromeric DNA and heterochromatin create structural domains that support the different functions of the centromere. Advances in molecular and microscopic techniques, coupled with rare centromere variants, have enabled us to gain fresh insights into the linear and 3D organization of centromeric chromatin.     

Epigenetics regulate centromere formation and kinetochore function

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.     

Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4

The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A^Cnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A^Cnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A^Cnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A^Cnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A^Cnp1 for assembly into central domain chromatin, resulting in less CENP-A^Cnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A^Cnp1 influence the extent of DNA at centromeres that is packaged in CENP-A^Cnp1 chromatin and the composition of this chromatin. Thus, CENP-A^Cnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A^Cnp1 and other core histones.     

Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere

Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis [1,2]. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region [3]. By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives [4], we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote. The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast [5], the requirements for cohesion are separable from those for kinetochore assembly.     

Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres.     

The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions

Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.     

Molecular cloning and sequence analysis of hamster CENP-A cDNA

Background  

The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A) is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA.     

Inner centromere formation requires hMis14, a trident kinetochore protein that specifically recruits HP1 to human chromosomes

Centromeric DNA forms two structures on the mitotic chromosome: the kinetochore, which interacts with kinetochore microtubules, and the inner centromere, which connects sister kinetochores. The assembly of the inner centromere is poorly understood. In this study, we show that the human Mis14 (hMis14; also called hNsl1 and DC8) subunit of the heterotetrameric hMis12 complex is involved in inner centromere architecture through a direct interaction with HP1 (heterochromatin protein 1), mediated via a PXVXL motif and a chromoshadow domain. We present evidence that the mitotic function of hMis14 and HP1 requires their functional association at interphase. Alterations in the hMis14 interaction with HP1 disrupt the inner centromere, characterized by the absence of hSgo1 (Shugoshin-like 1) and aurora B. The assembly of HP1 in the inner centromere and the localization of hMis14 at the kinetochore are mutually dependent in human chromosomes. hMis14, which contains a tripartite-binding domain for HP1 and two other kinetochore proteins, hMis13 and blinkin, is a cornerstone for the assembly of the inner centromere and kinetochore.     

The cenpB gene is not essential in mice

Centromere protein B (CENP-B) is a centromeric DNA-binding protein that binds to α-satellite DNA at the 17 bp CENP-B box sequence. The binding of CENP-B, along with other proteins, to α-satellite DNA sequences at the centromere, is thought to package the DNA into heterochromatin subjacent to the kinetochore of mitotic chromosomes. To determine the importance of CENP-B to kinetochore assembly and function, we generated a mouse null for the cenpB gene. The deletion removed part of the promoter and the entire coding sequence except for the carboxyl-terminal 35 amino acids of the CENP-B polypeptide. Mice heterozygous or homozygous for the cenpB null mutation are viable and healthy, with no apparent defect in growth and morphology. We have established mouse embryo fibroblasts from heterozygous and homozygous cenpB null littermates. Microscopic analysis, using immunofluorescence and electron microscopy of the cultured cells, indicated that the centromere-kinetochore complex was intact and identical to control cells. Mitosis was identical in fibroblasts derived from cenpB wild-type, heterozygous and null animals. Our studies demonstrate that CENP-B is not required for the assembly of heterochromatin or the kinetochore, or for completion of mitosis. Received: 17 September 1998 / Accepted: 9 October 1998     

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.     

Inhibitors of Histone Deacetylases Alter Kinetochore Assembly by Disrupting Pericentromeric Heterochromatin

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-? on pericentromeric heterochromatin in S-phase and G2, decreased pericentromeric targeting of Aurora B kinase, resulting in decreased pre-mitotic phosphorylation of pericentromeric histone H3(S10) in G2, followed by assembly of deficient kinetochores in M-phase. HP1-?, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.