The cid gene appears to have a very short transcriptional regulatory region, because only 406 bp upstream separate the ORF from an oppositely oriented ORF that is represented in the expressed sequence tag database. This ORF and the upstream region was cloned into a GFP fusion construct and the plasmid was introduced into D. melanogaster Kc cells by transient transfection. To characterize the cid promoter, the upstream promoter GFP construct was used to drive expression of Drosophila histones fused to GFP. Because histones are deposited at newly replicated DNA, cell cycle-limited expression might be revealed by restricted deposition of histone-GFP fusion protein observed at metaphase. When H2B-GFP was synthesized constitutively under control of the heat shock promoter, uniform GFP localization was seen consistently in mitotic figures of transiently transfected cells. This confirms that histone-GFP fusions can be deposited throughout chromatin. However, H3- and H2B-GFP constructs driven by the cid promoter show more limited localization: the euchromatic arms are labeled, but pericentric heterochromatin is not. Because constitutive expression gives uniform deposition, cid-driven expression must produce H2B-GFP and H3-GFP early in the cell cycle, when euchromatin is replicating, but these histones must have been used up by the time that pericentric heterochromatin replicated. Thus, the cid promoter drives early S phase-limited expression. This conclusion differs from the report that cell cycle-limited expression of CenpA mRNA occurs much later in synchronized HeLa cells and may reflect differences between Kc and HeLa cells or differences in procedures used to assess cell cycle-dependent expression (Henikoff, 2000).
Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID is depleted by RNAi, it is replaced by H3, demonstrating flexibility of centromeric chromatin organization. Finally, contrary to models proposing that H3 and CID/CENP-A nucleosomes are replicated at different times in S phase, it has been shown that interspersed H3 and CID/CENP-A chromatin are replicated concurrently during S phase in humans and flies. It is proposed that the unique structural arrangement of CID/CENP-A and H3 nucleosomes presents centromeric chromatin to the poleward face of the condensing mitotic chromosome (Blower, 2002).
Centromeric chromatin in both flies and humans is organized into a cylindrical 3D structure on metaphase chromosomes. This structure contains histones H2AB but appears to be devoid of H3 and PH3. This data suggests that the metaphase centromere is composed solely of CID/CENP-A-containing nucleosomes and that CID/CENP-A may replace all histone H3 in centromeric nucleosomes. Consistent with this data, CID mononucleosomes are homotypic in vivo and contain CID, H2A, H2B, and H4, as has been suggested by in vitro studies and the stoichiometry of CENP-A in human cells (Blower, 2002).
Previous 2D immunofluorescence studies in mammalian cells have suggested that CENP-A and CENP-C colocalize and are therefore positioned within the same region of the inner kinetochore. CENP-A localization within subkinetochore chromatin or the inner kinetochore plates using high resolution electron microscopy has not been previously reported. The 3D deconvolution studies described in this study demonstrate that while CENP-A and CENP-C are indeed closely juxtaposed, they show significant nonoverlap. In cross-section, the organization observed is consistent with human CENP-C forming a plate-like structure, as demonstrated in previous transmission EM studies. However, the data suggests that CENP-A chromatin is organized as a cylindrical structure, rather than a plate, and is predominantly located beneath the kinetochore, although some overlap with the inner plate cannot be excluded. CENP-A localization interior to CENP-C suggests that CENP-A nucleosomes are the physical foundation for kinetochore formation. CENP-A also serves as the functional foundation for kinetochore assembly, as all known kinetochore components are mislocalized in CENP-A disruptions, including CENP-C. CENP-C, in turn, is required for recruitment or maintenance of outer kinetochore proteins in mammals and worms. This data suggests that there is a specific order of recruitment and assembly of the kinetochore in all organisms, which mimics the 3D arrangement of the protein components (Blower, 2002).
It has been proposed that the kinetochore is composed of repeats of a functional base subunit. This conclusion was based on caffeine-induced kinetochore fragmentation in the absence of DNA replication, the ability of kinetochore fragments to move along spindle microtubules, and the discontinuous appearance of CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasias) antibody staining on mechanically stretched metaphase kinetochores. In contrast to the exclusion of H3 from metaphase kinetochores, blocks of CID/CENP-A nucleosomes and H3 nucleosomes are interspersed on chromatin fibers and long polynucleosomal fragments. Furthermore, because CENP-A nucleosomes are arranged in a discontinuous array and CENP-A is required for the recruitment of all other kinetochore components, it is concluded that CENP-A nucleosomes are the base subunit of the repeated centromere/kinetochore structure in diverse organisms (Blower, 2002).
Chromatin fiber analysis indicates variation in the number of CENP-A spots, particularly on fibers from human cells. These results are particularly provocative in that they suggest that sizes of CID/CENP-A-containing regions and/or numbers of CENP-A nucleosomes may vary widely, particularly among human chromosomes. CID/CENP-A-containing chromatin at Drosophila centromeres extends over 200-500 kb and over 500-1500 kb at human centromeres. The results are in agreement with recent findings indicating that the CENP-A binding domains at two human neocentromeres are 460 kb and 330 kb. In addition, CENP-A antibodies stain only one-half to two-thirds of alpha satellite DNA regions at normal human centromeres. This result suggests that the entire alpha satellite DNA array at a human centromere is not involved in kinetochore assembly and that alpha satellite DNA may have additional functions in centric regions (Blower, 2002).
How can the linear interspersion of CID/CENP-A and H3 be reconciled with the exclusion of H3 on metaphase chromosomes? It is proposed that centromeric DNA may form a spiral or loop, in which blocks of CENP-A nucleosomes are oriented on the poleward faces of chromosomes and blocks of H3 nucleosomes are located toward the inner chromatid region. At this time, it is not known whether there is any variation in the relative proportions of CENP-A and H3 nucleosomes in the centromere and whether the number of CENP-A or H3 nucleosomes varies from block to block. Does the conserved 3D structure and organization of centromeric chromatin play a role in centromere function? It is proposed that the purpose of the spiral or loop structure may be to 'present' centromeric chromatin to the exterior of the chromosome, where it can mediate kinetochore assembly and interactions with the spindle. If centromeric chromatin condenses along with the rest of the chromosome in a random fashion, this chromatin would most likely be hidden inside the chromatids (Blower, 2002).
In higher eukaryotes, centromeres are uniformly embedded in large blocks of repetitive DNA that are considered highly condensed, gene-poor, and inaccessible to transcription factors. However, flanking heterochromatin behaves as a domain that is structurally and functionally distinct from CENP-A-containing centromeric chromatin in S. pombe, flies, and humans. Further evidence for the existence of distinct domains within the centromere comes from the observation that chromatin immediately flanking CENP-A/CID chromatin appears to replicate at a different time than CID-containing chromatin. Discontinuity of replication has been seen in other genomic regions that are involved in epigenetic inheritance. It is possible that a replication boundary between centromeric chromatin and the flanking heterochromatin is important for establishment or maintenance of the two distinct chromatin states (Blower, 2002).
It is proposed that flanking heterochromatin may be required to organize the higher order structure of centromeric chromatin. Flanking heterochromatin may interact with the interspersed H3 domains to produce or maintain the CENP-A cylinder. This model predicts that the interspersed H3 may display heterochromatin-like properties, such as H3 methylation at lysine 9 and HP1 binding. Heterochromatin at the centromere may also be necessary to adopt a conformation that maintains cohesion between sister chromatids. Furthermore, it may define the borders of the centromeric chromatin domain and prevent CENP-A chromatin from spreading into adjacent regions, as observed for neocentromere formation in flies (Blower, 2002).
It appears that a single nucleosome containing the CENP-A homolog Cse4p is sufficient to nucleate microtubule interactions in S. cerevisiae. The point centromeres of S. cerevisiae are likely to represent the most basic iteration of a centromere, which expanded as organisms became more complex and evolved larger chromosomes. In S. pombe, the CENP-A homolog Cnp1 is present in an apparently uninterrupted stretch of ~5- 10 kb within the central core of the centromere, flanked by H3-containing chromatin. It is proposed that the different sizes and organizations of monocentric kinetochores, and even holocentric kinetochores, are produced by the same interspersed histone/higher order structure, present in different numbers and distributions. The holocentric kinetochores of C. elegans and other species on the surface appear to be quite different from monocentric kinetochores but could simply represent the broadest expansion of the functional centromere base unit. In C. elegans, CENP-A (HCP-3) is present in an unusually large number of discreet foci in interphase nuclei; during mitosis these foci coalesce into the thin kinetochore ribbon present on the poleward face of each chromosome. Thus, holocentric kinetochores appear to be organized into a 3D structure similar to humans and flies, in which centromeric chromatin is presented on the exterior face of metaphase chromosomes (Blower, 2002).
The plasticity of centromeric chromatin provides a plausible mechanism for how variations in the basic centromere unit may be established and maintained in different organisms. RNAi depletion of Drosophila CID results in reduced intensity and number of CID spots in fibers, an increase in the distance between spots, and an expansion of the H3 domains. Overexpression of CID results in continuous distribution of CID, rather than discrete arrays of CID chromatin. These results suggest that the interspersed organization of centromeric chromatin is plastic; in the absence of CID deposition, H3 chromatin is assembled on centromeric DNA, and vice versa. Reductions in the size and number of the CID blocks, and expansion of the H3 blocks, can account for the decreased recruitment of outer kinetochore proteins and increased chromosome segregation errors observed in an earlier previous study. In addition, variations in the amount of CENP-A in the nucleus, or the kinetics of H3 and CENP-A deposition, could be responsible for the evolution of different kinetochore sizes and interspersion patterns. The maximum extent of the centromeric chromatin could then be determined by altering the locations of specific boundary elements or the balance between the centromeric and flanking centric heterochromatin epigenetic states (Blower, 2002).
The plasticity of centromeric chromatin organization suggests that there must be an active mechanism to maintain the balance between CID/CENP-A deposition and H3 deposition. S. pombe Mis6 and S. cerevisiae ndc10 have been demonstrated to be required for localizing or maintaining CENP-A at centromeres (Ortiz, 1999; Takahashi, 2000), although Ctf3p, the budding yeast homolog of pombe Mis6, is not required for loading Cse4p onto centromeric DNA. Future studies of the determinants of centromere identity must identify the proteins required for CENP-A deposition in higher eukaryotes, and for assembly and maintenance of the linear arrangement and 3D structure of centromeric chromatin reported here (Blower, 2002).
Chromosome segregation during meiosis and mitosis depends on the assembly of functional kinetochores within centromeric regions. Centromeric DNA and kinetochore proteins show surprisingly little sequence conservation despite their fundamental biological role. However, identification in Drosophila of the most diverged orthologs identified so far, which encode components of a kinetochore protein network including the Ndc80 and Mis complexes, further emphasizes the notion of a shared eukaryotic kinetochore design. To determine its spatial organization, quantitative light microscopy was used to analyzed hundreds of native chromosomes from transgenic Drosophila strains coexpressing combinations of red and green fluorescent fusion proteins, fully capable of providing the essential wild-type functions. Thereby, Cenp-A/Cid, Cenp-C, Mis12 and the Ndc80 (CG9938) complex were mapped along the inter sister kinetochore axis with a resolution below 10 nm. The C terminus of Cenp-C was found to be near but well separated from the innermost component Cenp-A/Cid. The N terminus of Cenp-C is further out, clustered with Mis12 and the Spc25 end of the rod-like Ndc80 complex, which is known to bind to microtubules at its other more distal Ndc80/Nuf2 end (Schittenhelm, 2007).
Identification of Drosophila kinetochore proteins further exposes hidden similarities of kinetochore design in eukaryotes. In addition to the previously known, highly diverged Cenp-A/Cid and Cenp-C homologs, Drosophila expresses similarly diverged homologs of the Mis12 and Ndc80 complex network, which is also present in yeast, C. elegans, vertebrates, and presumably in plants as well. These ubiquitous CKC components have been localized along the intersister kinetochore axis with unprecedented spatial resolution. Early Drosophila embryos allow an efficient isolation of native mitotic chromosomes and thereby imaging with reduced background. Moreover, transgenic strains allow the expression of fluorescent fusion proteins, which were demonstrated to be fully functional by genetic complementation tests (Schittenhelm, 2007).
The position of fluorescent signal maxima has been determined within the kinetochore of native chromosomes released from embryos expressing fluorescent CKC fusion proteins. The CKC map is based on averaged data from hundreds of analyzed chromosomes. Therefore, its interpretation depends critically on the variability of kinetochore organization in individual chromosomes. For instance, in principle, a given component might be localized on the inner kinetochore side in 50% of the chromatids and on the outer side in the other half of the chromatids, resulting in a misleading central positioning in the CKC map. Theoretically, such variability should widen the distribution of the distances measured in individual chromosomes. However, kinetochore width is smaller than the spreading of the image of a point light source in the microscope, and several additional factors (like background, noise, pixelation) further limit the precision of the measurements. The effect of positional variability on distribution width of the measured values would therefore be very subtle. Moreover, none of the known CKC proteins has been firmly demonstrated to be a spatially invariable kinetochore component, precluding comparisons to an established standard distribution. However, the reproducible trilaminar structure of the kinetochore during prometaphase, that has been documented by EM, argues strongly against extensive organizational variability. It is emphasized that the difficulties in detecting subtle alterations in the distribution width of the measurements obtained for a given CKC component has important consequences even under the assumption that the spatial distribution of CKC components is essentially invariable in individual kinetochores. These difficulties prevent conclusions concerning the width occupied by a given CKC component within a kinetochore. For instance, Mis12 could either be confined to a single layer in the middle of the kinetochore or spread throughout the kinetochore, and both localization patterns would result in a central signal maximum. However, biochemical analyses of kinetochore proteins have so far revealed highly specific interactions, arguing strongly for a precise and restricted localization of CKC components. The following discussion is therefore based on the unproven but likely assumption that the kinetochore represents a precisely defined layered structure (Schittenhelm, 2007).
Based on previous analyses, Cenp-A, Cenp-C, and Mis12 are thought to be components of the inner plate of the characteristic trilaminar kinetochore structure apparent in the EM. The analyses indicate a significant separation between the inner most CKC component Cenp-A and all other CKC components analyzed here. Recently, Cenp-A nucleosomes purified from human cells were found to be intimately associated with the five proteins Cenp-M, Cenp-N, Cenp-T, Cenp-U, and Cenp-H in addition to Cenp-C. The apparent space between Cenp-A and Cenp-C might therefore be occupied by some of those proteins (Schittenhelm, 2007).
Many immunolocalization studies, including a recent study with Drosophila cells, have failed to detect a comparable extensive spatial separation between Cenp-A/Cid and Cenp-C. However, immunolocalization with human chromosomes also revealed little overlap between Cenp-A and Cenp-C, with the latter extending over the top and bottom of a Cenp-A cylinder. Antigen accessibility problems cannot affect the concurrent findings (Schittenhelm, 2007).
In this paper, Cenp-C is shown to be spread in a polar orientation across a central CKC region. The C-terminal domain of Cenp-C, which contains the most conserved region including the CENP-C motif, points toward the centromeric DNA. These C-terminal sequences are connected via minimally conserved spacer sequences to the N-terminal domain which is oriented toward the kinetochore spindle fibers. The N-terminal region of D. melanogaster Cenp-C contains some blocks which are highly conserved among Drosophilids. These blocks might be involved in recruiting the next layer of kinetochore proteins which are suggested to include the Ndc80 and Mis12 complexes. Mis12 is close to the N-terminal Cenp-C region. Moreover, the Ndc80 complex component Spc25 (CG7242) appears to be even a bit closer but well separated by about 20 nm from the other Ndc80 component Nuf2 (CG8902). Apart from a polar Cenp-C orientation, these analyses therefore also indicate a polar orientation for the Ndc80 complex (Schittenhelm, 2007).
The tetrameric Ndc80 complex has a highly elongated, rod-like structure in vitro. The globular N-terminal domains of Ndc80 and Nuf2 are present on one end of the rod. The remainder of these two subunits forms an extended coiled coil which is further prolonged at its C-terminal end by binding to the N-terminal coiled coil region of the Spc24/Spc25 dimer. Closely associated C-terminal globular domains of Spc24 and Spc25 form the other end of the rod. Scanning force microscopy and EM analyses have indicated that the coiled coil region separating the globular domains at the end of the Ndc80 complex has an extension of about 40 nm. This is twofold longer than the distance observed between fluorescent proteins at the N and C termini of Nuf2 and Spc25 in kinetochores of native Drosophila chromosomes. Many of the elongated Ndc80 complexes might not be perfectly oriented along the spindle axis, especially as the kinetochores in the preparations used in this study are not under tension. Such a nonuniform orientation could result in spatial distributions of the N and C termini of Nuf2 and Spc25, respectively, with signal maxima that are more closely spaced than their separation within an isolated complex. An analysis of the positions of CKC components in chromosomes that are bi-oriented within the spindle and under tension would clearly be of interest. However, the increased background levels present in living embryos have so far precluded such analyses (Schittenhelm, 2007).
The observed polar orientation of the Ndc80 complex within the kinetochore confirms the findings of a recent independent study. Moreover, the observation that Ndc80 and Nuf2 kinetochore localization is no longer observed in the absence of Spc24 or Spc25 is consistent but does not prove an orientation of the complex with inner Spc24/Spc25 and outer Ndc80/Nuf2 globular domains, because absence of Spc24 or Spc25 for instance might simply result in an instability of other complex components, as often observed in the case of stable complexes (Schittenhelm, 2007 and references therein).
In budding yeast, the Ndc80 complex has been proposed to function as a connection between the inner components (CBF3 complex, Cenp-A/Cse4 nucleosome, Cenp-C/Mif2, Mis12/MIND complex) and the Dam/DASH complex which is required for bi-orientation and appears to form a ring around the single microtubule attaching to a yeast kinetochore. More recently, bacterial expression of the C. elegans KMN network composed of the Spc105/KNL-1, Mis12 and Ndc80 complexes has led to a convincing identification of two independent sites in this protein network that can bind directly to microtubules in vitro. One of these microtubule binding sites is present within Spc105/KNL-1. The other is found within the globular N-terminal Ndc80 domain which is known to be within the outer kinetochore plates where kinetochore microtubules terminate. In vitro, the Ndc80 complex binds to microtubules at an angle. A corresponding orientation of the Ndc80 complex within the kinetochore is fully consistent with the finding that the separation of the terminal globular domains of Spc25 and Nuf2 along the intersister kinetochore axis appears to be less than their separation along the axis of isolated complexes. Accordingly, the 'barbed end' of microtubules decorated with the Ndc80 complex would be predicted to correspond to the plus end (Schittenhelm, 2007 and references therein).
In conclusion, in addition to the identification of Drosophila Ndc80 and Mis12 complex components, this work provides a highly resolved structural framework integrating the most widely studied ubiquitous CKC components and a precise method for a future incorporation of additional proteins (Schittenhelm, 2007).
The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, replication and chromatin assembly at centromeres in Drosophila cells was examined. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, centromeres were shown in this study to replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. It is suggested that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones (Ahmad, 2001).
Analysis of centromeres in complex eukaryotes has been hampered by the lack of sequence differences between the centromere and flanking heterochromatin, and the repetitive nature of these regions. These sequence commonalities have led to the attribution of heterochromatic features to the centromere, including late replication. The analysis performed in this study demonstrates that the replication of centromeres in Drosophila cells actually precedes that of pericentromeric heterochromatin. It is estimated that, on average, ~500 kb of centromeric DNA is replicated in the early S phase period. This size is in agreement with a genetically defined fully functional centromere in Drosophila, suggesting that the early replication domain corresponds to the complete centromere. The early timing of its replication distinguishes the centromere from other repetitive sequences and rules out models for defining centromeres that have invoked their very late replication. Early replication appears to be a general feature of centromeres, as Saccharomyces centromeres are known to replicate early in S phase (Ahmad, 2001).
The observations on the controlled assembly of conventional (H3-containing) and specialized (Cid-containing) nucleosomes at replicating centromeres suggests that chromatin assembly is a critical step in centromere maintenance. The cid promoter drives expression early in S phase (Henikoff, 2000), and centromeres are replicating during this time. Therefore, Cid synthesis and centromere replication appear to be tightly coordinated. In Schizosaccharomyces yeast, the Cnp1 gene (encoding the centromeric SpCenpA histone) is also expressed early in S phase (Takahashi, 2000), and it is expected that a similar coordination with centromeric replication will be found (Ahmad, 2001).
At the time that Cid is being deposited, H3 deposition is inhibited. It is striking that early replicating centromeres are typically surrounded by late-replicating heterochromatin, and it is suggested that inhibiting histone H3 incorporation at centromeres when they replicate is one function of this juxtaposition. Inhibiting histone H3 incorporation at centromeres requires the uncoupling of conventional chromatin assembly and DNA replication. These two processes are thought to be linked by interactions between replication machinery and the CAF1 chromatin assembly factor. Uncoupling may be accomplished if histone H3 or some component of its assembly machinery is excluded from the heterochromatic chromocenter early in S phase. Regions deficient in histone H3 would then be incorporated into Cid-containing nucleosomes by a replication-independent pathway (Ahmad, 2001).
The observation that centromeric histone H3-like proteins from worms and yeast preferentially localize to fly or human heterochromatin suggests that heterochromatin sequesters centromeric H3-like proteins in general. Sequestering Cid in the heterochromatic chromocenter would increase the local concentration of Cid around centromeres and thereby promote Cid deposition. Centromeres in many organisms are typically surrounded by heterochromatin, and genetic evidence suggests that heterochromatin is important for centromere function. The centromeres of Saccharomyces chromosomes are the only known exception to this rule, but in this organism centromeric activity is conferred by a specific DNA sequence and associated DNA-binding proteins. The importance of heterochromatin for the function of complex centromeres is reinforced by the finding that a human neocentromere shows M31 staining (a marker for mammalian heterochromatin), whereas the parental chromosomal region does not. Perhaps the exclusion of histone H3 during replication is one of the prerequisites for 'centromerization', thus necessitating that neocentromeres acquire heterochromatic proteins (Ahmad, 2001).
It is expected that centromeres must be protected from conventional nucleosome assembly pathways in all dividing cells, but heterochromatin may not always perform this function. For example, distinct heterochromatin does not form in the rapidly dividing nuclei of Drosophila syncytial embryos, and replication initiates throughout the chromosomes simultaneously. In these unusual nuclei, conventional nucleosome assembly might be prevented by excluding histone H3 from the apical edge of interphase nuclei, where centromeres lie. Similarly, varying local concentrations of proteins around nuclei have been proposed to explain progression of the syncytial cell cycle even though bulk cyclin levels are always high. In later cycles, it appears to be most efficient to produce Cid when centromeres replicate (Ahmad, 2001).
It has been of great interest to understand how the location of the centromere is stably maintained in successive cell divisions, because it does not appear that DNA sequence is responsible. Nucleosome particles form the fundamental unit of chromatin, and so an attractive alternative to DNA sequence-based inheritance of centromere identity is that centromeric nucleosomes participate in centromere maintenance. Replication initiation appears to depend on chromatin structure and it is suggested that Cid-containing nucleosomes predispose DNA to replicate early. This early replication and the exclusion of histone H3 in heterochromatin would preclude conventional chromatin assembly, thus allowing the assembly of Cid-containing nucleosomes and ensuring early replication again in the next cycle. This process would maintain centromeres (Ahmad, 2001).
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it has been proposed that centromeres are among the first or last sequences to be replicated. Centromere replication in Drosophila has been studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods have been used to evaluate replication timing of chromatin containing CID, a Drosophila homolog of CENP-A. Centromeres in Drosophila cell lines are replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo are replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres are replicated in late S phase, and those in which centric and surrounding heterochromatin have been partially or fully deleted are replicated earlier in mid S phase. This study provided the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin (Sullivan, 2001).
Centromere replication was visualized cytologically by correlating thymidine analog incorporation with CID antibody staining. For single labeling, Drosophila S2 and Kc tissue culture cells were treated with BrdU for increasing intervals to span S phase and then were blocked in metaphase to regressively determine when labeled sites had replicated. All chromosomes stain equally with CID antibodies, suggesting that the inherent aneuploidy of these tissue culture cells is not likely due to defective kinetochores but perhaps to spindle defects such as multipolar spindles. Kc cells contain 10-22 chromosomes and 5-11 CID-staining regions in nuclei, since homologs are paired in Drosophila nuclei. After 9 h of labeling, the entire dot-like 4th chromosomes, including the centromeres, were stained with BrdU. CID and BrdU colocalization was also observed on the metacentric third chromosomes and the acrocentric X chromosomes. The chromosome 2 centromere is not replicated in very late S phase, since CID staining at this time does not colocalize with BrdU staining (Sullivan, 2001).
Terminal labeling establishes a time period during which centromeres are replicated. However, it does not distinguish replication that occurs specifically in late S from DNA replication that initiates earlier and continues into late S. Thus, double labeling with iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) was used to view DNA replication in early and late S. In Kc cells, centromere replication occurs asynchronously throughout S phase. On average, only one centromere is replicated in early S, and two centromeres replicate in late S phase, indicating that centromeres in Kc cells are replicated primarily in mid S phase. In S2 cells, which are less aneuploid and contain 4-12 chromosomes, two centromere pairs on average are late replicating. Most CID antibody signals (8-11) do not overlap with either IdU (early S) or CldU (late S) staining, suggesting that most kinetochore-associated DNA in S2 cells is replicated in mid S phase. Thus, labeling experiments of interphase nuclei and metaphase chromosomes indicate that most centromere-associated DNA in tissue culture cells is replicated asynchronously in mid and late S phase. This finding agrees with studies in human tissue culture cells, showing that replication of centromeric DNA occurs in mid to late S phase. Taken together, these data argue against the hypothesis that replication of centromeric DNA occurs in a discrete time period in metazoan-cultured cells (Sullivan, 2001).
Studies of centromere replication in cultured cells may not reflect the in vivo process, particularly since Drosophila tissue cultured cells used in this and other studies are not diploid and may have defects in cell cycle regulation and progression. Therefore, centromere replication was studied in vivo. While studying endogenous centromere replication, the effects of flanking heterochromatin on centromeric replication timing was also tested using the Dp1187 deletion series of structurally distinct minichromosomes with functional kinetochores. Single labeling with BrdU for <=3 h progressively labels chromosomal regions that replicate from mid S (3 h before M) to very late S phase (60 min before M). Centromeres of the 3rd, 4th, and Y chromosomes are replicated very late (60 min before M). Although CID-associated chromatin of chromosome 2 is not replicated at this time, the surrounding heterochromatin shows BrdU staining. Centromeres of the X and 2nd chromosomes replicate during late S (1.5-2.5 h before M). After 3 h in BrdU, all Drosophila centromeres are labeled, indicating that in vivo centromere replication occurs primarily in late S phase. Noncentromeric labeling is observed on Drosophila chromosomes in very late S phase, arguing against models proposing that centromeres are the last to replicate in the cell (Sullivan, 2001).
To test if heterochromatin restricts centromeric replication to late S phase, replication of five structurally distinct minichromosomes was also studied. The centromere (CEN) of the parental minichromosome, Dp8-23, is surrounded by 400 kb of centric heterochromatin. Dpgamma238 was generated by an inversion in Dp8-23 so that its CEN is oriented in the opposite direction and is flanked by euchromatin on one side and 600 kb of heterochromatin on the other. Both Dp8-23 and Dpgamma238 show complete BrdU incorporation at the centromere and over the entire chromosome late in S phase, 1-3 h before M. Dp1187 was derived from the endogenous X chromosome, and consistent with its origin, intact minichromosome centromeres replicate coincident with the endogenous X centromere. Two deleted minichromosomes, Dp10B and Dpgamma1230, in which the only centric heterochromatin present corresponds to the functional centromere were completely labeled by BrdU in late S phase (Sullivan, 2001).
To address whether minichromosomes are replicated throughout S phase or only in a portion of S, neuroblasts were double labeled with IdU and CldU. In these experiments, Dp8-23, Dpgamma238, Dpgamma1230, and Dp10B were entirely late replicating. For example, Dpgamma238 is completely and exclusively labeled by CldU, the late S label. Therefore, these experiments corroborate that centromeres of Dp minichromosomes, even in the absence of flanking heterochromatin, are replicated late along with the endogenous X centromere and the other endogenous centromeres. Double labeling experiments ruled out the possibility that centromeres initiate replication in early S and continue throughout S phase (Sullivan, 2001).
Do sequences capable of supporting kinetochore assembly, although unrelated in DNA sequence, exhibit similar replication timing? DpJ21A and Dp26C, minichromosomes deficient for CEN DNA, allow this question to be addressed. Dp26C is a neocentromere, a normally noncentromeric 285-kb fragment that acquires centromere function by proximity to the Dpgamma238 centromere. Despite partial or total absence of CEN DNA, both minichromosomes contain functional centromeres and recruit CID and all known outer kinetochore proteins. These minichromosomes are propagated through meiosis and mitosis; slightly decreased mitotic transmission rates are due to their decreased size, which affects cohesion and antipoleward forces but not kinetochore assembly. By single labeling, DpJ21A and Dp26C were not stained until 4 h before M, suggesting that they replicate earlier than the large minichromosomes. In double labeling experiments, DpJ21A and Dp26C were typically unlabeled by either IdU or CldU, although in 20% of cells DpJ21A was late replicating. Replication of these minichromosomes occurs at the mid to late S transition. Similar to the larger Dp minichromosomes, DpJ21A and Dp26C are never observed to replicate in early S phase. Centromere replication in CEN DNA-deleted minichromosomes predominantly occurs in mid S phase and the beginning of late S phase, earlier than the larger minichromosome centromeres, which replicate within the last few hours of S (Sullivan, 2001).
Compartmentalized replication timing and/or marking of chromatin by CENP-A may specify centromere identity. Since CID/CENP-A is a conserved histone exclusive to functional centromeres and is required to recruit other kinetochore proteins, it is important to understand the mechanisms responsible for recruitment of CID/CENP-A solely to centromeres. Replication timing of Drosophila centromeres in vitro in cultured cells occurs asynchronously within the cell cycle from early to late S phase but primarily in mid S. In vivo replication of endogenous and defined minichromosome centromeres also occurs in mid to late S phase. Thus, Drosophila centromeres are neither the earliest or latest regions to replicate, ruling out models of centromere identity and propagation based on temporal separation of centromere replication from bulk chromatin. These in vivo findings agree with studies describing centromeric replication in mid to late S phase in human cells. Centromere replication in smaller deletion-derivative minichromosomes occursearlier in mid S, unlike late S replication of centromeres surrounded by heterochromatin. Asynchronous replication timing of different minichromosomes that all display centromere function further refutes models that require temporal sequestration of centromere replication (Sullivan, 2001).
The location of centromeres within the nucleus is thought to specify centromere identity and propagation. However, CENP-A/CID antibody spots are widely distributed throughout interphase nuclei in cultured cells. Within three-dimensionally preserved nuclei of S2 and Kc tissue culture cells analyzed by deconvolution microscopy, centromeres are present within multiple serial sections throughout S phase and do not appear to reside in a single nuclear location or domain. These findings are similar to the broad distribution of centromeres observed in human cells. Therefore, it is concluded that spatial sequestration of centromeres during S phase does not propagate centromere identity (Sullivan, 2001).
It is concluded that centromeres in tissue culture and in vivo replicate broadly across S phase and are not restricted to a single brief window of replication timing. Timing of centromere replication can occur differently in various cell types. Together with the results of minichromosome replication, it is concluded that timing of replication is unlikely to be a key determinant of centromere identity. These results support replication-independent incorporation of CID/CENP-A during centromere assembly. Self-propagation of centromere identity could occur through the action of proteins that incorporate CID/CENP-A into newly replicated regions by recognizing existing CID/CENP-A chromatin (Sullivan, 2001).
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, also termed Centromere identifier (CID) that provides a structural and functional foundation for the kinetochore. This study demonstrates that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. It is speculated 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 (Sullivan, 2004).
Post-translational modifications of histones are known to be biologically important in defining chromatin states, such as silent or active gene expression. Centromeric chromatin in flies and humans is defined as the full extent of staining for the centromere-specific histones CENP-A and CID, which contain interspersed subdomains of the CENP-A/CID and H3 nucleosomes. Immunofluorescence analysis of two-dimensional extended chromatin fibers and three-dimensional mitotic chromosomes demonstrated that H3 subdomains present within CEN chromatin are enriched for H3 Lys4-diMe, a modification associated with open but not active euchromatin. H3 subdomains within CEN chromatin do not contain the H3 Lys9 di- or trimethylation associated with heterochromatin, and lack acetylations at H3 Lys9 and H4 Lys5, Lys8, Lys12 and Lys16 that are generally found in euchromatin. Finally, the H3 Lys4-trimethylation associated with actively transcribed regions is also not present in CEN chromatin. It is concluded that the interspersed H3 present in fly and human CEN chromatin contains individual H3 and H4 modifications previously associated with both euchromatin and heterochromatin, but in a combined pattern that is distinct from each chromatin state individually. These results are unexpected; the fact that eukaryotic centromeres are embedded in heterochromatin has suggested that CEN chromatin should contain heterochromatic epigenetic imprints. This distinct pattern of histone modifications, which has been termed 'centrochromatin,' may contribute to the unique structure and function of the centromere, in combination with the presence of CENP-A/CID (Sullivan, 2004).
The regions that flank CEN chromatin in fly and human samples contained H3 Lys9-diMe and triMe, and hypoacetylation of H3 and H4, consistent with previous studies of pericentric heterochromatin. In fission yeast, tRNA genes seem to be associated with boundaries between CEN and flanking chromatin. It is unclear at this time whether the separation of CENP-A/CID and flanking heterochromatin domains in humans and Drosophila reflects the presence of a sequence-specific boundary, or a sequence-independent balance between the two epigenetic states. The pericentromeric regions of human metaphase chromosomes contained H3 Lys9-triMe, although this modification is under-represented, but is not completely deficient, in Drosophila pericentromeric heterochromatin. Fly pericentromeric regions showed a much more substantial enrichment for H3 Lys9-diMe in metaphase chromosome and chromatin fiber analysis. These results are consistent with previous reports showing that H3 Lys9-diMe is concentrated at heterochromatic chromocenters in Drosophila salivary glands. Pericentric regions in flies contain essential genes, whereas few genes have been reported in the pericentric regions of human chromosomes. Perhaps higher-order heterochromatin is regulated differently between humans and flies by other histone-modifying enzymes and heterochromatin proteins, to allow the expression of heterochromatic genes. Further investigations of the distributions of different histone modifications in pericentric heterochromatin are necessary to validate these observations, and to determine whether they have functional consequences (Sullivan, 2004).
Two recent studies have discovered correlations between distinct heterochromatic domains and different degrees of H3 Lys9 methylation in mouse embryonic stem cells and embryonic fibroblasts. It was demonstrated by indirect immunofluorescence that H3 Lys9-triMe is enriched in pericentric regions of mouse chromosomes and at DAPI-bright regions in interphase nuclei. These results agree with the current findings that H3 Lys9 methylation is present in pericentric regions of human and fly chromosomes. Chromatin immunoprecipitation (ChIP) was used analysis to identify patterns of H3 methylation within mouse chromosomes. In mice, the centromere and pericentromeric regions contain distinct, expansive arrays of satellite DNA. Major satellite comprises the largest region and is immediately adjacent to the functional kinetochore, and minor satellite is the region where mouse kinetochore proteins are located. It was recently reported that major and minor satellite DNAs are enriched primarily for H3 Lys9-triMe, and that both satellites are associated with H3 Lys9-diMe to a lesser extent (Sullivan, 2004).
The presence of H3 Lys9 methylation in mouse minor satellite suggests that CEN chromatin may be modified differently in mice, in comparison with the results reported for for humans and Drosophila in this study. However, human centromeres contain expansive, megabase-sized arrays of alpha-satellite DNA, and CENP-A localizes to only a portion of these arrays. The demonstration that CEN chromatin in humans and flies lacks H3 Lys9 methylation is consistent with a similar model for centromere organization in mice, in which minor satellite DNA contributes partly to CEN chromatin and partly to heterochromatin formation. Additional studies on mouse centromeres are necessary to specifically map H3 Lys9 methylation with respect to CENP-A and CEN chromatin (Sullivan, 2004).
These studies have focused on centromeric chromatin structure in human cells and Drosophila cultured cells and larval brains. Is CEN chromatin in other organisms marked by the same histone modifications? Centromeres in S. pombe consist of a basic unit of central core chromatin that contains CENP-A (Cnp1), flanked on both sides by heterochromatin that is marked by H3 Lys9 methylation. The initial finding that subdomains of CENP-A/CID and H3 are interspersed in fly and human centromeres produced the hypothesis that centromeres in larger eukaryotes might represent amplification of the basic CEN domain unit (heterochromatin - CENP-A - heterochromatin) found in S. pombe. However, in the present study, no H3 Lys9 methylation was observed in the regions between CENP-A/CID subdomains with the CEN regions. Thus, the current results argue that fly and human centromeres are not composed of multimers of units equivalent to S. pombe centromeres. However, the overall organization of the centromere region is conserved, such that the entire CENP-A/CID chromatin domain is flanked by heterochromatin that contains H3 Lys9 methylation (Sullivan, 2004).
Alternatively, it is possible that CEN chromatin does differ among organisms. Recently, ChIP analysis of rice centromeric regions suggested that H3 Lys9 di-Me is present within the CEN chromatin (defined by the presence of the CENP-A homolog CenH3). This result may reflect differences in CEN chromatin composition and organization between plants and flies, humans and S. pombe. However, a more extensive analysis of the spectrum of modifications, including cytological studies of the distributions of modifications in extended fibers and mitotic chromosomes, needs to be carried out in different plant species to test this hypothesis (Sullivan, 2004).
What are the functional roles of histone modifications in CEN and flanking chromatin? (1) Distinct chromatin states in the CEN region may contribute to the diverse properties of centromeric domains, such as differential replication timing of the CEN and flanking heterochromatin. Heterochromatic modifications may also maintain centromere size by creating a barrier against expansion of CEN chromatin. In Drosophila, CEN chromatin readily spreads into neighboring sequences when flanking heterochromatin is removed, allowing neocentromere activation. (2) The stacking and self-association of CENP-A nucleosomes, distinctly modified interspersed H3 nucleosomes and flanking heterochromatin may be responsible for the three-dimensional structure of CEN chromatin in mitosis. This organization could facilitate kinetochore assembly by orienting CENP-A/CID chromatin toward the outside of the chromosome, where it can interact with kinetochore proteins. CEN-specific combinations of histone modifications and the three-dimensional organization could also be important for recruitment of cohesion complexes to heterochromatin near sister kinetochores, while ensuring spatial separation of cohesion and kinetochore domains (Sullivan, 2004).
(3) Distinctly modified, interspersed H3 nucleosomes could participate in epigenetic propagation of centromere identity. As observed for other histone 'variants', CENP-A/CID assembly can be replication-independent, unlike that of canonical H3 nucleosomes. Specifically modified interspersed H3 subdomains could create a 'permissive' chromatin structure necessary for assembly of new CENP-A16 (Sullivan, 2004).
A new model for deposition of CENP-A specifically in centromeric chromatin is suggested by these observations. Perhaps the modification pattern of interspersed H3 nucleosomes and histone modification proteins (such as acetyltransferases, methyltransferases and kinases) helps propagate centromere identity, in lieu of (or in addition to) CENP-A-associated proteins. Future studies are necessary to address mechanisms responsible for formation, maintenance and separation of these distinct chromatin states, and well as their roles in centromere structure and function. It is also important to determine whether other functional domains embedded within heterochromatin, such as the nucleolus organizer - ribosomal DNA, show distinct patterns of histone modifications (Sullivan, 2004).
The centromere-specific histone H3 variant CENP-A plays a crucial role in kinetochore specification and assembly. A genetic approach was undertaken to identify interactors of the Drosophila CENP-A homolog CID. Overexpression of cid in the proliferating eye imaginal disc results in a rough eye phenotype, which is dependent on the ability of the overexpressed protein to localize to the kinetochore. A screen for modifiers of the rough eye phenotype identified mutations in the Drosophila condensin subunit gene Cap-G as interactors. Yeast two-hybrid experiments also reveal an interaction between CID and Cap-G. While chromosome condensation in Cap-G mutant embryos appears largely unaffected, massive defects in sister chromatid segregation occur during mitosis. Taken together, these results suggest a link between the chromatin condensation machinery and kinetochore structure (Jager, 2005).
Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, a soluble assembly complex has been biochemically purified that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription (Furuyama, 2006a; full text of article).
RbAp48 is sufficient for centromeric chromatin assembly in vitro, but is it necessary for this process in vivo? RbAp48 is found in various chromatin-associated protein complexes, where it is thought to play a common role in mediating their interactions with histones. Although no mutations have been reported to eliminate Drosophila RbAp48 (NURFp55), mutations in other components of RbAp48-associated complexes are lethal [Nurf-38, E(z), sin3, and many others]; therefore, it would be expected that removal of RbAp48 would have pleiotropic effects. Indeed, knock-down of RbAp48 by RNAi in Drosophila S2 cells results in S phase arrest and derepression of various Rb/E2F target genes. These pleiotropic effects caused by reduction in RbAp48 levels would mask any centromere defect, and, in any case, such a defect would not be expected to occur immediately, because disruption of fission yeast RbAp48 did not affect chromosome segregation until the second round of mitosis (Furuyama, 2006a).
The single chaperone purified by using tagged CID contrasts with the multiple subunits found in purified chaperone complexes using tagged H3.1 and H3.3. The H3.1-specific replication-coupled assembly complex contains more than seven nonhistone subunits, and the H3.3-specific replication-independent complex contains at least five. Furthermore, H3.1- and H3.3-specific assembly reactions were performed in the presence of crude lysates, suggesting requirements for additional components that might restrict deposition to polymerase-driven processes. In contrast, both purified and reconstituted CID/H4-RbAp48 are sufficient for chromatin assembly in the absence of any other processes (Furuyama, 2006a).
The formation of chromatin from histones and DNA is a thermodynamically favorable reaction, and it is thought that histone chaperones are needed to prevent nonproductive aggregation between highly positively charged histones and highly negatively charged DNA in a dense protein environment. Both replication-coupled assembly of H3.1/H4 and transcription-coupled assembly of H3.3/H4 take place in the highly dynamic context of multisubunit polymerase transit, and assembly in both cases might require a large number of subunits to facilitate tethering of assembly complexes for rapid histone deposition. However, the basic assembly reaction appears to have minimal requirements, and conventional nucleosomes can be assembled in the presence of the NAP1 protein chaperone, polyglutamate, or high concentration of salt. It is suggested that the simplicity of CID/H4-RbAp48 reflects a simple in vivo situation in which assembly occurs in the absence of rapidly transiting polymerases and associated factors. Although both H3.1- and H3.3-specific complexes also contain RbAp48 and RbAp48 alone can assemble H3 nucleosomes, other components in these complexes might prevent spontaneous deposition at gaps in chromatin due to steric hindrance, whereas the much simpler CID/H4-RbAp48 would gain access to these chromatin gaps without impediment. In other words, H3- and H3.3-specific chromatin assembly complexes may have evolved to strictly couple their activities to replication and transcription, respectively, to increase the efficiency of these cellular processes, and to delineate assembly pathways of different histone 3 variants. There is precedence for such a variant-dependent exclusion mechanism: H3 appears to be prevented from assembling by replication-independent deposition anywhere in the genome, whereas H3.3 appears to deposit anywhere except at centromeres. When overproduced, CID deposits in a euchromatic pattern that is similar to that seen for H3.3, suggesting that CenH3s have fewer constraints than either H3 or H3.3 and that other chaperones in these complexes are the best candidates for mediating differential exclusion. Any CenH3 that incorporates in euchromatin at transient gaps created by transcription would be continuously replaced by transcription-coupled assembly of H3.3; in this way, CenH3 would be passively retained at centromeres but actively removed from transcriptionally active regions (Furuyama, 2006a).
Exclusion of H3 and H3.3 but not CenH3 from centromeric chromatin, such as by steric hindrance or RNA-mediated targeting, might help account for the deposition of CenH3s at a wide variety of sequences within a genome, including human neocentromeres, nematode holocentromeres, and gene-rich rice centromeres. Furthermore, budding yeast CenH3 (Cse4p) can localize properly to human centromeres and rescue a CENP-A depletion phenotype. Because of the high degree of divergence between Cse4p and CENP-A relative to the near invariance of H3, it is unlikely that a protein complex that normally recognizes CENP-A can associate with Cse4p and deposit it only at the centromeres. Rather, assembly of CenH3-H4 into centromeric chromatin in other organisms might be achieved by a simple H4-binding chaperone, such as RbAp48. Perhaps what distinguishes a CenH3 from a canonical H3 is that it is not accepted by H3- or H3.3-specific chaperone complexes (Furuyama, 2006a).
The efficient propagation of centromeric chromatin domains during every cell cycle requires the correct localization of CenH3s. The robustness and precision of this process is extraordinary; for example, the location of centromeres have not changed in this lineage for 30 million years. It has been proposed that the compact structure of the CENP-A/H4 protein tetramer leads to the perpetuation of correct CENP-A localization, but it is not clear how compactness by itself can facilitate the faithful recruitment of additional CENP-A/H4 protein tetramers during every cell division. The apparent simplicity of CenH3 assembly can provide a mechanism to delineate this assembly pathway from that of H3 and H3.3. Torsional stress induced at centromeres at anaphase may be an efficient mechanism to clear H3 or H3.3 from centromeres and to create gaps for CenH3 deposition. Thus, the assembly of centromeric nucleosomes at gaps, which are created by the very process that requires CenH3, would provide a robust self-enforcing mechanism to maintain centromeres indefinitely (Furuyama, 2006a).
Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID was used to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. This study highlights the benefits of the improved biotin-mediated purification method, and addresses the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres (Furuyama, 2006b).
Centromere identity is determined by the formation of a specialized chromatin structure containing the centromere-specific histone H3 variant CENP-A. The precise molecular mechanism(s) accounting for the specific deposition of CENP-A at centromeres are still poorly understood. Centromeric deposition of CENP-A, which is independent of DNA replication, might involve specific chromatin assembly complexes and/or specific interactions with kinetochore components. However, transiently expressed CENP-A incorporates throughout chromatin indicating that CENP-A nucleosomes can also be promiscuously deposited during DNA replication. Therefore, additional mechanisms must exist to prevent deposition of CENP-A nucleosomes during replication and/or to remove them afterwards. This study used transient expression experiments performed in Drosophila Kc cells to show that proteasome-mediated degradation restricts localization of Drosophila CENP-A (CID) to centromeres by eliminating mislocalized CID as well as by regulating available CID levels. Regulating available CID levels appears essential to ensure centromeric deposition of transiently expressed CID as, when expression is increased in the presence of proteasome inhibitors, newly synthesized CID mislocalizes. Mislocalization of CID affects cell cycle progression as a high percentage of cells showing mislocalized CID are reactive against alphaPSer(10)H3 antibodies, enter mitosis at a very low frequency and show strong segregation defects. However, cells showing reduced amounts of mislocalized CID show normal cell cycle progression (Moreno-Moreno, 2006; full text of article).
Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3 -- Centromere identifier). However, the mechanistic basis for centromere maintenance is unknown. Biochemical properties were investigated of CenH3 nucleosomes from Drosophila cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical 'beads-on-a-string' appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity (Dalal, 2007; full text of article).
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