centromere identifier


Promoter Structure

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).

Organization of centromeric heterochromatin in Drosophila

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).

Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes

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).

Replication of centromeric chromatin in Drosophila

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).

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, 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).

Drosophila CENP-C is essential for centromere identity

Centromeres are specialized chromosomal domains that direct mitotic kinetochore assembly and are defined by the presence of CENP-A (CID in Drosophila) and CENP-C. While the role of CENP-A appears to be highly conserved, functional studies in different organisms suggest that the precise role of CENP-C in kinetochore assembly is still under debate. Previous studies in vertebrate cells have shown that CENP-C inactivation causes mitotic delay, chromosome missegregation, and apoptosis; however, in Drosophila, the role of CENP-C is not well-defined. This study used RNA interference depletion in S2 cells to address this question, and it was found that depletion of CENP-C causes a kinetochore null phenotype, and consequently, the spindle checkpoint, kinetochore-microtubule interactions, and spindle size are severely misregulated. Importantly, CENP-C was shown to be required for centromere identity, since CID, MEI-S332, and chromosomal passenger proteins fail to localize in CENP-C depleted cells, suggesting a tight communication between the inner kinetochore proteins and centromeres. It is suggested that CENP-C might fulfill the structural roles of the human centromere-associated proteins not identified in Drosophila (Orr, 2011).

Kinetochores are assembled at the centromere of each replicated sister chromatid and provide an essential protein interface to allow binding of spindle microtubules and consequent chromosome segregation during mitosis. This study found that in Drosophila, CENP-C plays a major role not only in kinetochore organization but also in the proper assembly/maintenance of important centromere components suggesting that communication between the inner kinetochore and the centromere is an essential step in determining centromere identity (Orr, 2011).

CENP-C inactivation in vertebrate cells has been performed by antibody microinjection in HeLa cells, using CENP-C knockout mice or by tetracyclin-inducible knockouts in DT40 cells, and all studies concluded that CENP-C is essential for cell viability and mitotic progression. Detailed immunofluorescence analysis in CENP-C-deficient DT40 cells revealed a partial disruption of the inner kinetochore accompanied by a BubR1-dependent mitotic delay. While CENP-C inactivation in vertebrate cells causes partial disruption of the inner kinetochore, in Drosophila, CENP-C appears to perform more important roles. Consistently, bioinformatic approaches directed at evaluating CENP-C conservation between species reveals that while CENP-C is highly conserved among other Drosophila species, it bears very limited homology with its counterparts in higher eukaryotes. These differences may reflect different functions for the Drosophila CENP-C homolog and argue in favor of a different centromere-kinetochore interface specific to Drosophila chromosomes (Orr, 2011).

This study shows that CENP-C is required for the loading/maintenance of all kinetochore proteins tested including the SAC proteins (Mad2, Bub1, BubR1, and Bub3), mitotic regulator Polo kinase, microtubule motor protein CENP-meta, and KMN proteins (Ndc80, Nuf2, and Mitch). Interestingly, the kinetochore null phenotype observed after CENP-C depletion appears to be specific to Drosophila and C. elegans chromosomes, since CENP-C has been shown not to be required for full kinetochore organization in higher eukaryotes. Similar to Drosophila, no Constitutive Centromere-Associated Network (CCAN) homologs have yet been identified in C. elegans, which suggests that in systems lacking CCAN, centromere function relies uniquely on the structural role of CENP-C. Different to what has been reported in vertebrate cells, the current results are consistent with a model in which CENP-C is required to lay the foundation for all components essential for kinetochore assembly (Orr, 2011).

Previous reports have shown that loss of CENP-C in mammalian cells causes a mitotic delay. In chicken cells, this mitotic delay is BubR1 dependent and associated to a 3-fold increase in the overall duration of mitosis. This study demonstrated that in the absence of CENP-C, Drosophila kinetochores are unable to recruit essential SAC proteins Mad2, Bub1, BubR1, and Bub3, even if mitotic exit is prevented and microtubules removed. Nevertheless, consistent with the observed loss of SAC proteins, these cells are insensitive to microtubule poisons and rapidly exit mitosis in the presence of spindle damage. As expected when analyzing SAC-deficient phenotypes, these cells undergo fast mitotic exit accompanied by premature sister chromatid separation. Cells exit mitosis with a mitotic timing similar to what has been observed after Mad2 depletion in the same cell line, which suggests that this 12-min period is the minimum time these cells require to complete all processes required for mitotic exit. Two possible hypotheses could explain why CENP-C inactivation in other systems causes cells to block in mitosis. Either CENP-C inactivation was not as efficient in other species as it is in Drosophila S2 cells or these discrepancies could reflect structural differences in kinetochore organization specific to Drosophila chromosomes. Interestingly, Drosophila CID mutants display mislocalization of several kinetochore components accompanied by a BubR1-dependent mitotic delay, which suggests that CID inactivation cannot account for the loss of SAC maintenance observed when disrupting Drosophila CENP-C. However, in the case of CID mutants, maternally contributed CID might have occluded phenotypes that may explain the SAC-dependent mitotic delay in these cells. This study shows that kinetochore null cells fail to maintain SAC activity even in the presence of microtubule poisons, which suggests that kinetochore inactivation is not compatible with a functional SAC. Taken together, these data demonstrate that CENP-C is essential for full kinetochore assembly, a pre-requisite for efficient SAC maintenance (Orr, 2011).

In Drosophila, the localization of all outer kinetochore proteins appears to be dependent on CENP-C. Moreover, CENP-C is an essential factor for CID assembly at Drosophila centromeres. In accordance, it was recently proposed that CCAN copy number at kinetochores varies between vertebrates and yeast, suggesting that although specific centromere/kinetochore assembly models appear to be conserved, differences in protein copy number may reflect structural discrepancies between phenotypic analyses. The current data confirm that efficient CENP-C depletion causes CID mislocalization at centromeres, and this appears to be specific to Drosophila centromeres as it has never been observed in other systems. Collectively, these studies highlight potential differences in kinetochore organization between Drosophila and vertebrate cells (Orr, 2011).

The data also demonstrate that in Drosophila, CENP-C is essential for the proper localization of other centromere-specific proteins including the cohesion protector MEI-S332 and the CPC components INCENP and Aurora B. Taken together, these results are consistent with the proposal that Drosophila CENP-C is essential for maintaining normal centromeric architecture and identity, which appears to be species specific. In vertebrates, however, a large cluster of constitutive centromere-associated proteins (CENP-C, CENP-H, CENP-I, and CENP-K to CENP-U, and CENP-X) was identified as the CCAN which associates with CENP-A throughout the cell cycle, although a recent report also identified CENP-W that forms a DNA-binding complex together with CENP-T, all of which have no identified Drosophila orthologs. However, similarly to CENP-C, many of the CCAN proteins may have failed to be detected in the Drosophila genome because they lack significant conservation. At this point, it is not possible to rule out this possibility, although it is clear that in Drosophila, CENP-C plays an essential role in overall centromere and kinetochore organization, a role that might be shared with the CCAN protein complexes in other systems (Orr, 2011).

Together with the cumulated published evidence on the functional analyses of CID and CENP-C, the data suggest that the Drosophila centromere/kinetochore interface is simpler than that of higher eukaryotes. It is proposed that CENP-C plays a direct role in maintaining centromere identity and may fulfill many of the structural roles of CCAN complex proteins present in other organisms. Importantly, it was shown that there is functional communication between the inner kinetochores and the centromere, and at this point, it would be essential to understand which proteins are responsible for performing analogous functions at centromeres of higher eukaryote cells (Orr, 2011).

Protein Interactions

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).

Chaperone-mediated assembly of centromeric chromatin in vitro

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).

Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells

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).

A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A

Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. This study shows that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, it was found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A (Lagana, 2010).

Epigenetic regulation of genome activity is critical during development and stem cell maintenance, and increasing amounts of evidence highlight its importance in cancers. However, mechanisms controlling epigenetic regulation during a single cell cycle are generally less well understood, compared with those involved in transcriptional programmes. Centromere specification is an epigenetic regulatory event that controls genome activity at singular chromosomal loci and occurs each cell cycle. Nucleosomes that contain CENP-A are thought to epigenetically define centromeres. During DNA replication, centromere identity is maintained by segregating CENP-A equally to the two daughter chromosomes. Before the subsequent S-phase, additional CENP-A must be incorporated at centromeres, thus propagating the centromere epigenetic mark. Critical to this cycle is maintenance of the proper amount of CENP-A; too little or too much CENP-A incorporation could result in either loss of centromere identity or errors in chromosome segregation. This study describes a mechanism to ensure maintenance of the proper CENP-A levels during the cell cycle regulated by a Rho family small GTPase molecular switch (Lagana, 2010).

Proteomics and quantitative imaging assays were used to identify a previously unknown step in centromere maintenance. MgcRacGAP, together with the GEF ECT2, and their cognate small GTPase Cdc42 (or possibly Rac) specifically maintain CENP-A at centromeres. MgcRacGAP localization to centromeres at the end of G1 is incongruous with a role in CENP-A loading and strongly suggests that MgcRacGAP acts in maintenance and not licensing or loading of CENP-A. Pulse-chase analysis revealed that MgcRacGAP is required specifically for maintenance of newly incorporated CENP-A as old CENP-A from the previous cell cycle was present at normal levels at centromeres. Reciprocal immunoprecipitation of MgcRacGAP did not isolate HsKNL2, probably because of a large excess of MgcRacGAP bound to other known interacting proteins in the cytoplasm (data not shown). These results support the conclusion that a minor subset of MgcRacGAP is bound to HsKNL2 for a brief period each cell cycle and imply that non-overlapping MgcRacGAP-containing protein complexes function in cells. Overall, this work defines a new event in epigenetic centromere regulation and reveals its control by a small GTPase molecular switch (Lagana, 2010).

A model is proposed wherein the HsKNL2–Mis18 complex licenses centromeres for loading of new CENP-A by the combined activities of HJURP and CAF1. After loading (approximately 8–12 h after anaphase onset), HsKNL2–Mis18 recruits Cdc42. The activity of Cdc42 is required for preservation of newly incorporated CENP-A and thus finalizes centromere repopulation. Cdc42 activity requires GTPase cycling facilitated by MgcRacGAP and the GEF ECT2. The results predict that newly incorporated CENP-A is distinct from CENP-A remaining from the previous cell cycle and can be recognized and removed. It is proposed that Cdc42 activity modifies (by either adding or removing a mark on) newly incorporated CENP-A, rendering it identical to old CENP-A. The manifestation of this mark could be any distinguishing modification, including but not limited to, recruitment of an additional protein, conformational change of the CENP-A nucleosome, or any of a range of post-translational modifications. New CENP-A that is not modified would be recognized as erroneously incorporated and removed from chromatin during a late-G1 surveillance step, or during DNA replication (Lagana, 2010).

In budding yeast, excess CENP-A (CSE-4) mislocalized to the chromosome arms is removed and selectively degraded through a proteasome-based mechanism. If this mechanism is conserved in human cells, it is expected to be less stringent, as overexpressed CENP-A localizes diffusely to chromosome arms without causing obvious defects in cell division. Alternatively or additionally, centromere maintenance could involve the chromatin remodelling protein RSF-1, which is required for CENP-A nucleosome stability. However, because RSF-1 is proposed to function in mid-G1 before MgcRacGAP and Cdc42 localize to centromeres, it is unlikely to be the downstream target of small GTPase activity at centromeres (Perpelescu, 2009). Regardless of the removal mechanism, it is proposed that a GTPase switch is spatially and temporally restricted through regulated localization to centromeres precisely after CENP-A doubling to promote the removal of spurious CENP-A (either excess at centromeres, or outside true centromere loci). By restricting centromere size, this 'quality control' mechanism helps to ensure proper centromere function and kinetochore assembly, thus preventing aneuploidy. Furthermore, it is possible that this mechanistic theme will apply to other epigenetic events that contribute to genomic regulation (Lagana, 2010).

Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, factors required for centromere propagation were isolated using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila. The proteins CAL1 and CENP-C were identified as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. The mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 were identified as regulators of centromere propagation. CYCA was shown to be centromere localized, and CYCA and RCA1/Emi1 were shown to couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. These findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division (Erhardt, 2008).

This is the first example of a genome-wide RNAi screen for mislocalization of an endogenous chromosomal protein and provides the distinct advantage that the primary screen output is a direct readout of the phenotype of interest. This approach identified novel and known factors that control the assembly of centromeric chromatin and link centromere assembly and propagation to the cell cycle (Erhardt, 2008).

Although centromere assembly has been described as a hierarchical process directed by CENP-A, the data show that CID, CENP-C, and CAL1 are interdependent for centromere propagation, which is consistent with experiments in vertebrate cells showing interdependence between the CENP-H-CENP-I complex and CENP-A. However, studies in C. elegans and vertebrates have not detected a role for CENP-C in CENP-A chromatin assembly, suggesting that CENP-C plays a more prominent role in regulating centromere propagation in flies. Collectively, these results suggest that CENPs that depend on CENP-A for their localization may 'feed back' to control CENP-A assembly. Histone variants are assembled into chromatin both by histone chaperones (e.g., the histone H3.3-specific chaperone HIRA [histone regulatory A] that provides specificity to the CHD1 chromatin-remodeling ATPase) and by histone variant-specific ATPases (e.g., Swr1 that can use the general chaperone Nap1 or the specific chaperone Chz1 to assemble H2A.Z). CENP-C or CAL1 might facilitate centromere-specific CID localization by providing centromere specificity to a chromatin-remodeling ATPase in a manner analogous to HIRA or might direct the localization of chromatin assembly factors to the centromere. It will be interesting to determine what factors associate with CAL1 and CENP-C as a route to elucidating the mechanisms of centromere assembly and propagation (Erhardt, 2008).

The loading of CENP-A in human somatic cells and in Drosophila embryos occurs after anaphase initiation when APCFZR/CDH1 activity is high. Ubiquitin-mediated proteolysis facilitates formation of a single centromere by degrading noncentromeric CENP-A, and subunits of the APC are localized to kinetochores. The results demonstrate that normal regulation of APCFZR/CDH1 activity is required for centromere propagation, providing a link between centromere assembly and cell cycle regulation (Erhardt, 2008).

Two alternative models are proposed for the role of APCFZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APCFZR/CDH1 and that the kinase activity of the CYCA-Cdk1 complex is required for the localization of CID, CENP-C, and CAL1 to the centromere. CYCA is normally degraded as cells proceed through mitosis, suggesting that CYCA-Cdk1 would likely act during G2 or early M to phosphorylate a substrate involved in centromere assembly. The CID and CENP-C localization defect caused by CYCA depletion was rescued by the simultaneous depletion of FZR/CDH1 even though the levels of CYCA protein remained low in the double depletion. The rescue of the CID and CENP-C localization defect in cells with low CYCA protein suggests that maintaining high levels of CYCA-Cdk1 activity is not required for centromere propagation, but it cannot be ruled out that the residual CYCA protein in these cells is sufficient to rescue the centromeric phenotype when APC activity is compromised by FZR/CDH1 depletion (Erhardt, 2008).

The second model that is consistent with these observations is that one or more APCFZR/CDH1 substrates (“X”) regulate the interdependent localization of CID, CENP-C, and CAL1 to the centromere. RCA1 and CYCA inhibit the APC in G2 to allow mitotic cyclin accumulation. An APCFZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APCFZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APCFZR/CDH1 activation by CYCA or RCA1 depletion might cause an increase of CID at centromeres as a result of premature assembly. It was observed that neither CDH1 nor CDC20 depletion alone impacted CID, CAL1, or CENP-C assembly at centromeres or the overall levels of these proteins but that premature APC activation resulted in failed centromere assembly (Erhardt, 2008).

A simple interpretation of the results is that CYCA-Cdk1 or another APCFZR/CDH1 substrate acts during G2/metaphase before APCFZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APCFZR/CDH1 substrate would cause failure to relicense the centromeres for assembly in the next G1 phase. When compared with the process of replication licensing, in which the positive regulator CDC6 and the negative regulators geminin and CYCA are all substrates of APCFZR/CDH1, the model of a single APCFZR/CDH1 substrate that controls centromere licensing or propagation may be oversimplified. This study observed that defective centromere localization of CID and CENP-C after CYCA or RCA1 depletion was not rescued by CDC20 depletion, but a role for APCFZY/CDC20 in centromere propagation cannot be ruled out because premature APCFZR/CDH1 activation could mask a subsequent role for FZY/CDC20, which is activated at the metaphase/anaphase transition (Erhardt, 2008).

It is not yet known whether the localization of CYCA at centromeres is important for the regulation of centromere assembly. In Drosophila, it has been demonstrated that the subcellular localization of CYCA is not important for proper progression through the cell cycle; however, these experiments did not directly address whether mislocalization of CYCA prevented the association of CYCA with centromeres. It will be interesting to determine whether CID, CENP-C, and CAL1 localization require centromere-localized CYCA-Cdk1 activity or whether any of these proteins are a direct target of CYCA-Cdk1 (Erhardt, 2008).

The results suggest that CID or CAL1 levels are indirectly controlled by APC activity. Interestingly, the human M18BP1 has recently been proposed to act as a 'licensing factor' for centromere assembly. Although no clear homologues of M18BP1/KNL2 have been identified in Drosophila, both CAL1 in flies and M18BP1/KNL2 in other species are interdependent with CENP-A for centromere localization. Strikingly, levels of CAL1 and M18BP1/KNL2 are reduced on metaphase centromeres and increase coincident with CENP-A loading in late anaphase/telophase. Further analysis is required to determine whether CAL1 and M18BP1/KNL2 function analogously in centromere assembly. It will be important to determine whether fly homologues of other Mis18 complex components are associated with CAL1 and important for centromere assembly. Identifying the APC substrates involved in centromere assembly will be necessary to distinguish between these models and to determine how these proteins epigenetically regulate centromere assembly and couple this essential process to the cell cycle (Erhardt, 2008).

Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1

Propagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere-specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp-A/Cid is required for the recruitment of Cenp-C, another conserved centromere protein. With yeast three-hybrid experiments, this study demonstrates that the essential Drosophila centromere protein Cal1 can link Cenp-A/Cid and Cenp-C. Cenp-A/Cid and Cenp-C interact with the N- and C-terminal domains of Cal1, respectively. These Cal1 domains are sufficient for centromere localization and function, but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores, it was demonstrates that centromeric Cal1 levels are far lower than those of Cenp-A/Cid, Cenp-C and other conserved kinetochore components, which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore, Cal1 limits centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis. The low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp-A/Cid and Cenp-C are present in excess (Schittenhelm, 2010).

The centromeric regions of chromosomes direct formation of kinetochores, which allow chromosome attachment to spindle microtubules. Centromeres and kinetochores are therefore of paramount importance for faithful propagation of genetic information. However, centromeric DNA sequences are not conserved. Most eukaryotes (including Drosophila melanogaster and humans) have regional centromeres with up to several megabases of repetitive DNA. Importantly, these repetitive sequences are neither necessary nor sufficient for centromere function, indicating that there is an epigenetic centromere specification (Schittenhelm, 2010).

A centromere-specific histone H3 variant (CenH3) is thought to be crucial for epigenetic centromere marking. CenH3 proteins are present in all eukaryotes (e.g. CENP-A in humans and Cid in Drosophila). They replace histone H3 in canonical nucleosomes or possibly variant complexes. Depletion of CenH3 results in a failure to localize most or all other centromere and mitosis-specific kinetochore proteins. Strong overexpression of Drosophila Cenp-A/Cid results in incorporation at ectopic chromosomal sites, which in part also assemble ectopic kinetochores during mitosis (Schittenhelm, 2010).

Ectopic kinetochores result in chromosome segregation errors and genetic instability. Ectopic CenH3 incorporation therefore must be prevented. Although still fragmentary, the understanding of the molecular mechanisms that regulate CenH3 incorporation is progressing rapidly. In proliferating cells, an additional complement of CenH3 needs to be incorporated during each cell cycle. In syncytial Drosophila embryos, this occurs during exit from mitosis. Similar findings were made in human cells, where Cenp-A deposition occurs during late telophase and early G1 phase. The number of factors shown to be required for normal CenH3 deposition is increasing rapidly, which suggests that there is an intricate control mechanism. Various and often dedicated chaperones, chromatin modifying and remodelling factors, as well as other centromere components are involved (Schittenhelm, 2010).

In Drosophila, Cenp-C is incorporated into centromeres concomitantly with Cenp-A/Cid. High-resolution mapping with native Drosophila chromosomes has indicated that these two proteins do not have an identical localization within the kinetochore. Although these localization studies cannot exclude an association between subfractions of Cenp-A and Cenp-C, direct molecular interactions between these centromere proteins have not yet been reported. Recently, however, Cal1 has been identified in Drosophila and shown to be required for normal centromeric localization of Cenp-A/Cid and Cenp-C. Moreover, these three Drosophila centromere proteins can be co-immunoprecipitated from soluble chromatin preparations. Cal1 might therefore provide a physical link between Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

This study reports that Cal1 has distinct binding sites for Cenp-A/Cid and Cenp-C. It can link these proteins together according to yeast three-hybrid experiments. However, the level of centromeric Cal1 is far lower than that of Cenp-A/Cid and Cenp-C. Cal1 therefore cannot function as a stoichiometric linker connecting each monomer or dimer of Cenp-C to Cenp-A within the centromere. But the low levels of Cal1 effectively protect cells against mitotic defects resulting from increased centromeric incorporation of excess Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

cal1 is an essential gene that is expressed specifically in mitotically proliferating cells. To provide its function, the protein product needs its N-terminal domain, which interacts with Cenp-A/Cid, as well as its C-terminal domain, which interacts with Cenp-C. By contrast, the most rapidly diverging middle region of Cal1 seems to be of lesser importance because expression of the N-C version, which lacks the M domain, is sufficient to prevent the characteristic defects in cal1 mutant embryos. The obvious functionality of the N-C version also emphasizes the importance of the centromeric localization of Cal1. The complete Cal1 protein is observed not only at the centromere, but also in the nucleolus. The M region is both sufficient and required for nucleolar localization. However, because this M domain is not required for cal1 mutant rescue, the significance of the nucleolar Cal1 localization remains unclear (Schittenhelm, 2010).

Rescue of cal1 mutants is not observed when the N- and C-terminal domains of Cal1 are expressed without a covalent linkage. The ability to recruit Cenp-A/Cid and Cenp-C into a complex, as clearly evidenced by yeast three-hybrid experiments, is therefore likely to be crucial for Cal1 function. Co-immunoprecipitation of Cal1, Cenp-A/Cid and Cenp-C has previously indicated that these components can associate in vivo. However, quantification of protein levels, which is largely dependent on the accuracy of EGFP signal quantifications, demonstrates that Cenp-C is not exclusively anchored to centromeric chromatin via persistent and stoichiometric Cal1-mediated links to Cenp-A/Cid. Centromeric Cal1 levels are more than 40-times lower than those of Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

The centromeric amount of Cal1 is also far lower than that of the other kinetochore components that have been quantified (Spc105, Spc25, Nuf2). Interestingly, per kinetochore, the copy numbers of these components appear to be scaling well with the number of kinetochore microtubules (kMTs) when comparing the current results from Drosophila with those described for budding and fission yeast. Spc25 and Nuf2 are constituents of the heterotetrameric Ndc80 complex, which binds directly to kinetochore microtubules (kMTs) (see Models of kinetochore assembly). Eight copies of the Ndc80 complex are thought to bind a single kMT to the budding yeast kinetochore. In Drosophila, where the number of kMTs per kinetochore appears to be around 11, about seven copies appear to be present per kMT according to this quantification. This quantification of kinetochore proteins fits very well with the notion that the kinetochores of higher eukaryotes might be composed of several copies of a module that is present in one copy in budding yeast. By contrast, the centromere proteins Cenp-A and Cenp-C are scaling less well with the number of kMTs. The increased complexity of lateral co-ordination within animal kinetochores and of epigenetic specification of centromere identity might explain the higher relative amount of centromere proteins apparent in Drosophila. Despite this relative increase, centromeric Cenp-A/Cid allows packaging of only about 5% of the centromeric DNA in Drosophila under the assumption that Cenp-A/Cid nucleosomes wrap about 200 bp of a 200 kb centromere (Schittenhelm, 2010).

Although these quantifications exclude the notion that Cal1 functions as a stable stoichiometric linker of Cenp-A/Cid and Cenp-C in mitotic kinetochores, the overexpression experiments provide further support for a role as a centromere protein-loading factor. Moreover, these experiments reveal additional layers of regulation that prevent excess incorporation of centromere proteins within the centromeric region. They also indicate that such excess incorporation is highly detrimental to kinetochore function. Previous work in Drosophila has demonstrated that strong overexpression of Cenp-A/Cid (about 70-fold) can lead to ectopic kinetochore formation. However, almost all Cenp-A/Cid that is incorporated ectopically within the chromosome arm regions is degraded rapidly, which is also observed in yeast. This study shows that the limiting amounts of Cal1 provide additional, highly efficient protection against excessive chromosomal incorporation of Cenp-A/Cid. After bypassing this protection by Cal1 overexpression, even low levels of Cenp-A/Cid overexpression (about 2.5-fold) result in increased incorporation into centromeres (about 1.6-fold). When, in addition to Cal1 and Cenp-A/Cid, Cenp-C is also mildly co-overexpressed (about 3.5-fold), the levels of centromeric Cenp-A/Cid are further increased (about 2-fold) along with those of Cal1 and Cenp-C. Importantly, co-overexpression of these centromere proteins resulted not only in increased centromeric levels, but also in severe mitotic defects (Schittenhelm, 2010).

Although other interpretations are not excluded, these findings strongly suggest that the mitotic defects observed after overexpression of Cal1 and Cenp-A/Cid, and even more strongly when Cenp-C was also overexpressed, reflect the consequence of the increase in the centromeric levels of these proteins. The increase in centromeric levels of centromere proteins was accompanied by a significant increase in kinetochore proteins (Spc105 and the Mis12 and Ndc80 complex) but only to a very limited extent and only when all three centromere proteins were co-expressed. The increased amounts of centromeric Cenp-A/Cid observed after co-expression of Cal1 and Cenp-A/Cid, which were not accompanied by a statistically significant increase in kinetochore protein levels, might therefore be sufficient to disturb the spatial organization of the kinetochore, leading to inefficient chromosome congression, spindle checkpoint hyperactivation and chromosome segregation defects in anaphase (Schittenhelm, 2010).

Experiments in stg mutant embryos, demonstrate that co-overexpression of centromeric proteins during interphase is not sufficient to cause excess centromeric incorporation, consistent with the previously demonstrated dependence of centromeric deposition of Cenp-A/Cid and Cenp-C on exit from mitosis. Indeed, forcing progression through mitosis (by hs-stg induction) was observed to be sufficient to cause centromeric deposition of the overexpressed proteins. Moreover, the fact that the excess centromere proteins that were not yet incorporated into the centromere did not disturb the hs-stg induced mitosis, further supports the suggestion that the mitotic defects observed after co-expression of centromeric proteins depend on excessive incorporation into the centromere (Schittenhelm, 2010).

The severe mitotic defects observed after co-overexpression of Cal1, Cenp-A/Cid and Cenp-C emphasize the importance of careful control of centromere protein deposition. Several levels of control are effective. The interdependence of Cal1, Cenp-A/Cid and Cenp-C functions in conjunction with cell cycle control to prevent detrimental excessive centromeric incorporation. The cell cycle regulators cyclin A, Rca1/Emi1 and Fzr/Cdh1 have recently been implicated in the control of deposition of Cenp-A/Cid and Cenp-C at the centromere. How these and possibly additional cell cycle regulators control centromere protein deposition has yet to be clarified (Schittenhelm, 2010).

A possible scenario for centromere protein deposition in Drosophila might include a release of nucleolar Cal1 at the onset of mitosis, followed by conversion into a form that associates with non-centromeric soluble Cenp-A/Cid during exit from mitosis. After binding of soluble Cenp-A/Cid to the N-terminal domain of Cal1, its C-terminal domain might become exposed so that it can bind to centromeric Cenp-C and promote Cenp-A/Cid transfer onto the neighboring centromeric chromatin and thereby indirectly also additional Cenp-C deposition (Schittenhelm, 2010).

The mechanisms and the extent of control of centromeric Cenp-A deposition appear to have evolved. In fission yeast, overexpression of Cenp-A/Cid alone is sufficient to obtain excess centromeric Cenp-A/Cnp1, and this excess does not result in increased kinetochore protein levels. Spreading of Cenp-A within centromeric chromatin has also been clearly demonstrated in human cells after mild overexpression of Cenp-A. Mitotic defects were not detected in this case, perhaps because of the very limited increase in centromeric Cenp-A. Cal1 homologs from non-Drosophilid genomes have not yet been identified so far. Conversely, with the exception of Cenp-C, homologs of the 15 components of the vertebrate centromere chromatin-associated network (CCAN), which is related to the yeast Ctf19 and Sim4 complexes, have not been revealed in Drosophilid genomes, neither by thorough bioinformatic analyses nor by genome-wide RNAi screens. The CCAN seems also to be absent in C. elegans. It is conceivable therefore that Cal1 is a functional analog of the CCAN, which has also been implicated in Cenp-A loading. However, because the evolutionary sequence conservation of centromere and kinetochore components is generally very low, it remains a possibility that Cal1 homologs also exist and function in centromere loading of human Cenp-A and Cenp-C (Schittenhelm, 2010).

Assembly of Drosophila centromeric chromatin proteins during mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. This study analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and Chromosome alignment defect 1 (CAL1), which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation (Mellone, 2011).

CID, CAL1 and CENP-C display different turnover and assembly dynamics, despite the fact that these essential centromeric components interact physically, and are interdependent for centromere localization. Epitope tagged SNAP-CID, SNAP-CAL1 and SNAP-CENP-C were expressed from the identical Copia promoter; thus it is unlikely that these distinctions are due to different rates of new protein synthesis. Using a pulse-chase strategy, it was shown that CID levels are reduced by ~50% after one cell cycle, which could result from semi-conservative distribution of pre-existing CID nucleosomes, or random redistribution of parental CID-H4 tetramers, to replicated sister chromatids. While CID and CENP-C display stable association with centromeres and 50:50 distribution after each cell cycle, 66% of TMR-CAL1 is replaced by new protein. Thus, CAL1 is either less stably bound, or its replenishment involves partial removal of pre-existing protein. Alternatively, CAL1 could undergo an even higher turnover and the quantification could be an underestimation; CAL1 could be entirely recruited de novo and the measured centromeric TMR-CAL1 could reflect recruitment from an initial soluble pool at the time of labeling (Mellone, 2011).

An additional difference is that while SNAP-CID and CAL1 are detectable at centromeres 1 h after quenching the SNAP epitopes, 10 h of chase time are necessary for CENP-C to be visible by fluorescent substrate tetramethylrhodamine (TMR) labeling. This suggests that at each cell cycle the recruitment of CID and CAL1 relies for the most part on newly-synthesized protein, while CENP-C recruitment also involves a pre-existing non-centromeric or soluble pool. Indeed, the cellular fractionation analysis demonstrated the presence of low levels of CENP-C in chromatin-free extracts, supporting the possibility that there is a soluble pool of CENP-C available to replenish the centromere-associated CENP-C diluted during the cell cycle (Mellone, 2011).

CENP-C is targeted to centromeres during multiple cell cycle stages, consistent with previous findings in human cells. In contrast, newly-synthesized CAL1 and CID are recruited to centromeres during discrete stages of mitosis. Using quench-chase-pulse time-courses in both asynchronous and arrested cultures, it was demonstrated that the contribution of interphase to CID loading is minimal, since the percent of interphase cells displaying newly-synthesized SNAP-CID and the signal intensity of TMR-CID differ dramatically from those measured for mitotic cells. These observations distinguish Drosophila from human HeLa cells, where CENP-A is recruited during G1, from fission yeast, where CENP-A assembles at centromeres in both S and G2 phases, as well as from plants and Dictyostelium (G2/prophase) (Mellone, 2011).

Both new CID and CAL1 are assembled at centromeres in mitosis, but each protein is recruited during discrete stages: prophase for CAL1 and metaphase for CID. It is possible that CID and CAL1 loading are initiated simultaneously in prophase, but CAL1 levels accumulate faster than CID at centromeres. Regardless, the observed temporal distinction suggests that CAL1 acts upstream of CID recruitment (see Model for centromere assembly in Drosophila cells.). Incorporation of nascent CAL1 at centromeres during prophase could be mediated by binding to pre-existing centromeric CID and CENP-C. This could in turn promote new incorporation of nascent CID during metaphase, either by gap-filling or exchange of space-holder histone H3 (Mellone, 2011).

Interestingly, a similar temporal distinction has been described for the human centromere proteins hMis18α, β and M18BP, which localize to centromeres in anaphase, before new CENP-A assembly in late telophase/G1. The lack of any physical interaction between hMis18α, β, M18BP and CENP-A, and the observation that hMis18α can localize to centromeres even if CENP-A is depleted, has led to the proposal that this complex may 'prime' centromeres to receive new CENP-A (Fujita, 2007) from the HJURP chaperone, whose centromeric targeting coincides temporally with deposition of new CENP-A. Homologs for hMis18 complex components and HJURP (or the budding and fission yeast Scm3 homologs) have not been identified in the Drosophila genome (Mellone, 2011).

Collectively these data support a model in which CAL1 performs functions attributable to both HJURP and hMis18, despite the lack of sequence homology. hMis18 proteins are recruited to centromeres before CENP-A, and CAL1 loading precedes CID assembly. However, the hMis18 complex does not interact with CENP-A, whereas CAL1 and CID are associated in chromatin-free extracts, identifying the first Drosophila protein that binds CID in its pre-nucleosomal form. HJURP also interacts with pre-nucleosomal CENP-A, and both HJURP and CAL1 strongly colocalize with nucleoli. Thus, CAL1 could 'prime' the centromere in prophase, and also mediate CID recruitment directly in metaphase (Mellone, 2011 and references therein).

It has been showm that gross-levels of centromeric GFP-CID and GFP-CENP-C did not visibly change through the cell cycle in time-lapse analysis, consistent with the 50:50 segregation observed in this study during one division. In contrast, GFP-CAL1 levels were significantly reduced in metaphase, increased again in telophase, and remained stable through interphase. The transient reduction in GFP-CAL1 levels at metaphase is intriguing, given that it coincides with new CID assembly. The observation that newly assembled TMR-CAL1 intensities were constant from prophase to cytokinesis suggests that most of the GFP-CAL1 reduction at metaphase and increase at telophase involves pre-existing protein. One model to account for these observations is that free CAL1 (not bound to CID) is recruited to centromeres in prophase where it performs a yet undefined ‘priming’ function; then, the subset of CAL1 bound to pre-nucleosomal CID escorts it to centromeres in metaphase while 'old' CAL1 is displaced (Model for centromere assembly in Drosophila cells.). The interdependency of CAL1, CID and CENP-C in centromere localization could be explained by the requirement of pre-existing CID and CENP-C for CAL1 assembly in prophase (Mellone, 2011).

The loading of CID and CAL1 in specific, early stages of mitosis also raises questions about the nature of the signal(s) that initiate assembly of centromeric chromatin. Centromere replenishment signaling by kinetochore-microtubule interactions is inconsistent with the demonstration that CID loading in metaphase is not affected by colchicine treatment, and therefore does not require spindles (as also observed in human cells), SAC inactivation, chromosome segregation, or inter-kinetochore tension. However, it has been shown that premature activation of the Anaphase Promoting Complex, by Cyclin A or RCA1 depletion, interferes with CID localization to centromeres, demonstrating that centromeric chromatin assembly is linked to key regulators of mitotic progression. Interestingly, Cyclin A localizes to centromeres and is degraded in metaphase; this study has demonstrated that metaphase loading depends on proteasome activity, which could include degradation of key mitotic regulators. MG132 treatment prior to BTP block prevented CID loading while transfecting cells with a non-degradable form of CYCA abrogated new CID recruitment in a subset of cells, and TMR-CID levels were significantly reduced in most cells. One possibility to explain the stronger impact of proteasome inhibition is that proteasome targets in addition to CYCA need to be degraded for efficient CID deposition. Alternatively, the presence of centromeric endogenous CYCA, which is probably degraded normally in the presence of excess ND-CYCA, might trigger a sufficient signal to initiate CID incorporation in some cells. Interestingly, Cyclin A is degraded in the presence of microtubule drugs and escapes inhibition of the APC by the SAC, which would explain why new CID recruitment takes place efficiently in the presence of colchicine. Proteasome and ubiquitin-ligase activities have been implicated in controlling proper CENP-A centromeric incorporation by degradation of euchromatic CENP-A in budding yeast and Drosophila. Understanding the relationship between the CENP-A degradation pathway and the implication of proteasome activity in the recruitment of nascent CENP-A will require further investigation (Mellone, 2011).

It is unclear at this point how degradation of CYCA contributes to CID assembly. One possibility is that high CDK-CYCA activity at the centromere inhibits CID recruitment, and that local inhibition of CDK activity through degradation of CYCA or other substrates triggers CID assembly. Understanding the role of degradation of Cyclin A and other APC and proteasome substrates in CID recruitment will be crucial to elucidating how centromere assembly is coupled to the cell cycle (Mellone, 2011).

The dynamics of centromere replenishment in Drosophila cultured cells differs from those observed in S. pombe and human HeLa cells. Early syncytial fly embryos display slightly later recruitment of new CID in anaphase, but this difference could be due to the unusually short nuclear cycles that lack G1 and G2 phases. Although CENP-A loading in HeLa cells is first observed in telophase, it is possible that the primary signal to initiate CENP-A loading (e.g. inhibiting local CDK-CYCA activity at the centromere) is conserved, and occurs during prophase or metaphase in both Drosophila and human cells (Mellone, 2011).

It is also puzzling that key proteins required in trans for CENP-A assembly, such as HJURP and CAL1, are not always conserved, in contrast to the universality of centromeric chromatin components such as CENP-A and CENP-C. It is possible that highly diverged proteins, such as CAL1, perform the same function(s) as human regulators such as HJURP and hMis18. Thus, although this data challenges the universality of centromere propagation dynamics in metazoans, it will be important to determine whether some mechanisms and signals required for CENP-A replenishment are conserved, despite different times of assembly in the cell cycle, and the lack of conservation for key regulatory proteins (Mellone, 2011).

Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division

Chromosome segregation requires centromeres on every sister chromatid to correctly form and attach the microtubule spindle during cell division. Even though centromeres are essential for genome stability, the underlying centromeric DNA is highly variable in sequence and evolves quickly. Epigenetic mechanisms are therefore thought to regulate centromeres. This study shows that the 359-bp repeat satellite III (SAT III), which spans megabases on the X chromosome of Drosophila melanogaster, produces a long noncoding RNA that localizes to centromeric regions of all major chromosomes. Depletion of SAT III RNA causes mitotic defects, not only of the sex chromosome but also in trans of all autosomes. It was furthermore found that SAT III RNA binds to the kinetochore component CENP-C, and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins. In conclusion, these data reveal that SAT III RNA is an integral part of centromere identity, adding RNA to the complex epigenetic mark at centromeres in flies (Rosic, 2014).

It is well-established that centromeric regions and their function are influenced by epigenetic mechanisms to maintain their identity throughout cell and organismal generations. The histone variant CENP-A has been singled out as a key player in determining centromeres in most organisms studied so far. However, diversity and differences within centromeres suggest that additional mechanisms also play a role in centromere determination. This study provides evidence that the SAT III transcripts from a highly repetitive region of the X chromosome of D. melanogaster are important to maintain correct centromeric function, and therefore normal chromosome segregation. SAT III RNA depletion causes severe chromosome segregation defects and a partial loss of essential kinetochore components that mediate the interaction with the mitotic spindle. Furthermore, SAT III RNA interacts with the inner kinetochore protein CENP-C. A model is proposed where SAT III RNA binds to CENP-C, which in turn is required to recruit or stabilize CENP-C and possibly CENP-C–interacting factors such as CENP-A at centromeres. When SAT III RNA is absent, the association of CENP-C with centromeres is destabilized or inhibited, which impairs the association of other proteins that are dependent on CENP-C for their centromeric localization. Reciprocally, in the absence of CENP-C, SAT III is absent from centromeres, which suggests an interdependence of SAT III RNA and CENP-C. CENP-C, together with CENP-A and CAL1, forms a platform for binding of KMN proteins (named for the Knl1 complex, the Mis12 complex and the Ndc80 complex), which are required for the attachment of chromosomes to the mitotic spindle. Therefore, it is proposed that as a consequence of the SAT III depletion, chromosome missegregation is caused by the destabilization of centromeric chromatin and therefore kinetochore formation during mitosis (Rosic, 2014).

SAT III is transcribed in D. melanogaster embryos and adult flies (Usakin, 2007; Salvany, 2009). Long centromeric transcripts have been identified in other species as well. Even though long SAT III transcripts are predominantly detected, the existence of smaller transcripts cannot be excluded, as rapid centromeric transcript turnover has been described previously. In maize, centromeric transcripts remain bound to the kinetochore after transcription, and are thought to participate in stabilization of centromeric chromatin. Maize RNA binds to centromeric protein CENP-C transiently, and promotes its binding to DNA. Therefore, noncoding RNA may play a role similar to a protein chaperone. Once CENP-C is localized to centromeres, DNA binding is facilitated with the help from RNA to stabilize its position. During interphase, SAT III RNA localizes to the nucleus, and forms a cluster in proximity to sites of centromeric clusters, perhaps at its transcription site. During mitosis, SAT III RNA is present at centromeric regions. It is suggested that satellite transcripts function in stabilizing the centromeric positioning of CENP-C, thereby facilitating the building of kinetochore structures, and in turn require CENP-C to localize to centromeres. This mechanism may be evolutionarily conserved, as CENP-C has been described to bind RNA from centromeric repeats in maize. In addition to SAT III RNA present at centromeres, some SAT III RNA is also detectable at pericentromeres of mitotic chromosomes and is non-chromatin-associated. SAT III RNA that is present at pericentromeres might also contribute to overall kinetochore structure, and signals distant from chromatin might represent distinct ribonucleoprotein particles. However, additional work is required to address these questions (Rosic, 2014).

Depletion of SAT III RNA in S2 cells caused severe mitotic defects, which indicates that SAT III RNA is crucial for cell division. The same phenotype was observed in vivo in D. melanogaster embryos. Importantly, flies carrying an X-Y translocation chromosome that has lost most of its SAT III DNA block do not transcribe any significant amount of SAT III RNA, and display segregation defects in early embryos similar to what what was described for S2 cells and SAT III LNA gapmer-injected embryos. Most of the Zhr1 flies are viable and fertile despite the segregation defects in early embryos. It is therefore suggeste that SAT III RNA function is only one part of a larger safeguard mechanism required for accurate chromosome segregation during mitosis. It has been shown that Zhr1 male flies rescue the female hybrid lethality in crosses between D. simulans females and D. melanogaster males. One of their hypotheses was that RNA originating from SAT III might be the cause of hybrid lethality in F1 daughters originating from these crosses. This study shows that Zhr1 flies do not have any SAT III transcripts, which indicates a possible incompatibility of SAT III RNA from wild-type D. melanogaster flies with either transcripts or the sequence of the X chromosome of D. simulans. However, this and other possibilities need to be tested in the future (Rosic, 2014).

A previous study showed that transcription of SAT III depends on the homeobox-containing transcription factor Hth, and mutations of hth lead to abnormal distribution of CENP-A (Salvany, 2009). Similarly, inhibition of transcription during mitosis resulted in a decreased level of centromeric α-satellite transcripts in human cells, which in turn resulted in lagging chromosomes and a reduction of CENP-C. Inhibition of transcription or mutations of transcription factors may, however, cause pleiotropic effects in cells; together with the results presented from a direct depletion of SAT III transcripts, this study concludes that the SAT III RNA directly influences centromere function and that satellite transcripts may have a conserved function in kinetochore formation (Rosic, 2014).

The inability of chromosomes to segregate properly in the absence of SAT III RNA is not restricted to chromosome X, the origin of SAT III transcripts. This indicates a trans-acting mechanism, as seen in dosage compensation and proposed for maize centromeric RNA. It has been suggested that each centromere is capable of producing RNA. Indeed, in D. melanogaster, active centromeric transcription by RNA polymerase II was observed on all chromosomes. This indicates that centromeric RNAs might have redundant functions, similar to what is described for the dosage compensation complex in Drosophila. Here, roX1 and roX2 RNA are required for spreading of the compensasome to the entire X chromosome. These two RNAs are redundant in their function, even though they have little sequence similarity. The presence of redundant RNAs may also explain why the majority of chromosomes usually segregate correctly upon SAT III RNA depletion, and why only some chromosomes are lagging (Rosic, 2014).

This study shows that SAT III RNA function is independent of heterochromatin formation. In support of this, Usakin (2007) reported that many D. melanogaster pericentromeric transcripts participate in heterochromatin formation, but SAT III transcripts were not among the RNAs that had an effect on the formation of centromeric heterochromatin. The observed heterochromatin defects in hth mutant embryos (Salvany, 2009) are, therefore, possibly caused by additional effects of depleting this transcription factor. Pericentromeric heterochromatin is required for sister chromatid cohesion and bipolar orientation during mitosis. However, the levels of cohesion proteins, as well as the heterochromatin markers HP1 and H3 lysine 9 methylation, are unaffected in SAT III–depleted cells. It is therefore concluded that the observed chromosome segregation defects after SAT III depletion are unlikely to be caused by a loss of sister chromatid cohesion or heterochromatin integrity (Rosic, 2014).

Levels of centromeric and kinetochore proteins were significantly reduced on mitotic chromosomes that failed to segregate properly in the absence of SAT III RNA, which implies a role of SAT III RNA in providing a competent centromere environment. Additionally, reducing the levels of CENP-C by RNAi caused a complete loss of SAT III from centromeres, which suggests that CENP-C and SAT III RNA are mutually dependent on each other for their centromeric localization. Because loading of CENP-C and CENP-A is mutually dependent as well, both proteins are reduced in the absence of SAT III, as expected. Spc105 is an essential component of Drosophila kinetochores; its localization is interdependent with MIS12 complex localization and required for localization of the NDC80 subcomplex, which directly binds microtubules. Hence, reduction of Spc105 protein at centromeres leads to severe defects in constructing a functional kinetochore, and provides an explanation for failures in chromosome segregation in the absence of SAT III RNA. Finally, SNAP tag experiments showed that loading of newly synthesized CENP-A and CENP-C proteins is also affected by the loss of SAT III, which suggests that SAT III plays an integral role in establishing and stabilizing centromeric chromatin. In conclusion, SAT III RNA was identified as an epigenetic factor involved in centromere regulation and function through interaction with the centromeric protein CENP-C, which suggests a vital and evolutionarily conserved role of noncoding RNAs in centromere determination and chromosome segregation (Rosic, 2014).

centromere identifier : Biological Overview | Evolutionary Homologs | Developmental Biology | References

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