InteractiveFly: GeneBrief

Centromeric protein-C: Biological Overview | References

BIOLOGICAL OVERVIEW

Faithful transmission of genetic information during mitotic divisions depends on bipolar attachment of sister kinetochores to the mitotic spindle and on complete resolution of sister-chromatid cohesion immediately before the metaphase-to-anaphase transition (see Organization of the animal kinetochore). Separase is thought to be responsible for sister-chromatid separation, but its regulation is not completely understood. Therefore, a screen was carried out for genetic loci that modify the aberrant phenotypes caused by overexpression of the regulatory separase complex subunits Pimples/securin and Three rows in Drosophila. An interacting gene was found to encode a constitutive centromere protein. Characterization of its centromere localization domain revealed the presence of a diverged CENPC motif. While direct evidence for an involvement of this Drosophila Cenp-C homolog in separase activation at centromeres could not be obtained, in vivo imaging clearly demonstrated that it is required for normal attachment of kinetochores to the spindle (Heeger, 2005).

Separase functions as a protease at the metaphase-to-anaphase transition of mitosis. At this crucial cell cycle transition, separase cleaves the α-kleisin subunit (Scc1/Mcd1/Rad21) of the cohesin complex and thereby promotes the final release of sister-chromatid cohesion. The careful control of separase activity during the cell division cycle involves regulatory subunits. Securin is a subunit that accumulates and associates with separase during interphase. It acts as an inhibitor of separase activity. Thus, the rapid degradation of securin at the metaphase-to-anaphase transition via the anaphase-promoting complex/cyclosome (APC/C) pathway of ubiquitin-dependent proteolysis results in separase activation. In Drosophila, securin is encoded by the pimples (pim) gene and the catalytic protease subunit by the Separase (Sse) gene. Drosophila Sse lacks the extensive N-terminal regulatory domain that is present in separases outside the dipterans because the corresponding gene region appears to have evolved into an independent gene, three rows (thr). Drosophila Thr binds to Sse and is required for sister-chromatid separation during mitosis (Heeger, 2005).

The precise role of Thr and the corresponding N-terminal domains in nondipteran separases is not understood. Moreover, Pim and other securins are not just separase inhibitors but also contribute in an unknown positive manner to sister-chromatid separation. In fission yeast, securin recruits separase to the mitotic spindle, and similar observations have been described in other organisms. Separase activation and transport on spindle microtubules might confine its action to the congressed chromosomes in metaphase plates and in particular to the pericentromeric region. This hypothetical scenario might explain why only a minute and preferentially pericentromeric pool of Scc1 appears to be cleaved by separase during mitosis of higher eukaryotic cells, while the large majority of Scc1 remains intact (Heeger, 2005).

To identify additional genes that might contribute to separase regulation and function, a screen was performed for chromosomal regions that act as genetic modifiers of the aberrant phenotypes resulting from overexpression of Pim or a dominant-negative Thr fragment during Drosophila eye development. Molecular characterization of an interacting locus revealed that it encodes a constitutive centromere protein. Mapping of its centromere localization domain in combination with sequence comparisons among Drosophilid orthologs allowed its identification as the most diverged Cenp-C homolog. Cenp-C was originally identified as a human autoantigen localized to centromeres and found to display limited sequence similarity to budding yeast Mif2, which was identified by mutations affecting the fidelity of chromosome transmission during mitosis. Homologs have also been described in nematodes (HCP-4) and plants. For simplicity, Cenp-C has been used as a designation for all these homologs. Interestingly, recent analyses have demonstrated that Cenp-C, as well as Cenp-A, a histone H3 variant present in centromeric nucleosomes, evolve rapidly and adaptively in many lineages, perhaps driven by the rapid evolution of centromeric satellite sequences, and in Drosophila, Cenp-C was supposed to be absent. Apart from providing further support for the striking sequence divergence of ubiquitous eukaryotic centromere components, these findings also raise the possibility that separase activity might be enhanced by such components (Heeger, 2005).

Identification of Drosophila Cenp-C closes a prominent gap in the arguments for homologous centromere organization. Centromeric DNA sequences have evolved extremely rapidly and appear to have driven the coevolution of centromeric proteins during eukaryote evolution. The resulting low sequence similarity between centromeric proteins has effectively concealed the existence of a common set of constitutive eukaryotic centromere proteins until very recently. The first features demonstrated to be shared among fungal, plant, and animal centromeres were centromere-specific histone H3 variants. In addition to Cenp-A homologs, only one further constitutive centromere component, Cenp-C, has so far been shown to be present in each of the three main eukaryotic branches. In combination with the recent identification of related Cenp-H-, Cenp-I-, and Mis12-like proteins in both vertebrates and yeast, these results provide strong support for the notion of a common set of constitutive centromere proteins. These proteins, which are centromeric throughout the cell cycle, appear to provide a foundation for kinetochore assembly and spindle attachment during mitosis by recruiting several distinct multisubunit complexes that also contain highly diverged proteins (Heeger, 2005).

The extensive sequence divergence characteristically observed among homologous eukaryotic centromere and kinetochore proteins is striking, especially in the light of their common fundamental cellular function. The average amino acid identity observed in a genome-wide comparison of D. melanogaster and D. pseudoobscura ortholog pairs is 77% and only 38% in case of the Cenp-C pair. Moreover, based on the ratio between radical charge mutations and conservative substitutions in D. melanogaster and D. pseudoobscura ortholog pairs, Cenp-C is one of 44 genes likely to have evolved under positive selection. Except for a few very restricted regions, comparison of D. melanogaster Cenp-C with the orthologs from D. erecta and D. yakuba, which are closer relatives than D. pseudoobscura and thus amenable to d_N/d_S analyses, did not reveal strong evidence for positive selection, in contrast to the recent findings in plant and mammalian lineages. However, these d_N/d_S analyses ignore insertions and deletions (indels), which have occurred considerably more often during Cenp-C evolution in Drosophilids than in the mammalian lineage. Most of the indels are observed within the minimally conserved central regions of Drosophilid Cenp-C. Similar variabilities resulting from recurrent exon duplications have been observed in the central region of the plant Cenp-C genes (Heeger, 2005).

The adaptively evolving regions of mammalian Cenp-C have been shown to bind to DNA in vitro, consistent with the proposed coevolution of centromeric DNA and protein sequences (Talbert, 2004). However, this DNA binding in vitro is not sequence-specific, suggesting that interactions with centromere-specific proteins contribute to centromere localization of Cenp-C. As in other organisms, Cid/Cenp-A is also required for centromere localization of Cenp-C in Drosophila. High-resolution light microscopy of mitotic chromosomes has indicated that human Cenp-C covers the poleward-oriented peripheral region of the Cenp-A-containing centromeric chromatin (Blower, 2002). Direct interactions between Cenp-A and Cenp-C have not yet been demonstrated in any organism. Attempts with yeast two-hybrid experiments were also unsuccessful (Heeger, 2005).

The CENPC motif has recently been identified as the only region conserved among the Cenp-C orthologs from fungi, plant, and animals (Talbert, 2004). In Drosophilids, even this short motif of ~24 amino acids is not fully conserved in its C-terminal part. These results suggest that this CENPC motif is crucial for centromere localizationA single-point mutation affecting one of the invariant positions in the CENPC motif interferes with centromere localization of the C-terminal domain of Cenp-C in a transfection assay. This mutation was identified as the only missense mutation interfering with centromere localization after extensive random mutagenesis. Further experiments will reveal whether and how the CENPC motif contacts Cid/Cenp-A nucleosomes. It is emphasized, however, that also in Drosophila Cenp-C, other regions than the CENPC motif clearly contribute to efficient centromere localization. Centromere localization of the CN subregion (1009-1205), for instance, is only detected in a transfection assay in live but not in fixed cells, while centromere localization of the larger C region (1009-1411) is resistant to fixation (Heeger, 2005).

The highest conservation among Drosophilid Cenp-C proteins is observed within the N-terminal third, which is neither required nor sufficient for normal centromere localization. Nevertheless, prolonged overexpression of this domain in proliferating eye and wing imaginal disc cells results in severe defects. The conserved N-terminal Cenp-C domains (R and DH) might bind to kinetochore proteins and titrate these away from the centromere when overexpressed. Biochemical and genetic characterizations in Saccharomyces cerevisiae and Caenorhabditis elegans have suggested that Cenp-C is not only associated with Cenp-A, but that it also recruits the next layer of kinetochore proteins, in particular the Mis12/Mtw1 and Ndc80 complexes, which remain to be identified in Drosophila (Heeger, 2005).

As in yeast (Meluh, 1995), chicken (Fukagawa, 1997; Fukagawa, 1999), and mice (Kalitsis, 1998), Cenp-C is also an essential gene in Drosophila. Antibody microinjection experiments in mammalian cells (Tomkiel, 1994); RNA interference in C. elegans (Moore, 2001; Oegema, 2001); and phenotypic analysis in yeast (Brown, 1993), chicken cells (Fukagawa, 2001), and mutant Drosophila embryos demonstrate that Cenp-C is required for normal chromosome segregation during mitosis. in vivo imaging in Cenp-C mutant embryos discloses these defects in detail. Previously, in vivo imaging has also been applied in C. elegans CENP-C(RNAi) embryos (Moore, 2001; Oegema, 2001). The formation of holocentric chromosomes and transient Cenp-C recruitment only during mitosis differentiates C. elegans from other metazoans like Drosophila and mammalian cells. Moreover, in contrast to the findings in C. elegans, chromosome congression into a central plane is still observed in the Drosophila Cenp-C mutants. Presumably, this chromosome congression reflects the function of residual maternally provided Cenp-C, which is still detectable at this stage of analysis. Moreover, time-lapse analyses demonstrate that chromosome congression is not entirely normal in the Cenp-C mutant embryos. Metaphase plate formation is delayed and often does not lead to the highly ordered arrangement of all chromosomes characteristically observed before anaphase onset in wild type. Occasional chromosomes fail to achieve bipolar attachment in the Cenp-C mutants. These chromosomes do not segregate normally during anaphase. Cenp-C is, therefore, clearly required for normal attachment of kinetochores to the mitotic spindle (Heeger, 2005).

Evidently, the insufficiently attached chromosomes in Cenp-C mutant embryos are unable to inhibit the onset of anaphase, even though the mitotic spindle checkpoint appears to be at least partially functional in Cenp-C mutants at the analyzed stage. However, assembly of mitotic spindle checkpoint proteins might fail, particularly on the kinetochores of those chromosomes that do not attach correctly to the mitotic spindle (Heeger, 2005).

In principle, the chromosome segregation defects observed in the Cenp-C mutants might not only reflect impaired interactions between kinetochores and spindle. Segregation of sister chromatids to the spindle poles also depends on complete resolution of sister-chromatid cohesion at the metaphase-to-anaphase transition. This final separation of sister chromatids is thought to be achieved by separase-mediated cleavage of the Scc1/Rad21 subunit of those cohesin complexes that perdure in the pericentromeric region until the metaphase-to-anaphase transition. Several observations are consistent with the idea that a localized full activation of separase might be assisted by centromeric proteins. Accordingly, mutations in Cenp-C might reduce separase activity and thereby explain the genetic interactions with the regulatory separase subunits Pim/securin and Thr that led to the identification of Drosophila Cenp-C. No coimmunoprecipitation of Cenp-C with separase complex proteins was observed and no effects of Cenp-C mutations on Pim and Thr levels was observed. This analysis has, therefore, not exposed clear evidence for separase activation at centromeres. In contrast, Cenp-C is clearly required for normal chromosome attachment to the mitotic spindle, and thus the observed genetic interactions most likely reflect a summation of negative effects on the efficiency of sister-chromatid separation (by separase) and segregation (by the spindle). However, it is emphasized that the results certainly do not rule out a local activation of separase within the centromeric region (Heeger, 2005).

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).

The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C

CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues is not understood in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. This study investigated the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila, using fixed and live imaging combined with genetic approaches. CID assembly was found to initiate at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, a novel decrease in CID levels was observed after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. It was also demonstrated that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, it was shown that the centromere proteins Chromosome alignment defect 1 (CAL1) and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. It is concluded that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote (Dunleavy, 2012).

This study reveals a surprising diversity of CID assembly timing in mitotic and meiotic tissues in the fruit fly Drosophila melanogaster. During mitosis, CID assembly initiates at late telophase and continues during G1 phase in somatic cells of the larval brain. These results are consistent with the timing and dynamics of CENP-A assembly reported for human cell lines and in general, with centromeric histone deposition outside of S phase, during mitosis and G1 phase. Notably, loading in mitosis was observed to occur at a later mitotic stage (telophase/G1 phase) than previously reported for cultured cells (metaphase) or fly embryos (anaphase). Interestingly, neuroblast stem cells display a subtle difference between cells derived from the same division; the mother cell, which will continue to act as a stem cell, starts CID loading at centromeres 3–6 min earlier than in the daughter cell that is committed to differentiation. It is currently unclear whether this difference in centromere assembly timing is due to differences in the regulation of mitotic exit between stem and daughter cells or is required for or a response to stem cell propagation mechanisms (Dunleavy, 2012).

It is proposed that such differences in timing reflect altered cell cycle regulation in cultured cells compared to animal tissues, and the results emphasize the importance of validating cell culture findings in animal models. It is important to note that despite similarities to the timing observed in human cultured cells (late telophase/G1 phase), the results in Drosophila raise questions about whether the analysis of cultured cells in humans and other species reflects the timing of CENP-A assembly in the organism (Dunleavy, 2012).

These results also show that the cell cycle timing for CID assembly in meiosis differs from mitosis. In male meiosis, there are two phases of CID assembly, at prophase of meiosis I and after exit from meiosis II, and two phases of chromosome segregation, resulting in haploid spermatids with nuclear CID levels equivalent to those observed at the beginning of meiosis. In meiosis in Drosophila females, CID assembly also occurs during prophase of meiosis I. Assembly in prophase provides another example of the restriction of CID assembly to a specific part of the cell cycle outside of S phase, but has not been observed previously in mitotic tissues or cultured cells from other organisms. It is also important to note that meiotic prophase in both male and female Drosophila occurs over days, indicating that CID assembly is gradual over this extended time period. Such slow assembly dynamics are unexpected, given that until now studies in mitotic cells indicate that CENP-A assembly is completed in the order of minutes to hours. How CID assembly is first initiated and then continues over such extended time periods awaits further investigation (Dunleavy, 2012).

It is likely that cell cycle regulators control CID assembly in meiosis as they do in mitosis. For example, a recent study showed that CDK activity inhibits CENP-A assembly in human cells and that blocking CDK activity results in precocious loading in S and G2 phases. Cyclin A is degraded during late prophase of meiosis I. This is consistent with the observed burst in CID assembly during a 10-min time window of late prophase/early prometaphase I, and the previous demonstration that Cyclin A degradation is required for mitotic CID assembly. However, CID assembly also occurs before Cyclin A degradation in meiosis I, implying that other unknown mechanisms initiate and continue assembly prior to late prophase I. Additionally, CID is not loaded between meiosis I and II, even though Cyclin A levels remain low. Instead, the partial degradation of Cyclin B to an intermediate level after meiosis I, which allows for spindle destruction but prevents a second round of DNA synthesis, could inhibit CID assembly between meiosis I and II. Moreover, the slow degradation of Cyclin B at the end of meiosis II could contribute to the gradual CID loading in spermatids, as the second phase of CID assembly after meiotic exit is more similar in terms of cell cycle regulation to the telophase/G1 loading observed in mitotic tissues in the animal and in human cells in culture. However, it was also observed that CID assembly occurs in prophase of meiosis I, when Cyclin B levels are high, but does not occur between meiosis I and II, despite low Cyclin A levels. This suggests that CID assembly in meiosis is regulated by other mechanisms in addition to the inhibition of Cyclin/CDK activities, as proposed for mammalian cells (Dunleavy, 2012).

Another striking observation from this study is that during meiosis I, CID assembly occurs prior to chromosome segregation, whereas most mitotic cells previously studied proceed through most of mitosis with half the maximal amount of CID at centromeres. In addition, a greater than 2-fold increase in CID intensity was observed at centromeres during prophase, even though a 2-fold increase would be sufficient to compensate for CID dilution in premeiotic S phase. What is the role, if any, of an increased level of CID at centromeres during the first meiotic division? In meiosis I, bivalent sister chromatid kinetochores are mono-oriented, instead of bi-oriented as they are in mitosis and meiosis II; combined with the maintenance of sister cohesion at centromeres, this ensures that homologs, and not sisters, segregate during meiosis I. It is speculated that extra CID may be required during the first meiotic division to assemble or maintain mono-oriented kinetochores and microtubule attachments. This hypothesis could also be extended to incorporate the surprising decrease in CID levels observed between the end of meiosis I and the beginning of meiosis II. Loss of CENP-A during normal cell divisions has only previously been observed as accompanying DNA replication and nucleosome segregation in S phase, events that do not occur between meiosis I and II. Thus, it is tempting to speculate that the additional loss of CID after meiosis I could contribute to the currently unknown mechanism responsible for reorganization of kinetochores from mono- to bi-orientation in preparation for meiosis II (Dunleavy, 2012).

Using targeted RNAi depletion of centromeric proteins during Drosophila male meiosis, this study found that both CAL1 and CENP-C are required for CID assembly in prophase of meiosis I. This is consistent with previous observations in mitotic cells, where CAL1, CENP-C, and CID are mutually dependent on each other for centromere localization. It was also found that depletion of CAL1 or CID in larval testes results in CENP-C delocalization from centromeres and sequestration in the nucleolus, again similar to observations in mitosis, possibly because it is no longer in a stable complex with CID or CAL1. The results also show that reduced CAL1 or CENP-C expression results in defective chromosome segregation and that both are required for normal progression through male meiosis. The finding that T1–T3 spermatids depleted for CAL1 or CENP-C have reduced CID at centromeres (although to a lesser extent in the case of CENP-C depletion) also suggests that CAL1 and CENP-C are required for CID assembly during the second phase of loading in spermatids. However, given that cells with reduced CAL1 or CID already show major chromosome segregation defects after meiosis I, meiosis-specific GAL4 drivers active in later stages of meiosis and spermatogenesis, which are currently lacking, are required to directly assay the requirements for CAL1 and CENP-C in the second phase of CID assembly or during fertilization. Requirements for CAL1 and CENP-C in both phases of meiotic CID assembly are surprising, given that centromeric CAL1 levels are greatly reduced during prophase I and at later stages of spermatogenesis and that CENP-C is not localized to centromeres after meiosis II. One intriguing possibility is that CID assembly requires CAL1 and CENP-C removal from centromeres (Dunleavy, 2012).

Another key observation from this study is the retention of CID at centromeres on mature spermatozoa in spite of an extensive period of chromatin remodeling and histone–protamine exchange during spermatocyte maturation. How CID is protected from histone removal prior to protamine exchange at centromeres remains to be investigated. It is possible that the local chromatin environment at centromeres is refractory to protamine exchange or that additional proteins present at centromeres could provide protection. Because fusion of male and female pronuclei does not occur until telophase of the first zygotic division, it is likely that paternal CID at centromeres is required for kinetochore formation and spindle attachment to paternal chromosomes. The amount of paternal CID at centromeres could be critical for the successful epigenetic inheritance of centromere identity and for the viability of the embryo, if paternal CID is diluted during subsequent zygotic divisions. Alternatively, maternal CID could compensate for a reduced level of CID on sperm or establish de novo centromeres on paternal chromosomes. Whatever the mechanism of CID maintenance in the zygote, the regulation of CID assembly on sperm is likely to prove very important in the transmission of epigenetic information and centromere specification into the next generation (Dunleavy, 2012).

Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. This study exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis (Unhavaithaya, 2013).

Localization studies demonstrated centromere pairing in yeast, Drosophila, and plants, and it showed that the centromeres cluster together in Drosophila meiosis I. This study has establish that centromere function is required for both pairing and clustering. Thus, centromeres are integrally involved in these two processes and not brought together solely by external factors. Because these events occur before assembly of the kinetochore, it is likely that the chromatin and associated proteins at the centromere are critical. The mutations in cenp-C reveal that functional CENP-C is necessary at a minimum for maintenance of centromere pairing and clustering in Drosophila oocytes. The noncomplementation between truncated CENP-C and CAL1 protein forms implicates CAL1 as also being crucial for centromere pairing and clustering. Given the role of CENP-C in recruiting proteins to the centromere, the requirement for this protein could reflect a direct role in centromere pairing and clustering or the need for a protein whose localization is dependent on CENP-C and/or CAL1. In the cenp-C mutant and the cenp-C cal1 double-heterozygous mutant, CID is still localized to the centromere, as evidenced by its presence at brightly DAPI-stained heterochromatin at levels that, by immunofluorescence, are not significantly lower than WT. Thus, CID presence is insufficient for centromere clustering and pairing. The reduced level of CID staining in the double-heterozygous mutant is nearly significant, however; thus, the possibility that reduced CID levels contribute to the mutant defects is not excluded (Unhavaithaya, 2013).

The proteins at the centromere may interact with nuclear structures to promote centromere clustering. This study identifies the nucleolus as a likely candidate. The centromere clusters are associated with the nucleolus in WT oocytes, and this association requires cenp-C and cal1 function. In Drosophila female meiosis, the nucleolus may serve as an anchor site for centromeres throughout prophase I. The SC also may cluster centromeres. Clustering has been shown to be disrupted in mutants for the SC transverse and central elements. The observation that the SC protein C(3)G fails to be retained at the centromere in cenp-C and cal1 mutants raises the possibility that the failure of clustering in these centromere protein mutants is a consequence of the absence of the SC. The hypothesis of this causality is consistent with the timing of defects; as early as pachytene, both centromere SC and clustering are absent. It remains to be determined how the SC, a structure contained between pairs of homologs, could gather centromeres into a cluster. In c(3)g mutants, more than four CID foci can be observed, indicating that both centromere pairing and clustering can be affected. Thus, failure of centromere retention of the SC also could account for the pairing defects in the centromere protein mutants (Unhavaithaya, 2013).

The allele-specific noncomplementation (type I second-site noncomplementation) between the mutations causing C-terminal truncations of CENP-C and CAL1 is unusual and informative. Such mutations that alter protein structure rather than simply reducing protein levels provide the opportunity to investigate genetic interactions. This allele-specific noncomplementation affects all the processes analyzed: centromere pairing, centromere clustering and nucleolar association, SC retention at the centromere, and meiotic segregation. The antagonistic genetic interaction requires the truncated protein forms, because deficiencies for each of the genes complement the truncation allele of the other for meiotic segregation and cause only slight defects in centromere pairing and clustering. This is also true for the cenp-CZ3-4375 allele that reduces protein levels. Thus, simply decreasing the levels of the proteins does not perturb these processes. The C-terminal region of CAL1 binds to CENPC, whereas the N terminus binds to CID; thus, the truncated form could have a dominant negative effect by binding CID and blocking its link to CENP-C. The C terminus of CENP-C is required for its localization to the centromere as well as binding to CAL1, whereas it binds the KNL-1/Mis12 complex/Ndc80 complex (KMN) kinetochore network via its N terminus. Thus, C-terminal truncated CENP-C also could act as a dominant negative to uncouple the KMN complex from a functional centromere association, particularly given that the N terminus alone can bind to kinetochore proteins but not to the centromere. Expression of the N terminus alone also can disrupt the spindle assembly checkpoint. The truncation alleles of cenp-C and cal1 each alone have slight semidominant effects on centromere pairing, clustering, and meiotic segregation, consistent with dominant negative activities. The combination of the two dominant negative effects could account for perturbation of the meiotic processes. It cannot be excluded, however, that these truncation alleles act as recessive neomorphs, conferring novel properties on the proteins (Unhavaithaya, 2013).

A critical question is whether centromere clustering is required for proper meiotic segregation. It remains to be determined whether the meiotic nondisjunction that occurs in these centromere protein mutants is linked to the failure of centromere clustering and/or centromere pairing. The meiotic segregation errors in oocytes affect both the X chromosome, which undergoes recombination, and the 4th chromosome, which is achiasmate and lacks SC. One way that meiotic segregation of both types of chromosomes could be dependent on clustering would be if association with the nucleolus is necessary for proper assembly of the kinetochore later in prophase I. It is notable, however, that the meiotic segregation errors in oocytes assayed for the X chromosome occurred exclusively in meiosis I; thus, a defect in kinetochore function necessary for both meiosis I and II was not evident. There are known meiosis I-specific requirements of the kinetochore, such as the need for the two sister kinetochores to co-orient in meiosis I, and establishment of these may require centromere clustering and/or nucleolar association (Unhavaithaya, 2013).

This proposal is consistent with the demonstrated effects of cenp-C mutants in meiosis in Saccharomyces pombe. An alternative possibility is that the centromere mutations have independent effects on centromere clustering and subsequent segregation. For example, the centromere clustering defects could result from failure to retain the SC at the centromere and the meiotic nondisjunction could be an independent consequence of improperly assembled kinetochores later in meiosis I. The centromere mutations clearly can affect meiotic segregation independent of centromere pairing and clustering, given the meiotic nondisjunction in males double-heterozygous for the cenp-C and cal1 alleles. In Drosophila male meiosis, centromere clustering, SC formation, and recombination do not occur (Unhavaithaya, 2013).

Although observed in yeast, plants, and Drosophila, a role for intrinsic centromere function in the nuclear localization of centromeres and associations between centromeres in meiosis has not yet been defined. The demonstration that proper centromere architecture is necessary for these interactions opens a path to define the molecular basis of centromere pairing and clustering across these species in meiosis (Unhavaithaya, 2013).

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).

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 (Kline, 2006; Vos, 2006). 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 (Foltz, 2006; Izuta, 2006; Okada, 2006). 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 (Maiato, 2006), 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 (Blower, 2002). Antigen accessibility problems, which were not excluded by Blower (2002), 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 (Talbert, 2004; Heeger, 2005), 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 (Heeger, 2005). 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 (Ciferri, 2005; Wei, 2005). 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 (Wei, 2006) 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 (Ciferri, 2005; Wei, 2005). 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 (Deluca. 2006). 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 (Cheeseman, 2006). One of these microtubule binding sites is present within Spc105/KNL-1. The other is found within the globular N-terminal Ndc80 domain (Cheeseman, 2006) which is known to be within the outer kinetochore plates where kinetochore microtubules terminate (DeLuca, 2005). In vitro, the Ndc80 complex binds to microtubules at an angle (Cheeseman, 2006). 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 (Ciferri, 2005; Wei, 2005). 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).

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 APC^FZR/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 APC^FZR/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 APC^FZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APC^FZR/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 APC^FZR/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 APC^FZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APC^FZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APC^FZR/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 APC^FZR/CDH1 substrate acts during G2/metaphase before APC^FZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APC^FZR/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 APC^FZR/CDH1, the model of a single APC^FZR/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 APC^FZY/CDC20 in centromere propagation cannot be ruled out because premature APC^FZR/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 the 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).

Drosophila CENH3 is sufficient for centromere formation

The centromere ensures correct segregation of chromosomes during mitosis by providing the site for kinetochore assembly and microtubule attachment. Most eukaryotic organisms contain only one centromere per chromosome, which is specifically positioned and faithfully propagated through each cell cycle. With the exception of Saccharomyces cerevisiae, DNA sequence is considered neither necessary nor sufficient to mark centromeres in most eukaryotes, suggesting that centromere identity in many organisms is determined epigenetically. CENH3 (CENP-A in mammals, CID in Drosophila) is a centromere-specific histone H3 variant that replaces canonical histone H3 in centromeric nucleosomes. It is essential for centromere function and kinetochore assembly and thus a prime candidate epigenetic mark for determining centromere identity (Mendiburo, 2011).

Global misincorporation of CID into chromosome arms leads to the formation of functional ectopic kinetochores only in a small subset of sites, hindering a direct correlation between CID presence and kinetochore formation. To determine whether CID is sufficient for directing kinetochore assembly and nucleate centromere identity, a CID-GFP-Lac Repressor (LacI) fusion protein was targeted to an array of lac operator (lacO) sequences stably integrated in Drosophila Schneider S2 cells. Inducible CID-GFP-LacI or GFP-LacI fusion proteins are efficiently targeted to lacO sequences only upon pulse induction, whereas low leaky expression of CID-GFP-LacI in uninduced cells correlates with an exclusive centromere localization (Mendiburo, 2011).

CENH3 has been shown to adopt a specialized nucleosomal structure, which is proposed to mark centromeric chromatin. To determine whether CID-GFP-LacI is incorporated in nucleosomes at the lacO, mononucleosomes from GFP-LacI and CID-GFP-LacI cells were separated on a sucrose gradient. Analysis of the fractions from both cell lines revealed that lacO DNA comigrates with fractions containing H3-nucleosomes, suggesting that they are chromatinized. GFP-LacI did not comigrate with nucleosomes, likely due to its release from lacO binding after micrococcal nuclease treatment. In contrast, CID-GFP-LacI was found in nucleosome-containing fractions, indicating that it can interact with chromatin independently of the LacI binding. Nucleosome incorporation was confirmed by coimmunoprecipitation of CID-GFP-LacI with histone H2A in the nucleosome fraction. To exclude that CID-GFP-LacI migration pattern is only due to the contribution of protein localized to endogenous centromeres, a mutant CID fused to GFP-LacI was created that does not target to centromeres and is therefore present only at the lacO array. In this mutated protein, three amino acids [G175S, L177V, and L178M (CIDsvm)] in the CENP-A targeting domain (CATD) of CID are replaced by the corresponding residues of histone H3.1, as shown for human CENP-A. The migration pattern of CIDsvm-GFP-LacI in sucrose gradients resembles that of CID-GFP-LacI, supporting the notion that CID-GFP-LacI is in nucleosomes also at lacO regions (Mendiburo, 2011).

Targeting of CID-GFP-LacI, but not GFP-LacI, to the lacO served to recruit kinetochore proteins, such as CENP-C and the microtubule-binding protein NDC80/HEC1, 1 day after pulse induction. Probing for the spindle assembly checkpoint proteins POLO kinase and MAD2 revealed the same pattern, suggesting that ectopically targeted CID is sufficient to direct kinetochore assembly (Mendiburo, 2011).

The formation of a functional ectopic kinetochore at the lacO is expected to generate a dicentric chromosome causing chromosome breakage and anaphase defects. Indeed, a higher number of aberrant mitotic chromosome configurations was observed in cells expressing CID-GFP-LacI compared with control GFP-lacI cells and containing the lacO array (Mendiburo, 2011).

The epigenetic model for centromere identity predicts that a functional centromere self-directs the loading of new centromeric marks after each cell division. If CID-GFP-LacI identifies the lacO as a centromere, CID molecules without a LacI fusion should also be recruited to lacO regions. To investigate this, a stable cell line was created containing both an inducible CID-GFP-LacI and a constitutively expressed CID-hemagglutinin (HA) HA construct. Upon pulse induction and targeting of CID-GFP-LacI, a low initial CID-HA recruitment to the lacO (9.7%) was found, increasing by two-fold 7 days later (24%). Higher-resolution analysis using stretched chromatin fibers revealed that at initial time points CID-HA localization is restricted to the lacO region, whereas 7 days after pulse induction, CID-HA or CID is also found spreading into adjacent regions. CID-HA or CID were never found in lacO sequences in the presence of GFP-LacI (Mendiburo, 2011).

CID-GFP-LacI targeting to an ectopic chromosomal locus results in mitotically unstable dicentric chromosomes, hindering the analysis of long-term inheritance of the centromere function. To directly test de novo centromere heritability, an analysis was carried out of whether CID-GFP-LacI targeting confers mitotic stability to a centromere-devoid episomal DNA element carrying lacO sequences and a G418-resistance cassette. This plasmid was transfected into S2 cell lines constitutively expressing low levels of either CID-GFP-LacI or GFP-LacI and kept under selection pressure for 28 days. The initial transfection efficiency was comparable in both cell lines. However, a decline was observed in the amount of replicated plasmids (+DpnI) in GFP-LacI-expressing cells, whereas CID-GFP-LacI cells maintained the plasmid until the end of the experiment. Efficient replication and proper segregation are the two major factors defining the stability of plasmids. For the first 7 days, both cell lines replicated the plasmid with similar efficiency, indicating that correct segregation is the main factor contributing to its maintenance in CID-GFP-LacI cells. This was also observed when selection was removed after 16 days or never applied. Furthermore, CENP-C, NDC80, and microtubule connections were found localizing to CID-GFP-LacI-bound lacO plasmids in mitosis. These plasmids separate earlier than endogenous chromosomes and generally display a symmetric distribution on the spindle poles. GFP-LacI cells grew poorly after transfection, and the few mitotic cells detected showed no colocalization of GFP-positive lacO plasmids with either CENP-C or NDC80. Further characterization of recovered plasmids showed that plasmid size remained stable except for variability in the repetitive lacO array and did not acquire endogenous Drosophila sequences. A minor fraction of concatenated plasmids did not correlate with efficient recruitment of kinetochore proteins (Mendiburo, 2011).

The centromeric mark can self-propagate in the lacO plasmid, as shown by the recruitment of CID-HA to targeted CID-GFP-LacI plasmids and the spreading of both into backbone sequences. To determine whether centromeric chromatin is inherited even after removal of CID-GFP-LacI targeting, the lacO plasmid and a CID-GFP-LacI expression construct were transiently cotransfected to deliver an initial expression pulse that is progressively lost. Episomal lacO plasmids were detected as nonchromosomal CID/CENP-C foci, with or without a GFP signal. Early after transfection, cells were found that contained both GFP-positive and -negative foci and a smaller cell population that had already lost CID-GFP-LacI expression but contained nonchromosomal CID/CENP-C foci. The percentage of these GFP-negative plasmids increased until day 27, when all detected CID/CENP-C foci were devoid of any GFP signal. It is concluded that an initial targeting of CID-GFP-LacI to the lacO plasmid enables the nucleation of centromere function, which is maintained independently of CID-GFP-LacI expression and targeting (Mendiburo, 2011).

In recent years, functional biosynthetic kinetochores have been constructed by targeting kinetochore components and chromatin modifiers to minichromosomes. Even though the possibility of bypassing CENH3 chromatin for the assembly of a kinetochore suggested that CENH3 is dispensable for this function, no evidence of centromere heritability was provided by these studies. Recent reports showing CENP-A-dependent kinetochore formation in vitro or by artificially targeting the CENP-A loading factor in humans (HJURP) further stress the importance of CENH3 in kinetochore assembly. The results presented in this study show that CENH3 behaves as a true centromeric epigenetic mark not only by being sufficient for the recruitment of kinetochore proteins during mitosis but also for directing its own incorporation and maintaining centromere function through each cell division (Mendiburo, 2011).

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively (Fachinetti, 2013).

A long-standing question in chromosome inheritance is the epigenetic mark of centromere identity. By artificially targeting components to chromatin, several groups have demonstrated that large artificial arrays of CENP-A-containing chromatin are sufficient to generate partially functional centromeres, but only after completely abrogating any epigenetic component. An earlier effort had reduced CENP-A levels using siRNA. Such approaches fail to test the epigenetic question as they are plagued by partial suppression that precludes testing epigenetic sufficiency. Indeed, this study now demonstrates that as little as 1% of the original CENP-A level is sufficient for retention of at least partial centromere function and assembly of kinetochore proteins (Fachinetti, 2013).

Use of conditional gene inactivation in human cells and in fission yeast has overcome the previous technical limitations and has now established that the CATD of CENP-A when substituted into histone H3 can template its cell-cycle-dependent, HJURP/Scm3-dependent centromeric loading at a constant level in the complete absence of CENP-A. Consideration of the number of molecules of CENP-A at the normal centromere offers strong support for this conclusion. The number of CENP-A molecules at the 40–500 kilobase chicken centromeres has been reported to be between 25 and 40. Increasing that by tenfold to account for the increased centromere size in humans yields a maximal estimate of ~ 250–400 CENP-A molecules per centromere, with a corresponding prediction of <1 molecule remaining per centromere within 9 divisions after CENP-A gene inactivation. Therefore, the current evidence demonstrates that H3^CATD continues to be loaded at its initial level at each centromere for 4–5 generations after the CENP-A level has fallen below ∼ 1 molecule per centromere. It is important to note that initial H3^CATD assembly occurs in the presence of endogenous CENP-A; therefore, the evidence offers no insight into de novo centromere formation (Fachinetti, 2013).

Despite its necessity and sufficiency for maintaining centromere identity in the absence of CENP-A, this study has shown that H3^CATD is not sufficient for long-term centromere function and cell viability because it does not nucleate kinetochore assembly. Rather, this study has identified two redundant pathways that function to initiate kinetochore assembly onto an epigenetically defined chromatin core containing the CATD. The evidence establishes that CENP-A-containing chromatin is the epigenetic mark that can identify, maintain and propagate human centromere function indefinitely through a conserved two-step mechanism in which it templates its own CATD-dependent replication and nucleates subsequent kinetochore assembly. Furthermore, it was demonstrated that the principles of epigenetic centromere inheritance and function are conserved from human to fission yeast (Fachinetti, 2013).

Centromeric α-satellite RNA is essential for the nucleolar localization of CENPC1 and INCENP

The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. This study demonstrates that centromeric α-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires α-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following α-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, the nucleolar localization sequences on CENPC1 and INCENP have been identified. Evidence that CENPC1 is an RNA-associating protein that binds α-satellite RNA in an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and 'RNA replenishment' experiments, α-satellite RNA is show to be a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. These data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis (Wong, 2007).

The centromere is a specialized structure on chromosomes for microtubule attachment to ensure the equal partitioning of chromosomes during cell division. This structure comprises two defined domains: the central core for the assembly of the kinetochore and the flanking pericentric heterochromatin for centromere cohesion. In Schizosaccharomyces pombe, the outer centromeric repeat sequences give rise to small interfering RNAs (siRNA) that participate in chromatin repression. The depletion of Dicer (a nuclease required for the processing of siRNAs) in a chicken cell line leads to the disruption of heterochromatin assembly and cohesion. However, Dicer depletion has no observable effect on the binding of the core kinetochore proteins CENPA and CENPC1, indicating that while the evolutionarily conserved RNA interference (RNAi) machinery is crucial for the establishment of the pericentric heterochromatin, it may not be essential for the core kinetochore region. A recent study in maize has further described the association of single-stranded centromeric transposable element and repeat RNA with the core kinetochore complex that is distinct from those at pericentric heterochromatin; however, the functional significance of the observed centromere RNA transcripts is unclear. Furthermore, little is known about the subnuclear distribution of centromere RNA, and the pathway and significance, if any, of such RNA in kinetochore formation and function (Wong, 2007).

The nucleolus is a specialized organelle of the interphase nucleus where ribosomal DNA is transcribed, pre-rRNA is processed, and ribosome subunits are assembled. In addition, this organelle is known to serve a variety of other essential cellular and cell cycle control functions by allowing the timely sequestration of specific trans-acting factors and the biogenesis of many cellular ribonucleoprotein particles (RNPs) including transfer tRNAs, small nuclear snRNAs, different mRNAs, and telomerase RNA. Specifically, numerous studies have directly demonstrated the accumulation of different centromere-associated proteins at the nucleolus, including borealin (See Drosophila Borealin-related), PARP1, and PARP2 (Wong, 2007 and references therein).

This study shows the enrichment of centromeric α-satellite RNA and centromere proteins CENPC1 and INCENP in the interphase nucleolus. Evidence is presented that the centromere satellite RNA is required for the assembly of centromere-associated nucleoprotein components at the nucleolus and kinetochore (Wong, 2007).

This study demonstrates the nucleolar accumulation of the constitutive centromere protein CENPC1, the centromere-associated chromosomal passenger protein INCENP, and centromeric α-satellite RNA. The nucleolus localization sequence (NoLS) motifs for both of these proteins coincide with the nucleus localization sequence (NLS) motifs, and are found at the N-terminal portions of the proteins. These NoLS motifs share significant homology with those of other nucleolus-associated proteins. Some of these proteins, including borealin, PARP1 and PARP2, and INCENP, also bind directly to the centromere (Wong, 2007).

The presence of centromeric α-satellite RNA is closely associated with the nucleolar localization of CENPC1 and INCENP, since RNase treatment induces a complete disruption of this localization. More specifically, in the RNA 'rescue' study, it was shown that the incubation of RNase-treated cells with α-satellite RNA results in the in situ targeting of myc-tagged CENPC1 and INCENP recombinant proteins to the nucleolus. Experiments involving ActD treatment further demonstrate that the nucleolar accumulation of α-satellite RNA, CENPC1, and INCENP is dependent on active RNA polymerase I transcription. Previous studies have similarly shown that the nucleolar localization of centromere proteins borealin and PARP1 and PARP2 is dependent on active nucleolar transcriptional activity. Recent studies in S. pombe have shown that RNA polymerase II and IV (a unique RNA polymerase in plants) are essential for the generation of siRNAs. The transcription machinery that modulates the transcription of centromere repeats or siRNA synthesis in vertebrates is still unknown. However, it is unlikely that RNA polymerase I is directly driving the transcription of the nucleolar α-satellite RNA, as indicated by nuclear run-on transcription data. Furthermore, the depletion of α-satellite RNA-FISH signals is unlikely to be due to the down-regulation of some specific proteins, since the conditions used for ActD treatment did not affect the expression levels of proteins in general, as exemplified by those of CENPC1 and INCENP. In this context, the mechanism of action for the RNA polymerase I remains undefined, although it may be directly involved in providing a structural network or complex that retains the various centromere components in the nucleolus (Wong, 2007).

The nucleolus serves the important function of ribosome synthesis. Increasing numbers of studies now show that it is also a multitasking organelle that engages in other important cellular functions. An example of the pluri-functionality of the nucleolus is the cell cycle-dependent nucleolar localization of the telomere components hTERT (reverse transcriptase catalytic subunit of telomerase), hTR (telomerase RNA), and telomeric DNA-binding protein TERF2, where these components have been shown to be released from the nucleolus at late S and G2/M phase at a time that coincides with telomere elongation. The data suggest that the nucleolus may similarly sequester centromeric components such as centromere RNA and proteins for timely delivery to the chromosomes for kinetochore assembly at mitosis. The centromere proteins, in particular the chromosomal passenger proteins such as borealin, PARP1 and PARP2, and INCENP, display dynamic changes in distribution patterns during various stages of the cell cycle. Although these proteins only bind the centromere following mitotic onset, they are expressed much earlier in mid-S to G2 phase and have been shown to accumulate in the nucleolus. During this period, the nucleolus may serve a repository role for the temporal storage of centromere RNA and proteins, and control the timely release of these components into the nucleoplasm for kinetochore assembly at M phase. It is also possible that the assembly of functional kinetochore nucleoprotein complexes may take place in the nucleolus (Wong, 2007).

Treatment of metaphase cells with single-stranded RNA-specific RNases (but not with a double-stranded RNA-specific RNase) results in the significant but incomplete delocalization of CENPC1, and the complete delocalization of two centromere-associated chromosomal passenger proteins INCENP and survivin, from the metaphase centromere. Similar treatments have no significant effect on the centromeric localization of CENPA, CENPB, and CENPE. These results indicate that the assembly of INCENP and survivin, and to an extent CENPC1, but not CENPA, CENPB, and CENPE, at the metaphase centromere is dependent on the presence of single-stranded RNA (Wong, 2007).

Several lines of evidence suggest that at least a component of this single-stranded RNA is directly transcribed from the centromeric α-satellite DNA, and that this α-satellite RNA transcript associates directly with CENPC1. (1) Recombinant GST-fusion protein truncation constructs and site-specific mutagenesis were used to show that CENPC1 binds single-stranded α-satellite RNA probes derived from chromosomes 2, 4, 9, or 13/21, and ranging in size from 167-342 nucleotides (nt). This RNA-binding activity resides within the central region (amino acids 426-551) as well as the C-terminal portion of the protein. (2) By performing RT-PCR on RNA isolated following RNA-ChIP using an anti-CENPC1 antibody, in vivo binding of single-stranded α-satellite RNA with the CENPC1-associated kinetochore protein complex has been demonstrated. The RT-PCR results indicate that the kinetochore-associated α-satellite RNA has a size range corresponding to multiples of the 171-nt α-satellite repeating unit. These studies show the presence of single-stranded α-satellite RNA of 171 nt and larger and belonging to the different chromosome-specific α-satellite subfamilies. However, the possibility cannot be excluded that shorter RNA or α-satellite RNA of other sequences may be present, as the present detection may bias against these RNAs (Wong, 2007).

(3) It has been shown that the in situ replenishment of single-stranded α-satellite RNA significantly restores the relocalization of myc-tagged CENPC1 and INCENP to the centromeres of some of the chromosomes following RNase treatment. The incomplete rescue of the recruitment of the myc-tagged recombinant proteins to the kinetochore could be due to the inappropriate folding or modification of the recombinant proteins or replenishing RNA used. Further investigations are needed to discern these possibilities and define the characteristics and possible heterogeneity associated with these RNA transcripts (Wong, 2007).

(4) It has been shown that wild-type GFP-CENPC1, but not the mutant construct with mutated residues within the NoLs domain, is able to partially restore the functionality of the CENPC1-depleted kinetochore in the CENPC1 RNAi-knockdown cells and rescue the cells from the inhibition of cell growth. The failure of the over-expression of the mutated GFP-CENPC1 protein in improving the rate of cell growth suggests that the NoLS domain is essential for the proper function of CENPC1 (Wong, 2007).

Together, this analysis indicates that, in addition to the important role played by the short, double-stranded siRNA at the pericentric heterochromatin, a distinct class of longer, single-stranded centromeric α-satellite RNA is an important structural component of the human centromere (Wong, 2007).

Central to this model is the finding that centromeric α-satellite RNA is essential for the enrichment of centromere proteins CENPC1, INCENP, and/or survivin at the nucleolus and mitotic centromere. It is proposed that the nucleolar sequestration of centromere-specific nucleoprotein components provides a regulatory mechanism for the timely release of these components into the nucleoplasm for kinetochore assembly at the onset of mitosis. The finding that CENPC1 binds centromere satellite RNA, whereas previous in vitro studies have shown that CENPC1 is also DNA-binding (Yang, 1996; Politi, 2002), together suggest that this protein has a dual RNA- and DNA-binding function. It is therefore possible that one pool of CENPC1 may play a constitutive centromere DNA-binding role that persists throughout the cell cycle, while a second pool of this protein may act directly as, or be part of, a chaperone mechanism to relocate centromeric α-satellite RNA and centromere proteins, including INCENP, from the nucleolus onto the mitotic centromere. The RNA dependence of the nucleolar localization of both CENPC1 and INCENP suggests that these proteins may associate with the centromeric α-satellite RNA and be assembled into a nucleoprotein complex in the nucleolus. This nucleolar chaperone complex may further include other centromere-associated proteins such as borealin, PARP1, and PARP2, since these components have also been shown to accumulate in the nucleolus. In further support of the proposed dual role of CENPC1, significantly higher quantitative immunofluorescence signals have consistently been observed for CENPC1 (but not other constitutive centromere proteins examined, including CENPA and CENPB) on the metaphase centromere compared with those of the interphase centromere. Of interest, a recent study (Kwon, 2007) has shown that CENPC1 is targeted to interphase or metaphase centromeres through interactions with different sets of centromere proteins, providing further evidence that CENPC1 is localized to the centromere via two independent pathways during interphase or the mitosis stage. The present study has shown that treatment with single-stranded RNA-specific RNases results in a significant, but not complete, delocalization of CENPC1 from the metaphase centromere, consistent with the idea that only the RNA-, but not the DNA-, associated pool of CENPC1 (and components of its complex, such as INCENP) is sensitive to the RNase treatment. The data demonstrate that survivin does not localize to the nucleolus, although its localization to the metaphase centromere is also RNA-dependent. This can be explained by the incorporation of survivin into the metaphase chromosomes following the breakdown of the nucleolar membrane, via INCENP and/or borealin; previous work (Klein, 2006) has shown that survivin forms a functional complex with these two proteins for targeting to the centromere (Wong, 2007).

CENP-C is a structural platform for kinetochore assembly

Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. This study has found that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, the Mis12 complex component Nnf1 was shown to interact directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, it was targeted to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis (Przewloka, 2011).

Centromeres are patches of chromatin defined by the presence of the specific histone H3 variant called centromeric protein A (CENP-A)/CenH3, also known as CID in Drosophila, Cse4 in S. cerevisiae, and Cnp1 in S. pombe. Shortly before each mitosis, kinetochores are assembled on centromeres in a process whose mechanism and regulation are largely unknown (Przewloka, 2011).

Recently published studies have pointed toward the centromeric protein CENP-C as a potential candidate for the regulatory role in kinetochore formation. CENP-C is localized at the centromeric chromatin throughout the cell cycle. It is embedded in the centromeric chromatin and, together with CENP-A, forms part of the core of the centromere. The carboxy-terminal part of CENP-C is known to be responsible for the binding to centromeres but by itself is insufficient to rescue the lack of CENP-C in vertebrate cells or Drosophila embryos. However, the exact function of CENP-C’s N-terminal part has not been defined. In vertebrates, CENP-C is also a component of the so-called constitutive centromere-associated network (CCAN) that comprises at least 14 proteins assembled in several subcomplexes (Przewloka, 2011).

Whereas CENP-C seems to be a component of the centromere in all organisms, the associated CCAN proteins appear to be absent from the centromeres in C. elegans and Drosophila. Because these organisms thus provide a fundamentally simplified centromere whose functions may be easier to dissect, the roles of CENP-C in kinetochore assembly were studied using D. melanogaster as a model (Przewloka, 2011).

In order to test the localization properties of the two functional parts of CENP-C, the open reading frame was arbitrarily divided, and both portions were expressed in Drosophila Dmel-2 culture cells. Stably transformed cell lines expressing a transgene encoding a fusion of EGFP to the C-terminal 623 amino acids of the 1411 amino acid CENP-C (which are referred to as EGFP::CENPC-C) were generated (Przewloka, 2011).

This transgene was under the control of the inducible metallothionein promoter to avoid potential dominant-negative phenotypes that might arise from constitutive expression. As expected, this CENPC-C fusion colocalized with the centromeric marker CENP-A/CID during the entire cell cycle. In contrast, a fusion of EGFP with the N-terminal 788 amino acids of CENP-C, EGFP::CENPC-N, localized to nuclei but not to centromeres when expressed from the metallothionein promoter. This suggests that this half of the protein must be transported actively into the nucleus, although a nuclear localization signal within this part was not identified previously. Moreover, its lack of centromeric localization suggests that this part of CENP-C is incapable of either dimerizing with full-length endogenous protein or interacting with other centromeric components (Przewloka, 2011).

This raised the question of what other proteins the N-terminal part of CENP-C might interact with. To address this, a stably transformed Drosophila cell line was built able to express a fusion of protein A::CENPC-N from the metallothionein promoter. After overnight induction of protein A::CENPC-N expression, the product of the transgene was affinity purified from cell extracts using IgG-coupled magnetic beads for analysis by mass spectrometry. No peptides from the centromeric proteins CENP-A/CID [18], CAL1 [19], or the C-terminal part of CENP-C were identified, in accordance with the observation that CENPC-N does not associate with centromeres. Surprisingly, however, it was possible to identify the entire set of kinetochore proteins belonging to theKMN (KNL1/Spc105, theMis12 complex, and the Ndc80 complex) network, including Spc105, Mis12, Nnf1, Spc25/Mitch, Nuf2, and Ndc80. In addition, proteins were identified belonging to the 14-3-3 family with high Mascot scores. The high levels of phosphorylation of CENP-C in cultured Drosophila cells and in Drosophila embryos would accord with being able to associate with 14-3-3 proteins that interact with phosphorylated targets.None of the abovementioned proteins copurified with protein A alone (Przewloka, 2011).

Because it was not possible to find any good candidate proteins that might provide a bridge between CENPC-N and KMN components, it was speculated that the interaction might be a direct one involving the innermost part of the kinetochore, the Mis12 complex. To test this possibility, the 35S-labeled Mis12 complex subunits Nnf1a and Nsl1 and also maltose-binding protein (MBP) as a negative control were synthesized in an in vitro transcription- translation system and whether they would interact with bacterially expressed CENP-C fragments was investigated. (Mis12 appeared to be either insoluble or very poorly expressed in this system, and hence its interaction could not be tested.) No binding of MBP, Nnf1a, or Nsl1 to GST::CENPC-C was detected. However, binding of Nnf1a to the N-terminal part of CENP-C was detected, whereas Nsl1 binding was poorly detectable. This suggests that this component of the Mis12 complex may be responsible for the association of the KMN network with CENP-C. It would accord with a recent study (Petrovic, 2010) that places Nnf1, of all the Mis12 complex components, as lying most proximal to the centromeric chromatin (Przewloka, 2011).

The finding that the N-terminal part of CENP-C can associate with all proteins of the KMN network led to a speculation about its potential role in kinetochore assembly and a test whether it would be sufficient to recruit the KMN network and other kinetochore proteins to ectopic sites. This part of CENP-C was targeted to centrosomes and whether the KMN network would be recruited to the same site was tested. To this end, a centrosome-targeting domain from Plk4 kinase was fused in frame with the EGFP::CENPC-N construct (this entire construct was termed ect-KTR₁, for ectopic kinetochore recruitment). As a control, the localization and recruitment capabilities were tested of the Plk4 centrosome localization domain (CLD) alone fused with EGFP. Expression of either construct in Dmel-2 cells from an inducible promoter resulted in the localization of EGFP at the centrosome, confirmed by costaining with the centrosomal markers dPLP/Cp309 (the Drosophila counterpart of pericentrin) and dSpd2/Cep192. The ect-KTR₁ fusion protein also localized to the nuclei of a substantial subset of cells during interphase, most likely reflecting the activity of its own strong nuclear localization signal. However, focus was placed on localization of these proteins in mitotic cells when kinetochores have normally assembled at the centromeres, thus allowing examination of the extent to which kinetochore proteins localized to either centromeres or centrosomes (Przewloka, 2011).

Because expression of either CENPC-N or ect-KTR caused frequent and severe mitotic phenotypes in which chromosomes became scattered so as often to lie in the vicinity of the poles, focus was placed on examining the localization of kinetochore proteins in cells where centrosomes and centromeres were well separated (Przewloka, 2011).

Cells were stained with several antibodies raised against kinetochore proteins. Expression of the CLD alone did not lead to any change of the localization of kinetochore markers. Kinetochore proteins belonging to the KMN network colocalized exclusively with CENP-A/CID in such cells. Expression of ect-KTR, on the other hand, led to the strong recruitment of all KMN network components tested to the centrosome and the frequent diminution of the levels of these proteins at the kinetochore. Thus, immunostaining clearly revealed Mis12, Nsl1, Nnf1a, and Spc105 to colocalize with ect-KTR and the centrosomal markers dPlp and Spd2. To determine the localization of the outer kinetochore plate Ndc80 complex, Dmel-2 cells were cotransfected with constructs expressing RFP-tagged ect-KTR and either EGFP-tagged Ndc80 or Nuf2. It was found in both cases that these components of the Ndc80 complex localized to centrosomes in ect-KTR-expressing cells. In contrast, neither of the centromeric markers CAL1 or CENP-A/CID colocalized with ect-KTR; they were found only at centromeres. Thus, the N-terminal part of CENP-C is not able to interact with centromeric proteins but is able to recruit the KMN network to an ectopic site in the cell (Przewloka, 2011).

It was also asked whether, in addition to the core KMN network proteins, other ancillary proteins known to associate temporarily with kinetochores might be relocated onto ectopically positioned CENPC-N. The localization was examined of the kinetochore-associated motor protein CENP-meta (the Drosophila counterpart of CENP-E) and the spindle assembly checkpoint proteins Mad2 and BubR1. It was found that, unlike the core KMN network proteins that were associated with centrosomes in all cells expressing ect-KTR, ancillary kinetochore proteins were only associated with centrosomes in a subset of ect-KTR-expressing cells. Thus, CENP-meta was predominantly centrosome associated in 78.8% of ect-KTR cells. Mad2 also showed similar localization to ectopic sites regardless of whether or not the checkpoint was activated by colchicine treatment. No strong signals of Mad2 were seen on scattered chromosomes in ect-KTR cells. This could be due to, for example, either depletion of a proper platform for binding of Mad2 on kinetochores such as the Ndc80 complex or the titration of Mad2 to centrosomes. This observation supports the idea that Mad2 binds to kinetochores, not centromeres. On the contrary, in the case of BubR1, its association with centrosomes in a subset of ect-KTR-expressing cells was much weaker than its association with centromeres in the same cells. This is in agreement with suggestions that BubR1 may have at least two independent binding sites: one on centromeres, which might be more exposed in cells expressing ect-KTR, and the other on kinetochores, which in cells expressing ect-KTR would be associated with centrosomes. In fact, this result supports the idea of BubR1 being recruited to centromeres rather than kinetochores. Because none of these ancillary kinetochore proteins associated with centrosomes in CLD-expressing cells, it is concluded that the ectopic localization of the N-terminal part of CENP-C was responsible for localizing the core KMN network to centrosomes, and that this then permitted recruitment of other kinetochore-associated proteins (Przewloka, 2011).

As indicated above, it was found frequent mitotic defects in cells expressing CENPC-N and ect-KTR, of which improperly congressed and scattered chromosomes were the most commonly observed aberrations. The scattering of chromosomes on elongated spindles was reminiscent of the phenotype observed after knockdown of components of the KMN network. It was observed that whereas spindles in CLD-expressing cells had a mean length of 6.83 that is typical of the parent Dmel-2 cell line, spindles in ect-KTR and CENPC-N cells were significantly elongated and had mean lengths of 8.7 and 8.04, respectively. To determine whether cells with elongated spindles were in anaphase, cells were costained with anti-CENP-A/CID and anti-cyclin B antibodies. This revealed that ect-KTR cells showing the mitotic phenotype had high levels of cyclin B and that the chromosomes had two centromeres and so were conjoined chromatids (Przewloka, 2011).

This strongly suggests that these cells were blocked in prometaphase with improperly congressed chromosomes and had not entered anaphase. Therefore, the chromosome scattering and the spindle elongation phenotype may be a consequence of the very long prometaphase block occurring as a result of the primary defect: severe congression problems (Przewloka, 2011).

It was hypothesized that the high incidence of mitotic defects present after ect-KTR or CENPC-N expression was caused by the titration out of one or more KMN network proteins by the ectopic construct. This interpretation was supported by the observation that immunolocalization signals of kinetochore proteins ectopically localized at centrosomes were usually stronger than the signals at centromeres in ect-KTR cells. To address this directly, fluorescence intensities of endogenous kinetochores were quantified after staining with anti-Mis12 and anti-Spc105 antibodies in relation to the intensities of the centromeric CENP-A/CID in CLD-, ect-KTR-, and CENPC-N-expressing cells. In ect-KTR- and CENPC-N-expressing cells, Mis12 levels at the kinetochore were reduced respectively to an average of 46% and 26.8% of the level in cells expressing the CLD construct alone. Similarly, levels of Spc105 were reduced to 29.2% and to 39.1% in ect-KTR and CENPC-N cells respectively. Thus, it is concluded that overexpression of ect-KTR or nontargeted CENPC-N indeed results in a depletion of kinetochore proteins from endogenous kinetochores, and that this in turn leads to mitotic defects (Przewloka, 2011).

Although the vast majority of CLD-expressing cells looked normal, a low level of background aberrations were noticed that may be attributable to the expression of the Plk4 fragment, which is able to drive production of extra centrosomes and which may affect the efficiency of spindle assembly. However, the finding of chromosomes scattered along elongated spindles was unique to CENPC-N- or ect- KTR-expressing cells and was not seen in CLD-expressing cells. This strong dominant-negative phenotype and the ectopic localization pattern of KMN network proteins in cells expressing ect-KTR lead to the unequivocal conclusion that the N-terminal domain of CENP-C is sufficient to recruit all of the major core kinetochore proteins, regardless of its localization in the cell (Przewloka, 2011).

Recent studies from a number of laboratories have provided a detailed description of the interaction network between subunits of the KMN supercomplex. This knowledge is key for reaching a full understanding of how kinetochores are formed early in mitosis and how they interact with microtubules of the mitotic spindle. Considerable progress has also been made in studies of centromere inheritance, composition, and function (Przewloka, 2011).

However, little is known yet about the interaction between these two complex structures. Previously, it was reported that kinetochore formation in Drosophila cells is absolutely dependent upon the presence of the centromeric protein CENP-C. The results presented in this study indicate that it is the N-terminal part of this protein that not only is responsible for binding to the KMN network but also is sufficient to recruit it to ectopic sites (Przewloka, 2011).

The consequential depletion of KMN proteins from the centromeres of mitotic chromosomes to centrosomes leads to strong mitotic defects. Together, these data support a model in which one face of CENP-C binds CENP-A-rich centromeric chromatin and the other binds components of the KMN network, providing a platform for kinetochore assembly at the onset of mitosis. That such a function of CENP-C may be conserved is strongly suggested from a contemporaneous study of its interacting partners in human cells (Screpanti, 2011). The length of the CENP-C molecule, the bifunctionality of its binding, and its potential for posttranslational modification together suggest that this protein might not only provide structural support for kinetochore assembly but also be a major hub for regulating centromere and kinetochore function. It will be important to learn more about such potential regulatory mechanisms, and also to determine whether CENP-C is the only component of the centromere to provide an interface between centromeres and kinetochores in either Drosophila or vertebrates (Przewloka, 2011).

CENP-C functions as a scaffold for effectors with essential kinetochore functions in mitosis and meiosis

The conserved kinetochore protein CENP-C plays a fundamental role in chromosome segregation, but its specific functions remain elusive. This study has gained insights into the role of CENP-C through identification of interacting effector proteins required for kinetochore function in fission yeast. Fta1/CENP-L is a primary effector that associates directly with Cnp3/CENP-C, and ectopic localization of Fta1 largely suppresses the mitotic kinetochore defects of cnp3Delta cells. Pcs1 functions downstream of Cnp3 to prevent merotelic attachment. In meiosis, Cnp3 further associates with and recruits Moa1, a meiosis-specific protein exclusively required for the mono-orientation of kinetochores. Genetic and biochemical analyses identified Cnp3 mutants that preserve intact mitotic kinetochore function but abolish the association with Moa1 and meiotic mono-orientation. Overall, therefore, these studies identify effectors of CENP-C in mitosis and meiosis and establish the concept that CENP-C serves as a scaffold for the specific recruitment of essential kinetochore proteins (Tanaka, 2011).

Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore

Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as the inner and outer kinetochore, respectively. The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multisubunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization. This study shows that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome missegregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and checkpoint impairment was observed in cells expressing the Cenp-C N-terminal segment. These studies unveil a crucial and likely universal link between the inner and outer kinetochore (Screpanti, 2011).

Dual recognition of CENP-A nucleosomes is required for centromere assembly

Centromeres contain specialized nucleosomes in which histone H3 is replaced by the histone variant centromere protein A (CENP-A). CENP-A nucleosomes are thought to act as an epigenetic mark that specifies centromere identity. CENP-N has been identified as a CENP-A nucleosome-specific binding protein. This study shows that CENP-C also binds directly and specifically to CENP-A nucleosomes. Nucleosome binding by CENP-C required the extreme C terminus of CENP-A and did not compete with CENP-N binding, which suggests that CENP-C and CENP-N recognize distinct structural elements of CENP-A nucleosomes. A mutation that disrupted CENP-C binding to CENP-A nucleosomes in vitro caused defects in CENP-C targeting to centromeres. Moreover, depletion of CENP-C with siRNA resulted in the mislocalization of all other nonhistone CENPs examined, including CENP-K, CENP-H, CENP-I, and CENP-T, and led to a partial reduction in centromeric CENP-A. It is propose that CENP-C binds directly to CENP-A chromatin and, together with CENP-N, provides the foundation upon which other centromere and kinetochore proteins are assembled (Carroll, 2010).

A model is proposed for human centromere assembly. A critical first step is the direct recognition of CENP-A chromatin by CENP-C. Because dimerization is important for CENP-C centromere targeting, but not for mononucleosome binding, it is speculated that each CENP-C dimer binds to two different, possibly adjacent, CENP-A nucleosomes within centromeric chromatin. A CENP-C dimer in conjunction with centromeric chromatin then provides the foundation upon which the rest of the Constitutive Centromere-Associated Network is assembled. CENP-N also binds directly to CENP-A nucleosomes, thus providing additional CENP-A nucleosome contacts that reinforce the specificity of the centromere assembly process (Carroll, 2010).

Search PubMed for articles about Drosophila Cenp-C

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Carroll, C. W., Milks, K. J. and Straight, A. F. (2010). Dual recognition of CENP-A nucleosomes is required for centromere assembly. J. Cell Biol. 189(7): 1143-55. PubMed ID: 20566683

Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. and Desai, A. (2006). The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127: 983-997. PubMed ID: 17129783

Ciferri, C., et al. (2005). Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore. J. Biol. Chem. 280: 29088-29095. PubMed ID: 15961401

Deluca, J. G., et al. (2006). Kinetochore microtubule dynamics and attachment stability are regulated by hec1. Cell 127: 969-982. PubMed ID: 17129782

Dunleavy, E. M., Beier, N. L., Gorgescu, W., Tang, J., Costes, S. V. and Karpen, G. H. (2012). The cell cycle timing of centromeric chromatin assembly in Drosophila meiosis is distinct from mitosis yet requires CAL1 and CENP-C. PLoS Biol 10: e1001460. PubMed ID: 23300382

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Fachinetti, D., Diego Folco, H., Nechemia-Arbely, Y., Valente, L. P., Nguyen, K., Wong, A. J., Zhu, Q., Holland, A. J., Desai, A., Jansen, L. E. and Cleveland, D. W. (2013). A two-step mechanism for epigenetic specification of centromere identity and function. Nat Cell Biol 15: 1056-1066. PubMed ID: 23873148

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date revised: 25 September 2023

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