BIOLOGICAL OVERVIEW

Centromeres are the chromosomal regions responsible for poleward movement at meiosis and mitosis, and are essential for the faithful segregation of genetic information. Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Centromeric chromatin is distinguished from bulk chromatin, most conspicuously by the presence of centromere-specific histone H3 variants (CenH3). A Drosophila CenH3, Cid (for Centromere identifier), the Drosophila homolog of the CENP-A centromere-specific H3-like proteins, localizes exclusively to fly centromeres. The function of Cid is highly conserved. This chromatin component probably plays a key role in assembling the kinetochore at meiosis and mitosis. Thus, CenH3 could be expected to both interact with the underlying centromeric DNA, as well as interact with other proteins (including other CenH3 molecules) to provide the foundation for the kinetochore (Malik, 2002).

Remarkably, when the cid upstream promoter region drives expression of yeast, worm, and human centromeric histone proteins, localization is preferentially within Drosophila pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Thus, these H3-like proteins from yeast and worms localize to pericentric heterochromatic regions surrounding fly and human centromeres. Preferential localization to heterochromatin in heterologous systems is unexpected if species specific centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, Cid is part of a specific conserved heterochromatic region surrounding centromeres (Henikoff, 2000).

Studies show that centromeres and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that Cid is required for normal kinetochore formation and function, as well as cell-cycle progression. Injection of Cid antibodies into early embryos, as well as RNA interference in tissue-culture cells, shows that Cid is required for several mitotic processes. Cid chromatin is physically separate from proteins involved in sister cohesion (Mei-s332), centric condensation (Prod, kinetochore function (Rough deal, Zeste-white 10 and Bub1) and heterochromatin structure (HP1). Cid localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of Polo, the kinesin kinetocore motor CENP-meta, Rough deal (Rod), Bub1 and Mei-S332, involved in sister chromatid cohesion) depends on the presence of functional Cid. It is concluded that Cid directs kinetochore formation and function by forming a unique heterochromatin within the centromere (Blower, 2001).

Immunolocalization experiments in mammals have shown that it is possible to distinguish between the inner and outer kinetochore by fluorescence microscope. To determine whether Cid is located in the inner kinetochore, Cid, spindle microtubules and many transient kinetochore proteins including Bub1, Zeste-white 10 (ZW10 -- accepted FlyBase name: Mitotic 15) and Rod were simultaneously localized. Each of these proteins would be expected to localize to the outer kinetochore plate or the fibrous corona, similar to other transient kinetochore proteins localized in mammals (for example, Bub1, CENP-E, Dynein) (Blower, 2001).

Simultaneous detection of Cid with outer kinetochore proteins showed that Cid is consistently separated from ZW10, Rod and Polo kinase, and is located closer to the chromosome and further from the kinetochore microtubules than these proteins. Cid is also offset from Bub1 (a component of the spindle assembly checkpoint) at unattached kinetochores, but Cid and Bub1 show significant overlap. This result is consistent with studies in mammals, which suggest that Bub1 may be located at both the inner and outer kinetochore plates. These results show that Cid is located in or near the inner plate of the kinetochore in Drosophila and is likely to be associated closely with centromeric DNA (Blower, 2001).

Previous work has shown that the outer kinetochore proteins ZW10 and Dynein are present on fully functional Drosophila minichromosomes (that is, 100% transmission through mitosis and meiosis), as well as structurally acentric minichromosomes that lack detectable centromeric sequence (neocentromeres). To determine the relationship of Cid-containing chromatin to the functional centromere, the localization of Cid protein was examined in a series of minichromosome derivatives of decreasing size and meiotic transmission efficiency (Blower, 2001).

Cid is present on all minichromosome derivatives that contain a fully functional centromere (gamma238, 31E, 10B and J21A, indicating that Cid colocalizes with the molecular-genetically defined centromere. Cid also is present on all of the neocentromeric derivatives that lack centric heterochromatin, including the normal minichromosome centromere (26C, J19B), consistent with the localization pattern of outer kinetochore components ZW10 and Dynein. It is concluded that Cid localization is correlated with centromere function, regardless of the composition of the underlying DNA (Blower, 2001).

The presence of Cid at the functional minichromosome centromere shows that Cid localization is correlated with centromere activity. To test the role of Cid in mitosis directly, affinity-purified chicken anti-Cid antibodies were injected into early embryos that express histone H2A/green fluorescent protein (GFP) and chromosome behavior was observed using time-lapse microscopy. Rhodamine-labelled anti-Cid antibodies bind specifically to centromeres in all stages of the cell cycle, and show no nonspecific crossreactivity with histone H3 either in vivo or by Western blot. Injected antibody binds centromeres in a gradient in which more antibody binds close to the site of injection (Blower, 2001).

Injection of Cid antibodies into early embryos results in a range of phenotypes affecting both cell-cycle progression and mitotic chromosome segregation. The phenotypic series is consistent with a gradient of Cid inhibition mirroring the gradient of antibody concentration. Nuclei closest to the site of injection arrest in interphase (13%), whereas nuclei further from the site of injection delayed entering mitosis and exhibited different mitotic defects: specifically, entry into prophase condensation followed by a loss of condensation (3.6%; metaphase arrest [15%]); and various anaphase chromosome segregation defects (failure to move toward the poles at anaphase onset, unequal chromosome segregation, failure to maintain spindle contact and karyokinesis defects at telophase, 20%) (Blower, 2001).

Cid function in Drosophila Kc tissue-culture cells is also disrupted using RNAi. Cells were treated with dsRNA corresponding to the whole Cid transcript; cells were then fixed and monitored for levels of Cid protein and aberrant chromosome behaviour every 24 h after adding dsRNA. Cells in a given treated population display a variable penetrance of Cid inhibition, which results in different phenotypes (Blower, 2001).

Mitotic defects in Kc cells are consistent with those observed after antibody injection into embryos, including aberrant prometaphase congression, precocious sister chromatid separation, kinetochore microtubule capture and anaphase segregation. Cells treated with dsRNA were no longer dividing 8-10 d after RNAi, suggesting that interphase arrest results from complete Cid disruption; however, without live analysis it is difficult to differentiate between interphase arrest and a terminal phenotype resulting from massive chromosome segregation defects (Blower, 2001).

The results of Cid disruption by both antibody injection into embryos and RNAi in tissue-culture cells show that Cid is directly or indirectly required for many aspects of kinetochore-mediated chromosome movement as well as cell-cycle progression (Blower, 2001).

Centromeres in most higher eukaryotes are embedded in centric heterochromatin, suggesting that both the structure and function of heterochromatin are required for centromere function. What are the structural relationships between centromeric chromatin, defined by Cid, and chromosomal proteins previously localized to the centromere region? This question was addressed using immunolocalization of three proteins and Cid on mitotic chromosomes from S2 and Kc tissue-culture cells (Blower, 2001).

Mei-S332 is required for sister chromatid cohesion during metaphase I of meiosis, and is present in the centromeric regions of meiotic and mitotic chromosomes. Simultaneous localization of Cid with Mei-S332 shows that Cid antibodies yield typical double-dot staining, whereas Mei-S332 is localized in two concentrated foci joined by a bridge of staining that connects the sister chromatids. Although Mei-S332 has been described as centromeric and possibly located to the inner kinetochore, the higher resolution localization of Mei-S332 presented in this study showed consistent offset of antibody staining to one side of the kinetochore and along the chromosome axis on all chromosomes. The offset localization is always to the same side of the kinetochore on each chromosome type. This is especially evident on the X chromosome, in which Mei-S332 is always located on the proximal long arm side of Cid, and for chromosomes 2 and 3, on the basis of colocalization with the sequence-specific satellite binding protein Prod. It is concluded that Mei-S332 is located near but not in the Cid chromatin, providing a physical basis for the previous observation that kinetochore function and Mei-S332-mediated cohesion can be separated using minichromosome derivatives (Blower, 2001).

proliferation disrupter (prod) mutant larval neuroblasts display hypo-condensation of the centromere region and metaphase/anaphase arrest. Consistent with the decondensation phenotype, the Prod protein localizes to the centromeric region of chromosomes 2 and 3 in mitosis, suggesting that it may be involved in kinetochore function on these chromosomes. However, simultaneous detection of Cid and Prod on mitotic chromosomes shows that Prod stains a more expansive portion of the chromosome than Cid, and is offset from the kinetochore in the same manner as Mei-S332. In fact, Prod and Mei-S332 are both localized to the same side of the kinetochore on chromosomes 2 and 3 (Blower, 2001).

HP1 mutants show dominant suppression of heterochromatin-induced position-effect variegation (PEV), and recessive telomere fusions and chromosome segregation defects. Human and mouse homologs of HP1 localize to the centromere region, and S. pombe Swi6, another chromodomain protein, is localized to the centromere and required for proper chromosome transmission. Simultaneous localization of Cid with HP1 revealed that HP1 is not present in centromeric chromatin in either interphase or metaphase. In metaphase chromosomes, HP1 is located throughout the pericentric heterochromatin, and is near but not in Cid chromatin (Blower, 2001).

It is concluded that Prod and HP1 are located in the pericentric heterochromatin and not in the centromeric chromatin. These results suggest that, although the centromere is embedded in large blocks of heterochromatin, centromeric chromatin is spatially separable from canonical centric heterochromatin (Blower, 2001).

Does the spatial separation of Cid chromatin, outer kinetochore proteins and centric heterochromatin proteins reflect functional independence? Cid localization was examined in larval neuroblasts from animals lacking either Prod, HP1, Mei-S332 or Polo kinase. In interphase nuclei and mitotic chromosomes from homozygous prod mutants, Cid remains localized in the typical punctate pattern observed in wild type, despite visible centromere region hypocondensation. Similarly, Cid was localized in the typical punctate pattern in interphase nuclei from homozygous mutant Su(var)2-5 (HP1) neuroblasts (Blower, 2001).

In mutant metaphase spreads exhibiting the Su(var)2-5 telomere fusion phenotype Cid still localizes in the characteristic double-dot pattern. Furthermore, Cid is also localized in the characteristic double dot pattern in homozygous mei-S332 mutant larval neuroblasts. Finally, in metaphases exhibiting circular spreads indicative of centrosome disorganization, characteristic of polo mutations, Cid remains localized in characteristic double dots. Thus, the analyses of Cid localization in mutant neuroblasts show that the assembly and maintenance of centromeric chromatin in interphase and metaphase is not dependent on the presence of proteins required for normal centromere region condensation (Prod), heterochromatin structure (HP1), centric cohesion (Mei-S332), or outer kinetochore function (Polo kinase) (Blower, 2001).

Although Cid localization is not dependent on the presence of Prod, HP1, Mei-S332 or Polo kinase, the mutant analyses did not determine whether the localization of these proteins depended on Cid. Therefore, Polo kinase, Mei-S332 and Prod localization were examined in embryos in which Cid function was inhibited. In embryonic nuclei close to the site of injection, where high levels of Cid antibody binding and the most severe mitotic defects are observed, Polo kinase localization is diffuse and apparently absent from kinetochores, as judged by counterstaining with Prod. Notably, in these same nuclei Mei-S332 was absent from the pericentromeric region, whereas Prod, a protein with sequence-specific satellite-binding properties, was still present in the pericentromeric region (Blower, 2001).

The localizations of Rod, CENP-meta outer kinetochore, CENP-E kinesin-like protein homolog, Polo kinase, Bub1 and Mei-S332, but not Prod or HP1, were also disrupted in Kc cells displaying mitotic defects as a result of RNAi inhibition of Cid expression. Quantitative deconvolution microscopy has revealed that transient kinetochore component recruitment is proportional to the amount of Cid present at the kinetochore, whereas Prod recruitment is independent of Cid levels. Thus, Cid function is required for the recruitment or maintenance of transient kinetochore components and a centric cohesion protein (Mei-S332), but is not required for the localization of Prod or HP1. These results also indicate that the pleiotropic mitotic defects observed in anti-Cid injection and RNAi are likely to be caused by a failure to recruit or bind transient kinetochore components and a centric cohesion protein (Blower, 2001).

CENP-A has been proposed to be an epigenetic mark required for determining centromere identity and thus the assembly of the kinetochore. This study has shown that Cid, the Drosophila CENP-A homolog, is associated with the inner kinetochore and that Cid localization correlates with centromere function, specifically the presence of the molecular-genetically defined centromere and neocentromere DNA (Blower, 2001).

The presence of Cid on neocentromeres shows that these structurally acentric yet functional chromosomes have acquired centromeric chromatin, consistent with the presence of outer kinetochore components. Furthermore, the presence of Cid on neocentromeres shows that the location of centromeric chromatin is independent of sequence, and that centromeric chromatin can confer centromere identity on normally non-centromeric DNA -- a state that is then propagated faithfully through replication and division (Blower, 2001).

Cid is required for kinetochore assembly and cell-cycle progression in early Drosophila embryos and Kc tissue-culture cells. Mitotic defects have been observed in human cells after CENP-A antibody injection and in the mouse CENP-A knockout; however, the 'live studies' reported in this study have allowed an examination of the temporal and cytological effects of CENP-A/Cid disruption in greater detail. Both antibody injection into embryos and RNAi inhibition in Kc cells results in several defects expected for centromere dysfunction: specifically, aberrant prometaphase congression; chromosome attachment to spindle microtubules; entry into anaphase; anaphase poleward segregation,and failure to resolve properly at telophase (Blower, 2001).

The mislocalization of Rod, Bub1, CENP-meta, Polo and Mei-S332 in nuclei displaying missegregation phenotypes shows that the defects are correlated with aberrant kinetochore structure and the recruitment of transient kinetochore proteins and other centromere region proteins. These results extend the earlier observation that the inner kinetochore protein CENP-C is mislocalized in the CENP-A knockout mouse to the location of outer kinetochore proteins. Notably, the amount of outer kinetochore components present at the kinetochore is proportional to the amount of Cid, suggesting that the kinetochore may be composed of a repeated substructure (Blower, 2001).

Cid disruption may decrease the number or size of functional subunits; this is sufficient to cause defects in mitosis because mitotic defects are observed in cells with decreased but visible amounts of Cid. Disruption of the centromere/kinetochore substructure may be responsible for different degrees of mislocalization of outer kinetochore components, which results in the observed pleiotropy of mitotic phenotypes. Karyokinesis defects observed after Cid inhibition may be the result of the failure of chromosomal passenger proteins to localize to the kinetochore and consequently to the spindle and midbody. On the basis of the mislocalization of several outer kinetochore components, it is concluded that Cid is epistatic to transient kinetochore components; these results support the hypothesis that CENP-A proteins are involved directly in the epigenetic marking of the site of kinetochore formation, and show conclusively that proper kinetochore function is required for many cell-division processes (Blower, 2001).

More complete Cid disruption in embryos results in a severe interphase arrest phenotype, showing that Cid function is also required before entry into mitosis, which is similar to one of the phenotypes observed after injection of anti-CENP-A antibodies into HeLa cells. The use of real-time analysis allows for an unambiguous conclusion that the nuclei are arrested before entry into mitosis. The interphase arrest phenotype suggests that Cid functions in interphase, where it is constitutively bound to centromeres, and that there may be another cell-cycle checkpoint that monitors kinetochore assembly and blocks entry into mitosis if this process is compromised (Blower, 2001).

This putative 'kinetochore assembly' checkpoint is also likely to be responsible for the delay in entering mitosis observed in nuclei just distal to the injection site. It is possible that disruption of an interaction between Polo kinase and Cid is responsible for this cell-cycle arrest because Polo is required for entry into mitosis. Clearly, further investigation is necessary to elucidate the precise pathway involved in this arrest, and to determine whether there is a checkpoint that monitors kinetochore assembly before entry into mitosis (Blower, 2001).

Genetic and protein localization studies have implicated several proteins in regulating centromere function. Until now, it has been difficult to determine whether these proteins are involved in kinetochore formation and function, other centromere functions, or functions independent of the centromere/kinetochore. The structural and functional analyses presented in this study support the hypothesis that distinct spatial and functional domains exist in the centromere and adjacent regions. Previously, cytological studies in humans have revealed the presence of spatially distinct protein domains in the mitotic kinetochore (Blower, 2001).

This study extends these observations by investigating the domain structure of the kinetochore and the centromere region, including the flanking heterochromatin, and has provided data that reveal the functional separation and interdependence of these structural domains. Centromeric chromatin is the central and most essential component of the centromere region, and is required for entry into mitosis and chromosome movement during mitosis, as well as for recruiting proteins to the kinetochore and flanking domains. The domain organization of the centromere observed in Drosophila is similar to the situation in S. pombe, where different proteins occupy distinct subdomains within the centromere region, and are required for separable chromosome segregation processes (Blower, 2001).

Studies in a variety of organisms have indicated that the centromere region is a site of specialized sister cohesion, not only in meiosis I but also in mitosis. For example, normal homolog disjunction requires that Mei-S332 functions to maintain sister chromatid cohesion in the centric regions throughout meiosis I and until anaphase of meiosis II. Although Mei-S332 is involved in sister chromatid cohesion, it is not a cohesin; moreover, its localization differs from that observed in S. cerevisiae, where cohesins are concentrated at the centromere and associated with centromeric chromatin (Blower, 2001).

Mei-S332 is required for proper chromosome inheritance in Drosophila, but surprisingly the chromosome still retains kinetochore protein localization and functions if Mei-S332 is eliminated. Despite the spatial and functional separation of Mei-S332 and Cid, Mei-S332 is dependent on functional centromeric chromatin for its recruitment to the centromere region: it is mislocalized in anti-Cid injected embryos and RNAi-treated Kc cells. Cid localization is not, however, dependent on the presence of Mei-S332. Therefore, Cid is epistatic to Mei-S332 in the pathway responsible for the assembly and/or maintenance of this physically and functionally distinct centromere region domain in mitosis. The relationship between Cid, kinetochore function and Mei-S332-mediated cohesion warrants further genetic and biochemical analyses. It will be particularly interesting to determine the significance of the consistent asymmetric positioning of Mei-S332 to only one side of the Cid chromatin, as well as its impact on Cid during meiosis, where mutant phenotypes are more severe (Blower, 2001).

Heterochromatin encodes several inheritance functions, including homolog pairing in meiosis I, sister chromatid cohesion and interactions with anti-poleward forces. The conserved location of centromeres in heterochromatin suggests that heterochromatin proteins, such as Prod and HP1, may be required for establishing or maintaining centromeres. Neither Prod nor HP1 are detectable in Cid chromatin and prod and Su(var)2-5 mutations do not affect the localization of Cid. Furthermore, neither Prod nor HP1 localization is affected in anti-Cid-injected embryos or RNAi-treated Kc cells (Blower, 2001).

It is concluded that Prod and HP1 function in the pericentromeric regions to promote normal condensation and chromosome segregation -- processes distinct from the centromere/kinetochore. Although the kinetochore is typically embedded in large blocks of heterochromatin, evidence has been provideed that it may be structurally and functionally distinct from the closely juxtaposed pericentromeric or centric heterochromatin (Blower, 2001).

If centromere chromatin structure is distinct from centric heterochromatin, why are centromeres embedded in heterochromatin in almost all multicellular eukaryotes? Perhaps the flanking heterochromatin does provide an environment that is necessary for the formation of a centromere-specific higher order chromatin structure. In addition, although Prod and HP1 are not necessary for Cid localization, they may encode functions redundant with other heterochromatic proteins that establish or maintain proper kinetochore structure. It will be interesting to determine whether protein localization and mutant analyses with other centric heterochromatin proteins are consistent with the results for prod and Su(var)2-5 (Blower, 2001).

Drosophila CENP-A/Cid is required for kinetochore formation and mitotic function, cell-cycle progression and recruiting transient kinetochore components and a sister chromatid cohesion protein. In contrast, Cid and proteins that function in the pericentric heterochromatin are physically and functionally independent. These results support the hypothesis that CENP-A proteins are central to many mitotic processes, and may be a component of the epigenetic mark responsible for centromere identity and function. Future studies should investigate what mechanism is responsible for loading CENP-A specifically into CENP-A chromatin in a replication-independent manner, since this process may be the key to understanding maintenance of the epigenetic mark and centromere identity (Blower, 2001).

PROTEIN STRUCTURE

Amino Acids - 225

Structural Domains

Mammalia CENP-A and yeast Cse4p share with histone H3 the ~100-aa nucleosomal core, but have N-terminal tails that are completely dissimilar from one another and from H3. In CENP-A, centromeric localization maps to the core, which is about equally divergent from both Cse4p and H3. Except for CENP-A and Cse4p, only three GenBank entries were found to have comparably diverged H3-like cores and dissimilar N-terminal tails. A 225-aa ORF that fits this profile has been found within a 'sequencing in progress' database entry (AC005652) deposited by the Berkeley Drosophila Genome Project. The possibility was considered that this ORF encodes a centromeric protein. An antiserum against a peptide predicted from the ORF was used for immunocytochemistry to D. melanogaster Kc cells. Intense, point-like signals were observed over interphase nuclei and exclusively over centromeric constrictions of all Drosophila mitotic chromosomes in both Kc tissue culture cells and larval neuroblasts. Similar results were obtained with antibody raised against a peptide predicted from another N-terminal tail region of the ORF. Therefore, these epitopes are present on a normal centromere component that is present throughout the cell cycle. Because the 225-aa protein behaves as a Drosophila homolog of CENP-A and Cse4p, the gene was named cid (for centromere identifier) (Henikoff, 2000).

date revised: 20 April 2002

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