The centromere-specific histone H3 variant CENP-A plays a crucial role in kinetochore specification and assembly. A genetic approach was undertaken to identify interactors of the Drosophila CENP-A homolog CID. Overexpression of cid in the proliferating eye imaginal disc results in a rough eye phenotype, which is dependent on the ability of the overexpressed protein to localize to the kinetochore. A screen for modifiers of the rough eye phenotype identified mutations in the Drosophila condensin subunit gene Cap-G as interactors. Yeast two-hybrid experiments also reveal an interaction between CID and Cap-G. While chromosome condensation in Cap-G mutant embryos appears largely unaffected, massive defects in sister chromatid segregation occur during mitosis. Taken together, these results suggest a link between the chromatin condensation machinery and kinetochore structure (Jager, 2005).
The majority of mutations that cause mitotic defects in Drosophila do not lead to developmental arrest until the late larval or early pupal stages of development. This is due to two developmental features of Drosophila: (1) during oogenesis large maternal stockpiles of mRNA and proteins needed for cell division are deposited in the egg, and (2) following 16 division cycles in embryogenesis, the majority of cells enter the endo cycle and become polytene. Thus during the larval stages mitosis takes place only in the developing brain and imaginal discs and these tissues are not essential until pupation (Dej, 2004).
Cap-G was identified in a mutagenic screen; this was carried out to identifying mitotic regulatory proteins that are turned over during the cell cycle and must be synthesized de novo in each new cycle. Focused on embryonic lethal mutations in which the first 13 nuclear divisions occur normally in a syncytium using maternal supplies, but embryos arrest in the postblastoderm divisions that follow cellularization. During these three divisions, cycles 14-16, zygotic gene expression occurs and proteins that are degraded at the end of one cell cycle are synthesized de novo from the zygotic genome in the next cell cycle. Mitotic arrest during the postblastoderm divisions is a rare phenotype, described for mutations in only four genes prior to this study. The other two mutations were in a single complementation group corresponding to Cap-G (Dej, 2004).
Deletion mapping suggested that a complementation group discovered in a mutagenic screen to discover mitotic regulators (see above) was the Cap-G gene, a hypothesis supported by the lethality of these two alleles in trans to an EP element inserted within the 5' UTR, 17 bp upstream of the start codon of Cap-G. The identity of these mutations were identified as novel Cap-G alleles by two approaches: (1) the lethality of the EP element insert was reverted by precise excision of the element from the chromosome; (2) the Cap-G gene was sequenced in each of the two EMS mutations, dcap-gK1 and dcap-gK2, and point mutations were identified that generated premature stop codons in the Cap-G coding region (Dej, 2004).
The EP-element insertion was used to generate additional Cap-G alleles by imprecise excision. Seven new lethal alleles and one semilethal, female-sterile allele were derived. One lethal allele, dcap-gK4, is a deletion of 1162 bp at the 5' end of the Cap-G gene from the site of the EP-element insertion to within the third exon (e3; 1060 bp from the start codon). The female-sterile allele, dcap-gK3, is the result of the deletion of most of the P element without any loss of Cap-G sequence. This allele is not fully viable and shows lethality at the larval stage of development. Viability is reduced to 23% of the expected number of adult males, 64% of adult females (Dej, 2004).
The syncytial divisions of the early Drosophila embryo are not affected in any of the Cap-G embyonic lethal alleles, probably due to the presence of maternal stockpiles from the heterozygous mother. The Cap-G alleles do not show any mitotic defects until cycle 15 of the postblastoderm embryo. In wild-type cells, chromosomes begin the process of chromosome condensation during prophase. At prometaphase, chromosomes are condensed and rod shaped so that individual chromosomes can be visualized with a DNA stain. Stages were identified in wild-type and mutant cells by the pattern of tubulin staining. These chromosomes labeled strongly with an antibody to phosphorylated histone H3 (phospho-H3) that correlates with condensed chromosomes in mitosis. However, in dcap-gK4 mutant embryonic cells (dcap-gK4/Df(2R)vg56), prophase and prometaphase chromosomes show a range in the level of chromosome condensation. In prophase, some nuclei contain chromatin that appear condensed with the DNA stain, but label only weakly or not at all with the antibodies to phospho-H3. As the cells proceeded into prometaphase, the chromosomes became increasingly condensed, but condensation and the accumulation of phospho-H3 was not uniform within the same nucleus. These observations suggest that either the dcap-gK4 mutants have a prolonged prophase-prometaphase period that allows visualizualization of normal events in the process of chromatin condensation or an unusual level of condensation occurs in the absence of a fully functional condensin complex. However, even the most fully condensed chromosomes observed in prometaphase in the dcap-gK4 mutants were nonuniformly stained with a DNA dye and unevenly labeled with phospho-H3. In embryos carrying mutations in both dcap-gK1 and barrenL305, a similar defect in the process of chromosome condensation was observed. In prophase, condensed regions showed little or no phospho-H3 staining. In prometaphase, chromosomes were abnormally condensed, were not organized into compact rod structures, and showed a nonhomogeneous staining with phospho-H3 (Dej, 2004).
To test whether prometaphase was prolonged, the number of prometaphase figures was assayed in dcap-gK4 mutants compared to those in wild type in representative embryos. Wild-type prometaphase figures were identified by the appearance of condensed rod-shaped chromosomes that stained uniformly with phospho-H3. The mutant prometaphase figures were classified by the appearance of any condensed chromosome arms and some degree of labeling with phospho-H3. Appearance of these figures within a single field of cycle-15 mitotic divisions was scored in similar mitotic domains within the dorsal ectoderm of stage-10 embryos. The Cap-G mutant embryos have an increased number of prometaphase figures. This suggests that the length of prometaphase may be increased. A similar observation was made for the barrenL305 dcap-gK1 double mutant (Dej, 2004).
At metaphase, condensed chromosomes in wild-type cells align upon the metaphase spindle. Tubulin staining of the mitotic spindle was used to identify metaphase cells. Apparently normal chromosome condensation was observed at metaphase in dcap-gK4 mutant embryos, despite the fact that the pathway leading up to this point was perturbed. Metaphase figures in dcap-gK1 and barrenL305 double-mutant embryos also shows normal condensation, although the chromosome alignment on the metaphase spindle may be slightly disrupted (Dej, 2004).
At anaphase, chromosomes appear normally condensed, but show defects in sister-chromatid separation. The hypomorphic alleles, dcap-gK1 and dcap-gK2, exhibit bridging of one or two chromosome arms in cycle 15 of embryogenesis. This is similar to the observed phenotype of the embryonic lethal barren and gluon alleles, although the defect is observed in cycle 16 for mutations in these genes (Bhat, 1996; Steffenson, 2001; Hagstrom, 2002; Hudson, 2003). However, the dcap-gK4 allele exhibited a more severe defect in sister-chromatid separation. In dcap-gK4 mutant embryos, all of the chromosomes fail to segregate to the spindle poles except for the small 4th chromosome, which can be seen to separate and segregate and appears as a small dot at each of the poles. This severe separation defect is also seen in barren dcap-gK1 double-mutant embryos. The phenotype of apparently normal condensation at anaphase but a failure of sister-chromatid separation has been observed for mutations in condensin subunits in several metazoans (Bhat, 1996; Steffenson, 2001; Hagstrom, 2002; Hudson, 2003). However, the defect seen in the dcap-gK4 and barrenL305 dcap-gK1 double-mutant embryos, in which none of the major chromosomes were able to separate, was much more severe than that reported for other condensin mutants (Dej, 2004).
As cells enter telophase, the chromosomes gradually decondense and lose phosphorylated histone H3. In Cap-G mutants, persistent labeling of the bridging chromosomes was observed with antibodies to phospho-H3. A similar observation was made in barren mutant embryos (Bhat, 1996) and in SMC4-depleted S2 cells. This may represent a defect in the process of decondensation or it may represent a normal stage in the process of chromosome decondensation in which the phosphorylation of histone H3 is lost progressively, beginning at the centromeres and moving along the arms. This is consistent with a model in which the arms fail to separate and form the bridges while the centromeres separate appropriately (Dej, 2004).
These observations reveal a role for the condensin complex in condensation, but show that the cells can compensate to achieve condensation by metaphase. This suggests that surveillance mechanisms may prolong prometaphase until condensation is complete or has reached a sufficient level (Dej, 2004).
In chicken cells, mutation of the SMC2 subunit of the condensin complex disrupts the localization of nonhistone chromosomal proteins to the kinetochore. In C. elegans the localization of kinetochore proteins is aberrant in the absence of a functional condensin complex (Hagstrom, 2002; Stear, 2002), and in Xenopus egg extracts immunodepletion of condensin causes disorganized kinetochore structure and function (Wignall, 2003; Ono, 2004). In Drosophila SMC4-depleted S2 cells, centromeres and kinetochores are able to segregate, while the sister-chromaid arms show bridging at anaphase (Coelho, 2003). To test whether mutations in Cap-G affect the kinetochore and surrounding centromeric chromatin, the localization of two proteins, CID and MEI-S332, was examined. MEI-S332 is a centromeric protein that localizes to condensed chromosomes at prometaphase, but concomitant with the separation of sister chromatids, delocalizes from the centromeres at anaphase. In dcap-gK1 mutant embryos, MEI-S332 localizes normally onto prometaphase chromosomes and properly delocalized at anaphase. Similarly, MEI-S332 localizes to prometaphase and metaphase chromosomes in larval imaginal discs and delocalizes at anaphase despite the apparent failure of sister-chromatid separation as evidenced by persistent bridging. CID, a Drosophila CENP-A homolog, localizes to centromeres throughout the cell cycle. The localization of CID is normal during mitosis in dcap-gK3 [dcap-gK3/Df(2R)vg56] mutant larval imaginal discs. Centromeres were labeled with CID during prometaphase despite abnormal chromosome condensation. Centromeres were also labeled in metaphase and anaphase. Thus centromere structure is not detectably perturbed by loss of Cap-G function in Drosophila. CID localization also revealed that in mutant anaphases there is normal separation of centromeres despite the failure to separate sister-chromatid arms (Dej, 2004).
To elucidate further the role of Cap-G in chromosome dynamics, the mitotic divisions were examined in squashed preparations of the third instar larval brain of animals that were hemizygous for the semilethal dcap-gK3 allele; defects were found in chromosome morphology. There were several anomalies in mitosis, including aneuploidy, the aberrant separation of centromeres, the failure to resolve sister-chromatid arms, and an increase in the axial length of the chromosomes (Dej, 2004).
Larval brains from mutant and wild-type animals were treated with a hypotonic solution. In wild type this has the effect of separating the sister-chromatid arms at metaphase while maintaining centromere attachment, thus creating stereotypical mitotic figures. In the brains of larvae that were hemizygous for dcap-gK3, many metaphase figures had a fewer number of chromosomes than in wild type, while others were polyploid. Strikingly, in these metaphase figures the arms failed to separate and, instead, the DAPI-bright foci at the centromeres appeared to be dissociated. Anaphase figures that showed chromatid bridging were also observed. The centromere separation defect was verified using antibodies to the centromeric protein MEI-S332. MEI-S332 localized to the separated centromeres in the mutant metaphase figures. While this confirmed the identity of the centromere, it also suggested that these nuclei had not yet entered anaphase and that the separation of the centromeres occurred aberrantly in metaphase. This aberrant separation of centromeres was not observed in the gluon/smc4 larval lethal mutations (Steffenson, 2001), nor was it apparent in the RNAi depletion of Smc4 or Barren (Dej, 2004).
In addition to abnormal centromere separation, the metaphase figures contained sister-chromatid arms that failed to resolve and therefore the two sister-chromatid arms could not be distinguished. This suggests that the process of chromatid resolution, a process that normally occurs during prophase as chromosomes condense and the bulk cohesin is released, was disrupted. The failure in sister-chromatid resolution is the likely upstream defect that leads to the segregation errors in anaphase, such as lagging chromosomes, bridging, and, ultimately, aneuploidy (Dej, 2004).
Mutations in gluon/smc4 show minor defects in chromosome condensation during the larval mitotic divisions in brains, in that the width of the chromosomes is broader in mutants (Steffenson, 2001). The dcap-gK4 mutants showed no measurable change in the width of the mitotic chromosomes, yet measurements of the length of the X chromosome, which was easily identified due to the presence of the DAPI-bright heterochromatin at one end of the chromosome, revealed a slight increase in axial length from 2.79 µm in wild type. A greater defect in axial condensation was observed with the two autosomes, chromosomes 2 and 3. These were identified by the DAPI-bright heterochromatin in the center of the arms. The combined average length of these chromosomes was 4.25 µm in wild type and 7.8 µm in Cap-G mutants. However, there is no complete loss of the chromosome condensation at metaphase. This difference in the gluon and Cap-G phenotypes may reflect the roles of the SMC vs. non-SMC subunits in the condensin complex, differences in allele strengths, or differences in protein stability during the cell cycle. An examination of the DAPI-bright heterochromatic DNA on the autosomes revealed that this region was longer in the dcap-gK4 mutants. DAPI preferentially stains repetitive, AT-rich sequences of the DNA that are found on all Drosophila chromosomes at the centromeres and along the Y chromosome. This suggests that condensation of the heterochromatic DNA at the centromere is disrupted, although this defect alone does not account for the overall increase in chromosome length. The Cap-G mutations reveal distinct, but perhaps interrelated, roles for Cap-G: resolution of sister-chromatid arms, association of sister centromeres, and a contributing, but not exclusive role, in axial chromosome condensation (Dej, 2004).
The differential requirement of the condensin complex in chromosome condensation, as suggested by the condensin mutant phenotypes in Drosophila and C. elegans and the in vitro studies in Xenopus extracts, may be due to the different source of the chromosomes in the different systems. While in the Drosophila and C. elegans studies the endogenous chromosomes were analyzed, the Xenopus experiments used exogenous sperm chromatin in egg extracts. Sperm chromatin contains protamines that must be replaced by histones before undergoing condensation. In addition, in the Xenopus studies condensation is measured in the absence of replication, and thus single sister chromatids are condensed. Toposisomerase II (TopoII) is a component of the chromosome scaffold and TopoII mutants show a similar phenotype to condensing mutants. Recent studies in Xenopus have shown a role for DNA replication in the recruitment of topoisomerase II to the chromosomes to facilitate condensin assembly and condensation. Furthermore, in S2 cells depleted of SMC4, topoisomerase II is not localized normally and Barren is not loaded onto chromsomes (Dej, 2004).
It was of interest to test whether the presence of a replicated sister chromatid could augment condensation, to explain why the condensin complex was not essential for condensation in the Drosophila mutants. The requirement for Cap-G for condensation was analyzed in the absence of a sister chromatid by employing a mutant in an essential replication initiation factor, double parked (dup/cdt1). Mutant alleles of dup block replication in cycle 16 of the postblastoderm divisions. dupa1 mutants fail to replicate in S-phase, yet proceed into mitosis and often appear clustered at the spindle equator in a pseudometaphase due to the attachment of the single kinetochore to microtubules emanating from both spindle poles. Cells accumulate in mitosis, but fail to complete anaphase, because the single kinetochores are incapable of a normal bipolar attachment and induce the spindle checkpoint. The chromosomes, although composed of single sister chromatids at cycle 16, condense appropriately and show robust labeling with phospho-H3. Condensation in the dup mutant is dependent on a functional Cap-G protein. In dcap-gK1 dupa1 double mutants, cells at cycle 16 contain unreplicated chromosomes that failed to condense into discernible metaphase chromosomes and showed punctate labeling with phospho-H3. This is in striking contrast to chromosomes composed of two replicated sister chromatids and shows that, in the absence of replicated sister chromatids or the process of DNA replication, the condensin complex is essential for chromosome condensation (Dej, 2004).
Adult flies carrying the dcap-gK3 [dcap-gK3/Df(2R)vg56] allele exhibit phenotypes suggestive of defects in gene expression such as wing notches and rough eyes. While staining with acridine orange revealed comparable levels of cell death in the imaginal discs of male and female larvae, the wing notches appeared only in the adult male flies. This suggests that the defects in this tissue may be the result of disrupting male-specific gene regulation. The role of Cap-G in regulating gene expression was tested by examining the effect of Cap-G alleles on position-effect variegation (PEV). PEV is the effect on gene expression mediated by the chromatin structure associated with heterochromatic regions. The whitem4h allele is the result of a genomic inversion that places the white gene next to heterochromatic DNA and results in a downregulation of gene expression to produce white patches in the eye. It was found that embryonic lethal alleles of barren and Cap-G exhibit a dominant suppression of PEV at the whitem4h locus. Thus, one copy of dcap-gK2 or dcap-gK1 or barrenL305 results in an increase in red pigment due to an increase in white gene expression (Dej, 2004).
Barren has been shown to interact with the Polycomb complex, a protein complex that maintains a repressive chromatin structure (Lupo, 2001). This complex acts at specific recognition elements, one of which is the FAB-7 Polycomb response element (PRE). FAB-7 PRE represses expression of an adjacent white gene in a transgene construct, but this repression is alleviated by mutations in members of the Polycomb complex or by mutations in barren. As an additional test of the role of Cap-G in interphase gene expression, it was asked whether Cap-G mutations affect repression by FAB-7 PRE, but no such effect was seen (Dej, 2004).
Reference names in red indicate recommended papers.
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date revised: 15 August 2005
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