InteractiveFly: GeneBrief

Cap-G: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - Cap-G

Synonyms -

Cytological map position - 49F2--4

Function - chromatin condensation

Keywords - condensin complex, mitotic chromosome condensation

Symbol - Cap-G

FlyBase ID: FBgn0259876

Genetic map position - 2R

Classification - Armadillo-like helical HEAT domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Piskadlo, E., Tavares, A. and Oliveira, R. A. (2017). Metaphase chromosome structure is dynamically maintained by Condensin I-directed DNA (de)catenation. Elife 6. PubMed ID: 28477406
Summary:
Mitotic chromosome assembly remains a big mystery in biology. Condensin complexes are pivotal for chromosome architecture yet how they shape mitotic chromatin remains unknown. Using acute inactivation approaches and live-cell imaging in Drosophila embryos, this study dissects the role of condensin I in the maintenance of mitotic chromosome structure with unprecedented temporal resolution. Removal of condensin I from pre-established chromosomes results in rapid disassembly of centromeric regions while most chromatin mass undergoes hyper-compaction. This is accompanied by drastic changes in the degree of sister chromatid intertwines. While wild-type metaphase chromosomes display residual levels of catenations, upon timely removal of condensin I, chromosomes present high levels of de novo Topoisomerase II (TopoII)-dependent re-entanglements, and complete failure in chromosome segregation. TopoII is thus capable of re-intertwining previously separated DNA molecules and condensin I continuously required to counteract this erroneous activity. It is proposed that maintenance of chromosome resolution is a highly dynamic bidirectional process.
BIOLOGICAL OVERVIEW

Chromosomes are dynamic structures that are reorganized during the cell cycle to optimize them for distinct functions. Structural maintenance of chromosomes (SMC) and non-SMC condensin proteins associate into complexes that have been implicated in the process of chromosome condensation. The roles of the individual non-SMC subunits of the complex are poorly understood, and mutations in the CAP-G subunit have not been described in metazoans (Dej, 2004).

A role for Cap-G in chromosome condensation and cohesion has been demonstrated in Drosophila. The requirement of Cap-G for condensation during prophase and prometaphase is demonstrated; however, alternate mechanisms are demonstrated to ensure that replicated chromosomes are condensed prior to metaphase. In contrast, Cap-G is essential for chromosome condensation in metaphase of single, unreplicated sister chromatids, suggesting that there is an interplay between replicated chromatids and the condensin complex. In the Cap-G mutants, defects in sister-chromatid separation are also observed. Chromatid arms fail to resolve in prophase and are unable to separate at anaphase, whereas sister centromeres show aberrant separation in metaphase and successfully move to spindle poles at anaphase. A role for Cap-G during interphase in regulating heterochromatic gene expression is demonstrated (Dej, 2004).

Chromosomes undergo dynamic behaviors during mitosis to enable the precise separation of the two replicated sister chromatids. It is vital that the replicated sister chromatids are separated successfully. There are two crucial prerequisites for accurate segregation: (1) cohesion between the replicated chromatids must be maintained until anaphase and (2) compaction of the chromosomes into a manageable form, condensation, must be completed prior to metaphase. These processes require two major protein complexes, the cohesin and condensin complexes. Each of these complexes is founded upon a heterodimer of SMC proteins, which are chromosome-associated ATPases (Hirano, 1998; Hirano, 2002). Also within each complex are two or three non-SMC subunits, which contribute specific functions to the SMC holocomplex. Despite a similar structural paradigm, the condensin and cohesin complexes are functionally distinct. Although each complex was originally identified for unique functions during mitosis, it is now clear that both complexes are involved in a wide array of activities, including DNA repair, chromatid separation, and the regulation of gene expression (reviewed in Jessberger, 2002; Hagstrom, 2003; Legagnex: 2004; Dej, 2004 and references therein).

The structure and function of the cohesin complex is understood in the most detail and its structure has been elucidated (reviewed in Hirano 2000; Lee, 2001; Nasmyth 2002). The SMC subunits, SMC1 and SMC3, form two antiparallel coiled-coils (Hirano 2002). One of the two non-SMC subunits, SCC1/Mcd1/Rad21, associates the ends of the SMC coiled-coils into a ring structure. This ring structure holds the two sister chromatids together, perhaps by encircling them after S-phase. Cohesin is necessary for holding replicated sister chromatids together from S-phase until anaphase. The complex accumulates on chromosomes prior to S-phase and is maintained and activated through the process of replication. By the end of S-phase, replicated sister chromatids are associated through the cohesin complex at sites along the length of the arms. In yeast, the cohesin complex is maintained until anaphase along the chromosome. In metazoans, the bulk of the cohesin complex is displaced at prophase, but a subset of cohesin complexes is maintained at the centromere and perhaps other sites. This final population of cohesin complexes is lost at anaphase as the sisters separate (Dej, 2004).

The condensin complex, the subject of this biological overview, is a second SMC complex that is found in yeast and metazoans and is involved in chromatid segregation. It also contains two SMC subunits, SMC2 and SMC4 (Hirano 2002), and three non-SMC subunits, CAP-H, CAP-G, and CAP-D2 (Swedlow, 2003). These three subunits form an 11S regulatory subcomplex that is required to activate the SMC ATPases and to promote mitosis-specific chromatin binding of the holocomplex (Kimura, 2000). However, the individual functions of the non-SMC subunits within the complex remain undefined. Recent studies have identified another condensin complex containing alternate non-SMC subunits, CAP-G2, CAP-H2, and CAP-D3 (Ono, 2003). While there is a single condensin complex in both budding and fission yeast, condensin I and condensin II complexes are found in Xenopus and humans (Ono, 2003). Within the Drosophila genome, genes coding for a second CAP-H and a second CAP-D2 are found, but there appears to be only a single CAP-G protein (Ono, 2003; Dej, 2004 and references therein).

The condensin I complex was first identified biochemically in Xenopus extracts (Hirano, 1997). Sperm chromosomes in egg extracts depleted of condensin complex subunits assume a dispersed interphase organization. When the condensin complex is added back, the chromatin reorganizes into condensed chromosomes. This suggests a role in chromosome condensation supported by genetic analyses in yeast. In HeLa cells, depletion of condensin I or II complex subunits disrupt chromosome condensation, but depletion of subunits from both complexes has a more profound effect (Ono, 2003). Mutations in condensin subunits in yeast show precocious separation of sister chromatids in addition to defects in chromosome condensation (Saka, 1994; Strunnikov, 1995; Freeman, 2000; Ouspenski, 2000; Lavoie, 2002). Condensation defects in budding yeast were revealed through the use of fluorescent in situ hybridization (FISH) probes to rDNA, which appeared more dispersed in the mutants (Strunnikov, 1995; Freeman, 2000; Lavoie, 2002). In addition, FISH to euchromatic sites in fission yeast revealed loci to be more separated in condensin mutants (Saka, 1994) than in wild type (Dej, 2004).

In contrast, genetic analyses in metazoans to date have not delineated an essential role for the condensin complex in chromosome condensation. Embryonic lethal mutations in barren, the gene coding for the Drosophila homolog of CAP-H, show a failure to separate sister chromatids, but no described defect in condensation (Bhat, 1996). Animals with larval lethal mutations in gluon/smc4 also show defects in sister-chromatid separation. A partial effect on condensation is seen by an increase in chromosome width, but no change in the compaction along the length of the chromosomes (Steffenson, 2001). Further complicating the analysis of the role of condensin is the observation that in Drosophila S2 cells depleted of Barren by RNAi, chromosomes are poorly condensed with sister chromatids that are fuzzy and indistinct (Somma, 2003). Similarly, depletion of SMC4 by RNAi results in chromosomes that are undercondensed with sister chromatids that are unresolved (Coelho, 2003). In Caenorhabditis elegans, mutations in SMC4 show condensation defects at prometaphase, but little effect on condensation at metaphase and anaphase (Hagstrom, 2002). This is similar to observations in chicken cells lacking ScII/SMC2 in which chromosome condensation is delayed, but eventually reaches normal levels (Hudson, 2003). Together, these observations suggest that the condensin complex is not the only mechanism for compacting chromosomes in mitosis (Dej, 2004).

The genetic analysis of several mutations in the Cap-G gene was used to understand the role of Cap-G in Drosophila. Chromosome condensation was found to be compromised during mitosis in Cap-G mutant cells, but normal levels of condensation can be attained by metaphase. This suggests that there is a second pathway for condensing chromosomes that can compensate for a compromised condensin complex. Insight into this pathway comes from observations that, in the absence of replication, the Cap-G protein is required for chromosome condensation. In addition, in cells mutant for Cap-G, sister-chromatid arms are unable to resolve at prophase and sister chromatids show massive bridging defects at anaphase. While there is appropriate assembly of at least two centromere components, aberrant separation at the centromere is observed. Finally, it is shown that the Cap-G protein and perhaps the entire condensin complex may be required for chromatin-mediated gene expression in heterochromatic sequences (Dej, 2004).

These studies have demonstrated a role for Cap-G in chromosome condensation and cohesion in Drosophila. There is a requirement for Cap-G in the process of condensation during prophase and prometaphase; however, compensatory mechanisms ensure that chromosomes condense prior to metaphase. Reorganization of chromatin into condensed chromosomes is a process that involves the prior replication of chromatids and the condensin complex. Anaphase defects are also observed; specifically, sister-chromatid arms fail to separate. The separation defect is likely the result of the defect in sister-chromatid resolution during prophase that was evident in neuroblast mitotic squashes. In contrast, centromere separation is observed at anaphase and, in larval neuroblast preparations, this separation occurs aberrantly in metaphase figures. The role of Cap-G, and possibly of the condensin complex, is not limited to mitosis. Mutations have been identified that reveal roles for Cap-G during interphase in heterochromatic gene expression (Dej, 2004).

There is a striking condensation phenotype early in the cell cycle in Cap-G mutants. In prophase and prometaphase, condensation is nonuniform. By metaphase, condensation has achieved apparently normally levels, suggesting that a prolonged prometaphase enables chromosomes to achieve a high degree of condensation in the absence of a fully functional condensin complex. The role of the condensin complex in chromosome condensation prior to metaphase has been observed in other organisms. Mutations in the C. elegans smc-4 gene diminish chromosome compaction at prometaphase, but chromosomes are highly condensed by metaphase (Hagstrom, 2002). RNAi depletion of SMC4 in Drosophila S2 cells also shows aberrant condensation at prometaphase (Coelho, 2003). Similarly, in chicken cells lacking the condensin subunit ScII/SMC2, chromosome condensation is delayed, but chromosomes ultimately reach nearly normal levels of condensation (Hudson, 2003). Recently it has been shown that Xenopus and humans have two sets of condensin subunits (Ono, 2003). It is possible that in some organisms when one complex is unable to function the other can compensate partially and complete chromosome condensation by metaphase. However, while there are two CAP-H and CAP-D2 condensin subunits in Drosophila, there is only a single gene coding for a CAP-G subunit (Ono, 2003). This single Cap-G protein may be required in both complexes; therefore no such compensation is expected to occur in Drosophila. The significance of the alternate splice forms of the Cap-G gene is not known, although the two predicted proteins are similar across most of their lengths (Dej, 2004).

The prolonged prometaphase may be the result of activating the spindle checkpoint. This checkpoint may be used to monitor the degree of condensation at prometaphase to prevent sister-chromatid separation prior to complete condensation. The spindle checkpoint monitors the kinetochore-spindle attachment and delays anaphase until the appropriate bipolar connections are achieved and the chromosomes are congressed at the metaphase plate. It is thought that this checkpoint might monitor the tension at the kinetochores. It is possible that chromosome condensation might be monitored through this pathway, as a hypocondensed centromere and/or chromosome might reduce tension. In this way, the spindle checkpoint may delay progression through the cell cycle until the chromosomes are sufficiently condensed. The spindle checkpoint would require two sister chromatids for bipolar attachment, tension, and congression to occur (Dej, 2004).

The observation of a severe condensation defect at metaphase in other systems may be due to the absence of an active spindle checkpoint. For example, RNAi of Barren, the CAP-H homolog in Drosophila, demonstrated chromosome condensation defects at metaphase (Somma, 2003), but S2 cells have weak checkpoints controlling behavior in mitosis (Dej, 2004 and references therein).

Cap-G;double parked double mutants show that the condensin complex is dispensable for chromosome condensation by metaphase except in the absence of a replicated sister chromatid. What could replication provide to the condensation process? The cohesin complex could compensate for a faulty condensin complex, and replication is required to assemble the cohesin complex and establish cohesion. In this model the unreplicated sister chromatids in double parked mutants would contain inactive cohesin complex that would be unable to compensate for a faulty condensin complex in the process of chromosome condensation. Alternatively, experiments in Xenopus show that TopoII activity during replication is a prerequisite for setting up a structural axis required for the mitotic chromosome assembly (Dej, 2004).

Although there are no mutations in TopoII in Drosophila, some insights into whether TopoII could play the same role during replication in establishing a condensation-competent chromosome axis emerge from RNAi ablation in Drosophila cell culture. In S2 cells depleted of TopoII, mitotic chromosomes condense, but chromosomes are less compact at the metaphase plate (Chang, 2003). In these TopoII-depleted cells, Barren loading to centromeres and its dissociation at anaphase are normal and chromosome decondensation begins at anaphase (Chang, 2003). In SMC4-depleted S2 cells, TopoII localization is aberrant. In cells containing SMC4, TopoII appears in discrete regions along a defined chromatid axis, while in SMC4-depleted cells TopoII is associated diffusely with the chromosomes (Coelho, 2003). Thus, perhaps a condensation defect is seen at metaphase in the dup dcap-g double mutants because there was no replication, TopoII was unable to establish the appropriate architecture prior to mitosis, and the condensin complex is faulty during mitosis (Dej, 2004).

These studies present evidence that there is a distinct role for Cap-G in centromere segregation. The Cap-G, barren, gluon, as well as yeast and C. elegans condensin mutants exhibit the segregation of centromeres at anaphase, but a failure to separate arms. Aberrant separation of centromeres in Cap-G mutants were found at metaphase, prior to anaphase. Few other mutants show this defect in sister-chromatid centromere association. Mutations in cohesin subunits show premature separation of both the arms and the centromeres of chromatids (reviewed in Lee, 2001). In Drosophila, mutations in wings apart-like (wapl) cause aberrant separation of centromeres. However, in wapl mutants, the arms are resolved appropriately. This cytological observation suggests that there is a role for Cap-G and perhaps the condensin complex in mediating centromere association via heterochromatin. An effect on heterochromatic chromosome condensation in Cap-G mutants is further suggested by the observation that DAPI-bright repetitive sequences at the centromere appears to be expanded. A distinct role for Cap-G at the centromeres of the chromosomes is consistent with observations in other organisms that the condensin complex accumulates at centromeres (Dej, 2004 and references therein).

Consistent with the concept of a specific role for condensin in centromeric genomic regions is the observed role of condensin in regional gene regulation. Cap-G is required for the transcriptionally repressive state of centromere-proximal heterochromatin. Mutations in wapl not only show premature separation of centromeres at metaphase, but also, like Cap-G, act as dominant suppressors of variegation at the white locus. This emphasizes the intrinsic relationship between mitotic centromere structure and interphase heterochromatic organization. This is consistent with the widely held notion that the transcriptionally inactive state of mitotic condensation may be similar to the transcriptionally repressed heterochromatic regions of the genome. It is now becoming clear that the same proteins may establish both chromatic states (Dej, 2004).

Global gene repression in C. elegans is observed in XX hermaphrodites that downregulate gene expression from both X chromosomes. Dosage-compensation factors that resemble condensin subunits form a complex that associates with the chromosomes and mediate this chromosome-wide gene regulation. In C. elegans, a condensin complex containing MIX-1, SMC-4, and HCP-6 mediates mitotic chromosome condensation and a condensin-like complex containing MIX-1, DPY-26, DPY-27, and DPY-28 is required for dosage compensation (reviewed in Hagstrom, 2003). Silencing at the mating-type loci in Saccharomyces cerevisiae has also been found to require condensin subunits, specifically, CAP-D2 and SMC4, but not SMC2 (Bhalla, 2002). Perhaps in yeast, where there is a single condensin complex, a subset of condensin proteins assembles into a distinct condensin-like complex that is required for transcriptional silencing (Dej, 2004).

These studies reveal several distinct roles for the Cap-G condensin protein. In addition to its role in condensation and sister-arm resolution, these observations highlight the role of Cap-G and perhaps the condensin complex in centromere organization. This role is important for the association of sister chromatids during mitosis and for the regulation of heterochromatin-mediated gene expression during interphase (Dej, 2004).


REGULATION

Protein Interactions

Cap-G interacts with the centromere-specific histone H3 variant CID

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).


DEVELOPMENTAL BIOLOGY

Embryonic

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).


EFFECTS OF MUTATION

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).


REFERENCES

Search PubMed for articles about Drosophila Cap-G

Bhalla, N., Biggins, S. and Murray, A. W. (2002). Mutation of YCS4, a budding yeast condensin subunit, affects mitotic and nonmitotic chromosome behavior. Mol. Biol. Cell 13(2): 632-45. 11854418

Bhat, M. A., Philp, A. V., Glover, D. M. and Bellen, H. J. (1996). Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II. Cell 87(6): 1103-14. 8978614

Chang, C. J., Goulding, S., Earnshaw, W. C. and Carmena, M. (2003). RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate. J. Cell Sci. 116(Pt 23): 4715-26. 14600258

Coelho, P. A., Queiroz-Machado, J. and Sunkel, C. E. (2003). Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J. Cell Sci. 116(Pt 23): 4763-76. 14600262

Dej, K. J., Ahn, C. and Orr-Weaver, T. L. (2004). Mutations in the Drosophila condensin subunit dCAP-G: defining the role of condensin for chromosome condensation in mitosis and gene expression in interphase. Genetics 168(2): 895-906. 15514062

Freeman, L., Aragon-Alcaide, L. and Strunnikov, A. (2000). The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J. Cell Biol. 149(4): 811-24. 10811823

Hagstrom, K. A., Holmes, V. F., Cozzarelli, N. R. and Meyer, B. J. (2002). C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis. Genes Dev. 16(6):729-42. 11914278

Hagstrom, K. A. and Meyer, B. J. (2003). Condensin and cohesin: more than chromosome compactor and glue. Nat. Rev. Genet. 4(7): 520-34. 12838344

Hirano, T., Kobayashi, R. and Hirano, M. (1997). Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89(4):511-21. 9160743

Hirano, T. (1998). SMC protein complexes and higher-order chromosome dynamics. Curr. Opin. Cell Biol. 10(3): 317-22. 9640531

Hirano T. (2000). Chromosome cohesion, condensation, and separation. Annu. Rev. Biochem. 69: 115-44. 10966455

Hirano T. (2002). The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair. Genes Dev. 16(4): 399-414. 11850403

Hudson, D. F., Vagnarelli, P., Gassmann, R. and Earnshaw, W. C. (2003). Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev. Cell. 5(2): 323-36. 12919682

Jager, H., Rauch, M. and Heidmann, S. (2005). The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID. Chromosoma 113(7): 350-61. 15592865

Jessberger, R. (2002). The many functions of SMC proteins in chromosome dynamics. Nat. Rev. Mol. Cell. Biol. 3(10): 767-78. 12360193

Kimura, K. and Hirano, T. (2000). Dual roles of the 11S regulatory subcomplex in condensin functions. Proc. Natl. Acad. Sci. 97(22): 11972-7. 11027308

Lavoie, B. D, Hogan, E. and Koshland, D. (2002). In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin. J. Cell Biol. 156(5): 805-15. 11864994

Lee, J. Y. and Orr-Weaver, T. L. (2001). The molecular basis of sister-chromatid cohesion. Annu. Rev. Cell. Dev. Biol. 17: 753-77. 11687503

Legagneux, V., Cubizolles, F. and Watrin, E. (2004). Multiple roles of Condensins: a complex story. Biol. Cell 96(3): 201-13. 15182703

Nasmyth, K. (2002). Segregating sister genomes: the molecular biology of chromosome separation. Science 297(5581): 559-65. 12142526

Ono, T., et al. (2003). Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115(1): 109-21. 14532007

Ono, T., et al. (2004). Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells. Mol. Biol. Cell 15(7): 3296-308. 15146063

Ouspenski, I. I., Cabello, O. A. and Brinkley, B. R. (2000). Chromosome condensation factor Brn1p is required for chromatid separation in mitosis. Mol. Biol. Cell 11(4): 1305-13. 10749931

Saka, Y., et al. (1994). Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J. 13(20): 4938-52. 7957061

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Stear, J. H. and Roth, M. B. (2002). Characterization of HCP-6, a C. elegans protein required to prevent chromosome twisting and merotelic attachment. Genes Dev. 16(12): 1498-508. 12080088

Steffensen, S., et al. (2001). A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis. Curr. Biol. 11(5): 295-307. 11267866

Strunnikov, A. V., Hogan, E. and Koshland, D. (1995). SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family. Genes Dev. 9(5): 587-99. 7698648

Swedlow, J. R. and Hirano, T. (2003). The making of the mitotic chromosome: modern insights into classical questions. Mol. Cell 11(3): 557-69. 12667441

Wignall, S. M., Deehan, R., Maresca, T. J. and Heald, R. (2003). The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts. J. Cell Biol. 161(6): 1041-51.


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