barren: Biological Overview | References
Gene name - barren
Synonyms - CAP-H
Cytological map position - 38B1-38B2
Function - chromatin protein
Keywords - mitotic chromosome condensation, promotion of sister chromatid resolution, maintainance of structural integrity of centromeric heterochromatin
Symbol - barr
FlyBase ID: FBgn0014127
Genetic map position - 2L:20,058,164..20,060,975 [-]
Classification - Condensin, subunit H, Barren protein
Cellular location - nuclear
|Recent literature||Beck, E. A. and Llopart, A. (2015). Widespread positive selection drives differentiation of centromeric proteins in the Drosophila melanogaster subgroup. Sci Rep 5: 17197. PubMed ID: 26603658
Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led to a hypothesis that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. This study reports evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.
During cell division, chromatin undergoes structural changes essential to ensure faithful segregation of the genome. Condensins, abundant components of mitotic chromosomes, are known to form two different complexes, condensins I and II. To further examine the role of condensin I in chromosome structure and in particular in centromere organization, the Drosophila CAP-H homologue Barren, a subunit exclusively associated with condensin I was from depleted form S2 cells. In the absence of Barren/CAP-H the condensin core subunits DmSMC4 (Gluon) and DmSMC2 still associate with chromatin, while the other condensin I non-structural maintenance of chromosomes family proteins do not. Immunofluorescence and in vivo analysis of Barren/CAP-H-depleted cells showed that mitotic chromosomes are able to condense but fail to resolve sister chromatids. Additionally, Barren/CAP-H-depleted cells show chromosome congression defects that do not appear to be due to abnormal kinetochore-microtubule interaction. Instead, the centromeric and pericentromeric heterochromatin of Barren/CAP-H-depleted chromosomes shows structural problems. After bipolar attachment, the centromeric heterochromatin organized in the absence of Barren/CAP-H cannot withstand the forces exerted by the mitotic spindle and undergoes irreversible distortion. Taken together, these data suggest that the condensin I complex is required not only to promote sister chromatid resolution but also to maintain the structural integrity of centromeric heterochromatin during mitosis (Oliveira, 2005).
The genome of eukaryotic proliferating cells undergoes programmed structural changes in order to ensure the integrity of genetic material and cell viability during cell division. First, during S phase, when DNA is duplicated, sister chromatid cohesion is established along the entire length of DNA molecules and is maintained until entry into mitosis. Subsequently, during the early stages of mitosis, chromosomes condense into higher-order levels of chromatin organization, leading to the resolution of chromosome arms, a prerequisite for genome stability. Although mitotic chromosomes were one of the first subcellular structures observed, the mechanisms underlying their establishment have only recently begun to be unveiled (Oliveira, 2005).
A major contribution was the identification of the multiprotein condensin complex, initially purified and characterized from Xenopus extracts and later shown to be highly conserved. Condensin is composed of two subcomplexes: a core heterodimer formed by the chromosomal ATPase SMC family (structural maintenance of chromosomes) proteins SMC2 and SMC4 and a regulatory subcomplex formed by three non-SMC proteins, CAP-D2, CAP-G, and CAP-H. All the members of the condensin complex have been shown to be essential for cell and organism viability (Oliveira, 2005).
Mutation analysis of condensin subunits in both Saccharomyces cerevisiae and Schizosaccharomyces pombe show defects in chromosome condensation and segregation. However, genetic analyses in multicellular organisms such as Drosophila revealed that loss of condensin subunits leads to strong defects in segregation but had only partial effects on chromosome condensation. Mutation of Drosophila SMC4/gluon was shown to severely compromise sister chromatid resolution but not longitudinal axis shortening. Mutation of barren, the Drosophila CAP-H orthologue, does not affect chromosome condensation but impairs sister chromatid segregation (Bhat, 1996). More recently, genetic analysis of Drosophila CAP-G shows that chromosome condensation is perturbed in prometaphase but normal condensation levels can be achieved at metaphase (Dej, 2004). Consistently, depletion of scII/SMC2 in DT40 chicken cells showed that chromosome condensation is delayed, however, normal levels are eventually reached. Similar results were obtained after depletion of SMC4 and MIX-1 in Caenorhabditis elegans. These data suggest that the condensin complex might not be the major factor required for the organization of the mitotic chromosome (Oliveira, 2005 and references therein).
Indeed, recent studies have identified a new condensin complex in HeLa cell extracts named condensin II. Condensin II shares the core SMC proteins with condensin I but has different regulatory subunits. It has been suggested both condensin complexes contribute distinctly to the metaphase chromosome architecture in vertebrate cells. However, not all organisms appear to have the two types of complexes and different condensin complexes might be required for different tissues or at different developmental stages. Condensins I and II were shown to display different spatial and temporal chromatin localizations. Condensin II was shown to be predominantly nuclear during interphase, and it was suggested to contribute to early stages of chromosome assembly in prophase, whereas condensin I was described to access chromatin only after nuclear envelope breakdown. Moreover, in HeLa cell chromosomes at metaphase, condensin II is enriched at the primary constriction. Previously, studies in Drosophila revealed a strong localization of condensin I at the centromere (Steffensen, 2001). These findings raise the hypothesis that condensin complexes play a specific role in the organization of centromeric chromatin (Oliveira, 2005).
The centromere plays an essential role in chromosome segregation. First, it underlies the organization of the kinetochore and thereby the attachment and movement of chromosomes along spindle microtubules. Second, it ensures sister chromatid cohesion until metaphase-anaphase transition. In that way centromeres contribute to bipolar attachment of chromosomes, essential for the proper partitioning of the genome in cell division. In most higher eukaryotes, centromeres are formed by large arrays of tandem repeated sequences. Moreover, centromere inheritance appears to be dependent on the presence of specialized centromeric nucleosomes containing CENP-A (centromere protein A), a specific histone isoform that belongs to the histone H3 protein family. In the holocentric chromosome of C. elegans, several studies indicate that CENP-A colocalizes with the condensin subunits along the entire chromosome length and can play a role in centromere organization. Indeed, it has been shown that SMC-4 and MIX-1 are required for proper centromere biorientation and segregation (Oliveira, 2005 and references therein).
These results could be attributed to the particular features of C. elegans holocentric chromosomes. However, there is increasing evidence that condensin might have a role at the centromeres of monocentric chromosomes. In agreement, a genetic and physical interaction between Drosophila CAP-G and the centromere-specific CID/CENP-A has recently been reported (Jager, 2005). Also, in S. pombe, chromatin immunoprecipitation assays showed that condensin localizes to CEN DNA. However, little is known about the role of condensins in the centromere structure (Oliveira, 2005).
This study evaluated the role of condensin I upon the organization and segregation of mitotic chromosomes by depleting Barren/CAP-H from Drosophila S2 cells. It was shown that depletion of Barren/CAP-H compromises the binding to chromatin of the other condensin I regulatory subunits, DmCAP-D2 and DmCAP-G. However, the absence of Barren does not interfere with the binding to chromatin of the DmSMC4/2 core heterodimer, demonstrating the ability of the heterodimer to associate with chromatin independently of the regulatory subcomplex. It was also shown that S2 cells depleted of Barren/CAP-H display abnormal sister chromatid resolution and segregation (Oliveira, 2005).
In vivo analysis of Barren/CAP-H-depleted cells expressing green fluorescent protein (GFP)-histone H2B shows that chromosomes are unable to align at the metaphase plate and exhibit chromatin bridges as soon as anaphase starts. Immunofluorescence analysis also indicates that although chromosomes show bipolar attachment, intercentromere distances are unusually large. Moreover, centromeric markers appear distorted and the cohesin protein DRAD21 shows an abnormally broad distribution. Furthermore, it was found that the heterochromatin- specific K9 dimethylated histone H3 is also abnormally distributed. Taken together, these results suggest that condensin I plays a major role in the organization of centromeric heterochromatin in order to maintain its elastic properties, which are essential to withstand the forces exerted by the mitotic spindle (Oliveira, 2005).
Evidence is provided that condensin I is absolutely required for mitotic chromosome resolution and cell viability. In the absence of condensin I DNA bridges are observed during anaphase and telophase. Importantly, it was shown that condensin I depletion results in congression defects associated with alterations in the structural integrity of the centromere-proximal chromatin (Oliveira, 2005).
Depletion of Barren/CAP-H, a condensin I-specific subunit in Drosophila S2 cells, leads to the formation of chromosomes that cannot resolve their sister chromatids. Nevertheless, DmSMC2 and DmSMC4, the two core proteins shared by both condensins I and II, are able to localize to Barren/CAP-H-depleted chromosomes. However, DmSMC2/4 subunits were found diffuse over the chromatin and are not confined to a well-defined central axis. This strongly suggests that condensin I depletion results in mitotic chromosomes with a poorly defined axial organization (Oliveira, 2005).
The identification of a second condensin complex in HeLa cells, named condensin II, suggested the two condensin complexes could play distinct roles in mitotic chromosome organization (Ono, 2003). The authors reported that complete disruption of chromatid axial organization was achieved only when both condensin I and condensin II were absent. While the contribution of condensin II to mitotic chromosome structure in Drosophila cells remains undetermined, previous studies in S2 cells have shown that if both condensin complexes are removed by depleting one core subunit (DmSMC4), sister chromatid resolution is specifically affected (Coelho, 2003). Accordingly, depletion of Barren/CAP-H results in a chromosome structure phenotype similar to that described previously for depletion of DmSMC4. These observations suggest that in S2 tissue culture cells, if a condensin II complex does exist, it does not play a significant role in mitotic chromosome organization (Oliveira, 2005).
Additionally, it was observed that in the absence of a regulatory subunit of condensin I, the DmSMC2 and DmSMC4 core proteins are able to localize to chromatin. This suggests that the DmSMC2/4 heterodimer binds DNA independently of the regulatory subunits. In agreement, in vitro studies have shown that the core SMC heterodimer alone has DNA binding properties. Furthermore, short interfering RNAi depletion of human CAP-D2 from HeLa cells does not alter the levels of human CAP-E/SMC2 on mitotic chromosomes. In contrast, studies in budding yeast revealed that only the entire condensin complex is able to associate with DNA. This diversity probably results from species differences in the mechanism responsible for loading condensin to mitotic chromosomes (Oliveira, 2005).
The condensin I regulatory subunits DmCAP-D2 and DmCAP-G do not localize to Barren/CAP-H-depleted mitotic chromosomes. These data indicate that loading of the regulatory subcomplex to mitotic chromosomes requires all non-SMC subunits to be present. Interestingly, a homologue for CAP-G2 was not found in Drosophila melanogaster, and it has been suggested that the DmCAP-G subunit could be shared by both condensins I and II in this organism. The absence of DmCAP-G in Barren/CAP-H-depleted chromosomes clearly reveals that either the Drosophila DmCAP-G equivalent in condensin II has not been identified yet or the condensin II complex is totally absent from mitotic chromosomes in S2 cells (Oliveira, 2005).
One of the consequences of depleting condensins from mitotic chromosomes is the consistent presence of DNA bridges formed during anaphase that remain unresolved until telophase or even further. This study by in vivo time-lapse microscopy that the chromatin bridges observed in Barren/CAP-H-depleted cells must already be present at the metaphase-anaphase transition and are likely to be due to the inability to resolve catenated sister chromatids. Moreover Barren/CAP-H depletion can disrupt cytokinesis. Cytokinesis failure has already been correlated with condensin depletion in other studies (Bhat, 1996, Hudson, 2003). This correlation is more likely related to a physical incapacity in completing cell division due to DNA bridges at the cleavage furrow than to a direct role of condensin in cytokinesis (Oliveira, 2005).
In vivo analysis of condensin I-depleted cells in mitosis also revealed that chromosome congression is abnormal. Indeed, Barren/CAP-H-depleted chromosomes are unable to align at the metaphase plate even when extra time is provided by preventing anaphase onset with the proteosome inhibitor MG132. Studies in HeLa cells have also pointed out abnormal chromosome alignment after depletion of condensin I and it has been suggested that condensin is required for normal centromere/kinetochore function. However, the current results show that in the absence of condensin I, the centromere supports the formation of a functional kinetochore as revealed by the normal localization of POLO and the correct kinetochore-microtubule bipolar attachment. Previous experiments in which DmSMC4 was depleted in S2 cells also reported a normal kinetochore organization and function. These findings show that in Drosophila, the organization of the kinetochore does not require the underlying chromatin to contain condensins (Oliveira, 2005).
Indeed, the results demonstrate that the abnormal chromosome congression observed in Barren/CAP-H-depleted cells is likely to be related to the loss of centromere elasticity rather than kinetochore malfunction. In the absence of Barren/CAP-H, after bipolar attachment is established, the centromeric region elongates nearly twice the distance observed in control chromosomes. In agreement, abnormal centromere separation has also been recently reported when CAP-G is mutated in Drosophila (Dej, 2004). Also, several studies in C. elegans have suggested a role for condensin II (the sole condensin complex in this organism) in centromere resolution and integrity. The current observations also show that the normal organization of pericentric heterochromatin is not restored after removal of microtubules. This suggests that Barren/CAP-H is essential to prevent irreversible loss of centromere integrity after bipolar attachment (Oliveira, 2005).
A possible explanation for the abnormal separation of sister centromeres could be due to an altered cohesion between sister chromatids in the absence of Barren/CAP-H. However, this study clearly shows that despite its broad distribution pattern, SCC1/DRAD21 is still present between the abnormally apart sister centromeres in metaphase arrested cells. Additionally, cohesin follows a normal dynamics during mitosis in DmSMC4-depleted cells (Coelho, 2003). Thus, the structural alterations observed after depletion of Barran/CAP-H are unlikely to result from abnormal cohesin distribution. Moreover, it was found not only that the pairing domain of sister chromatid is altered, but also that the pericentric heterochromatin-associated dimethylated K9 histone H3 is irregularly distributed and the centromere marker CID appears distorted (Oliveira, 2005).
It has been demonstrated that centric and pericentric heterochromatin shows stronger attachment to a central proteinaceous scaffold or matrix. Reciprocally, chromatin immunoprecipitation experiments in S. pombe revealed a preferential association of condensin subunits with central centromeric sequences (Aono, 2002). Recently, a genetic and direct interaction between Drosophila CAP-G and the centromere-specific histone H3 variant CID was reported (Jager, 2005). These observations, taken together with the current data, strongly support the idea that the association between the centromere/pericentromere chromatin and the chromosome axis is required for the establishment of an elastic but rigid structure able to resist the forces exerted by the spindle upon sister centromeres during congression (Oliveira, 2005).
Several studies regarding the longitudinal elastic properties of mitotic chromosomes have shown that the elastic behavior depends strongly on the continuity of the DNA chain. However, the contribution of the protein scaffold to the elastic response of chromatin is controversial. It has been shown that the elastic and bending properties of mitotic chromosomes are inconsistent with the existence of a well-defined central chromosome scaffold, and alternatively, it has been suggested that the mitotic chromosome is essentially a chromatin network. Other studies revealed that the elastic properties depend on a mitotic chromosome protein scaffold, in particular on SMC proteins, as domains containing SMC proteins were shown to exhibit a higher elastic response (Oliveira, 2005).
While most studies have concentrated on the elastic properties of the arms, much less is known about the elasticity of the centromere region. Several studies pointed out the elastic properties of the centromere-proximal chromatin. Indeed, this study has showm that the absence of condensin I compromises the elastic properties of centromeric chromatin. Therefore, the data favor the hypothesis that the elastic properties of the chromosome are indeed dependent on a proteinaceous structure, at least at the centromeric region (Oliveira, 2005).
In summary, this study has shown that Barren-CAP-H is essential to allow the organization of a defined chromosome axis and to resolve sister chromatids. Furthermore, condensin I is not required for the organization of functional kinetochores but is essential to maintain the structural integrity of the centromeric region during mitosis (Oliveira, 2005).
During mitosis, faithful inheritance of genetic material is achieved by chromosome segregation, as mediated by the condensin I and II complexes. Failed chromosome segregation can result in neoplasm formation, infertility, and birth defects. Recently, the germ-line-specific DEAD-box RNA helicase Vasa was demonstrated to promote mitotic chromosome segregation in Drosophila by facilitating robust chromosomal localization of Barren (Barr), a condensin I component. This mitotic function of Vasa is mediated by Aubergine and Spindle-E, which are two germ-line components of the Piwi-interacting RNA pathway. Faithful segregation of chromosomes should be executed both in germ-line and somatic cells. However, whether a similar mechanism also functions in promoting chromosome segregation in somatic cells has not been elucidated. This study presents evidence that belle (vasa paralog) and the RNA interference pathway regulate chromosome segregation in Drosophila somatic cells. During mitosis, belle promotes robust Barr chromosomal localization and chromosome segregation. Belle's localization to condensing chromosomes depends on dicer-2 and argonaute2. Coimmunoprecipitation experiments indicated that Belle interacts with Barr and Argonaute2 and is enriched at endogenous siRNA (endo-siRNA)-generating loci. These results suggest that Belle functions in promoting chromosome segregation in Drosophila somatic cells via the endo-siRNA pathway. DDX3 (human homolog of belle) and DICER function in promoting chromosome segregation and hCAP-H (human homolog of Barr) localization in HeLa cells, indicating a conserved function for those proteins in human cells. These results suggest that the RNA helicase Belle/DDX3 and the RNA interference pathway perform a common role in regulating chromosome segregation in Drosophila and human somatic cells (Pek, 2011).
Although Vasa and Belle have been implicated in the piRNA and endo-siRNA pathways, respectively, it is not known whether DDX3 is also involved in the RNAi pathway. The fact that DDX3 is involved in viral RNA sensing offers the possibility that DDX3 may be a component of the RNAi pathway in humans. Furthermore, the DCR-dependent localization of DDX3, both during interphase and prophase, suggests that DDX3 may function downstream of DCR. Further investigation into the nature of the genomic loci and RNAi pathway components that associate with DDX3 and the nature of the noncoding RNAs involved in this process will provide greater insight into its molecular mechanism in human cells (Pek, 2011).
This study has indicated that the robust chromosomal localization of Barr/hCAP-H is regulated by the Vasa/Belle/DDX3 class of DEAD-box RNA helicases in both germ-line and somatic Drosophila cells and human somatic cells. This finding suggests the possibility of a common pathway that regulates chromosome segregation by the Vasa/Belle/DDX3 class of RNA helicases. Although chromosome segregation appears to be regulated by RNAi machinery, the necessary small RNA pathway components vary notably between the germ-line and somatic cells. The piRNA pathway components are required in the germ-line cells, whereas the endo-siRNA pathway components function as their somatic counterparts. This finding suggests that various cell types can use the existing small RNAs and RNAi factors to achieve a common goal of robust Barr/hCAP-H localization. This study also provides a framework for future studies investigating the molecular mechanism of the cooperation between the Vasa/Belle/DDX3 RNA helicases and the RNAi factors to ensure proper chromosome segregation (Pek, 2011).
Vasa (Vas) is a conserved DEAD-box RNA helicase expressed in germline cells that localizes to a characteristic perinuclear structure called nuage. Previous studies have shown that Vas has diverse functions, with roles in regulating mRNA translation, germline differentiation, pole plasm assembly, and piwi-interacting RNA (piRNA)-mediated transposon silencing. Although vas has also been implicated in the regulation of germline proliferation in Drosophila and mice, little is known about whether Vas plays a role during the mitotic cell cycle. This study reports a translation-independent function of vas in regulating mitotic chromosome condensation in the Drosophila germline. During mitosis, Vas facilitates robust chromosomal localization of the condensin I components Barren (Barr) and CAP-D2. Vas specifically associates with Barr and CAP-D2, but not with CAP-D3 (a condensin II component). The mitotic function of Vas is mediated by the formation of perichromosomal Vas bodies during mitosis, which requires the piRNA pathway components aubergine and spindle-E. These results suggest that Vas functions during mitosis and may link the piRNA pathway to mitotic chromosome condensation in Drosophila (Pek, 2010).
In Drosophila, the association of Barr with mitotic chromosomes is highly dynamic, and Barr is loaded primarily at the centromeric regions before spreading distally. How such a dynamic event is regulated remains unclear. It is suggested that during mitosis, Vas forms bodies that are in close proximity to pericentromeric piRNA-generating loci, where they function to facilitate chromosomal recruitment of Barr to participate in the robust condensation of chromosomes. Alternatively, despite the localization to pericentromeres, Vas may promote the long-range stable association of Barr with entire mitotic chromosomes by a yet unknown mechanism. Barr appears to be a principal effector of Vas, because ectopic expression and enhanced localization of Barr to mitotic chromosomes suppresses, to a large extent, chromosomal defects in vas mutants. Furthermore, mitotic localization of Vas correlates with the timing of active chromosomal loading of Barr during prometaphase, and Vas interacts specifically with Barr and CAP-D2 (condensin I components). Interestingly, relocalization of Vas bodies to regions between segregating chromosomes were also observed during anaphase. In a small fraction of vas mutant germline cells (3.2%), segregation defects are not suppressed by ectopic expression of Barr, suggesting a condensin I-independent role for Vas during anaphase. It would be interesting to examine whether Vas also functions in a tether-based mechanism to facilitate the chromosome segregation seen in neuroblasts (Pek, 2010).
Studies have shown that loss of condensin function triggers the spindle assembly checkpoint (SAC). Consistent with the observation that vas promotes robust chromosomal localization of condensin I components, it was observed that the delay in mitotic progression in vas mutants is partially rescued by reducing a copy of the SAC genes bubR1 or zw10, suggesting that the SAC may be activated in vas mutants. However, it cannot be completely excluded that Vas may also regulate other factors that trigger or suppress the SAC (Pek, 2010).
Because Vas has a wide range of functions, vas mutants exhibit a pleiotropic phenotype, including showing a mild defect in stem cell maintenance and germline differentiation. In aub and spn-E mutants, in which the mitotic function of Vas is perturbed, less robust Barr localization was observed and a delay in chromosome condensation during prometaphase, but germline differentiation was unaffected. Similarly, cap-g (condensin I) mutants are female sterile and have impaired Barr localization and delayed chromosome condensation during prometaphase. In these mutants, although the dynamics of the synaptonemal complex during meiosis are perturbed, progression of oogenesis is unaffected. Moreover, introduction of the vasΔ617 or barr-GFP transgene into vas mutants restores mitotic defects of prometaphase delay and lagging chromosomes, but not the later-stage germline differentiation defects. Taken together, these data suggest that the function of vas in chromosome condensation is not required for progression of germline differentiation but is required to ensure the fidelity of chromosome segregation, which may contribute in part to the age-dependent atrophy, oocyte differentiation defect, and sterility in vas mutants. Analysis of a Vas variant that specifically abrogates the interaction of Vas with Barr would give insight into the molecular function of Vas in regulating Barr (Pek, 2010).
This study identifies a possible link between the piRNA pathway and chromosome condensation and segregation during mitosis. This is particularly intriguing because recent studies in C. elegans have shown that 22G-RNAs and several P granule components function to organize chromosomes with holocentric centromeres during mitosis. Together with this study in Drosophila, these studies raise an interesting possibility that components of the nuage and small RNA pathways may play a unique role in organizing chromosomes during the cell cycle in eukaryotes. It will be interesting to dissect the molecular involvement of piRNAs, siRNAs, and other small RNA pathway components in regulating mitotic chromosome condensation and segregation in germline cells (Pek, 2010).
It is proposed that Vas has a role in regulating chromosome condensation during mitosis of germline cells at least in part by facilitating robust chromosomal localization of Barr. This process is mediated by the formation of Vas perichromosomal bodies during mitosis and depends on aub and spn-E (Pek, 2010).
The condensed state of mitotic chromosomes is crucial for faithful genome segregation. Key factors implicated in the formation of mitotic chromosomes are the condensin I and II complexes. In Drosophila, condensin I appears to play a major role in mitotic chromosome organization. To analyze its dynamic behavior Barren, a condensin I non-Structural Maintenance of Chromosomes subunit, was expressed as a fully functional enhanced green fluorescent protein (EGFP) fusion protein in the female, and it was followed during early embryonic divisions. Barren-EGFP was found to associate with chromatin early in prophase concomitantly with the initiation of chromosome condensation. Barren-EGFP loading starts at the centromeric region from where it spreads distally reaching maximum accumulation at metaphase/early anaphase. Fluorescence Recovery After Photobleaching analysis indicates that most of the bound protein exchanges rapidly with the cytoplasmic pool during prometaphase/metaphase. Taken together, these results suggest that in Drosophila, condensin I is involved in the initial stages of chromosome condensation. Furthermore, the rapid turnover of Barren-EGFP indicates that the mechanism by which condensin I promotes mitotic chromosome organization is inconsistent with a static scaffold model (Oliveira, 2007).
The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. This study analyses the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. It is demonastrated that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits. CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they start to decondense late in mitosis. The consequences of CAP-D2 loss after dsRNA-mediated interference was analyzed, It was discovered that the protein is essential for chromosome arm and centromere resolution. The loss of CAP-D2 after RNAi has additional downstream consequences on the stability of CAP-H, the localization of DNA topoisomerase II and other condensin subunits, and chromosome segregation. Finally, it was discovered that even after interfering with two components important for chromosome architecture (DNA topoisomerase II and condensin), chromosomes are still able to compact. This work paves the way for the identification of further components or activities required for this essential process (Savvidou, 2005).
Chromosomal DNA, if stretched end to end, measures about 2 metres in any one cell of the human body and therefore must be highly folded to fit into a nucleus of only 5 µm diameter. DNA has to condense even further prior to cell division in order to form the highly compact metaphase chromosome comprising two sister chromatids, capable of withstanding anaphase segregation. Thus, mitotic chromosome condensation involves a process in which interphase chromatin is (1) compacted, and (2) resolved into two distinct rod-shaped sister chromatids. Recent studies have contributed to the unravelling of this process. A five subunit 'condensin' complex was initially identified biochemically in Xenopus, and comprises two SMC (structural maintenance of chromosomes) proteins (SMC2 and SMC4) and three non-SMC proteins [CAP-D2, CAP-G or CAP-H (Barren in Drosophila)]. All five subunits have been identified in the eukaryotes examined to date. The SMC proteins belong to a superfamily of highly conserved and ubiquitous chromosomal ATPases (Cobbe, 2004). The non-SMC subunits have been proposed to have dual roles in the regulation of condensin function: one is to activate the SMC ATPases to perform ATP-dependent supercoiling activity, and the other is to allow the holocomplex to associate with chromatin in a mitosis-specific manner (Kimura, 2000). Each of the condensin subunits is essential and required for proper organisation and segregation of mitotic chromosomes, but exactly how the complex contributes to these processes is still unclear. Recently a second condensin complex (Ono, 2003) has been identified, with a different complement of non-SMC subunits (Savvidou, 2005).
Studies of condensin function have resulted in conflicting hypotheses as to the role of this complex. Cells of Schizosaccharomyces pombe mutant in Cut3 and Cut14 (the SMC4 and SMC2 subunits, respectively) exhibited chromosome condensation defects and displayed a 'cut' phenotype where septation (cell division) occurred with incompletely separated chromosomes. Fluorescence in situ hybridization in these mutants demonstrated that the length of mitotic chromosome arms increased while the centromeric DNA could separate and move to the opposite poles normally. In S. pombe and Saccharomyces cerevisiae, studies have shown that all members of the condensin complex are required for proper chromosome condensation and segregation. In Xenopus, when condensin was depleted from mitotic extracts before or after condensation, unreplicated chromatin failed to assemble into individual chromatids or became completely disorganised, suggesting that the complex was required both for assembly and maintenance of mitotic chromosomes. In contrast, genetic studies in Drosophila and C. elegans showed that resolution between the sister chromatids rather than condensation of the chromosomes was compromised when the condensin complex was disrupted (Bhat, 1996; Hagstrom, 2002; Steffensen, 2001). In both organisms, a high degree of compaction relative to interphase and a bipolar metaphase plate formed. However, severe chromosome segregation defects and chromosome breakage were observed in anaphase and telophase. DmSMC4 RNAi in Drosophila cultured cells confirmed the requirement of this protein for chromosome organisation and segregation (Coelho, 2003). In both flies and worms, the absence of condensin subunits results in the formation of chromosome bridges because of incomplete resolution and ensuing failure of sister chromatids to separate completely during anaphase (Hagstrom, 2003). Particular insight was gleaned when the robustness of chromosome architecture was assessed after SMC2 knock-out in chicken DT40 cells: while chromosomes still reached normal levels of condensation, clear defects in association of other non-histone chromosomal proteins was observed (Savvidou, 2005 and references therein).
Recently, a second condensin complex, called condensin II, was identified in vertebrates (Ono, 2003). This complex shares the same two SMC subunits as the original complex, now named condensin I, but appears to contain different non-SMC subunits (named CAP-D3, CAP-G2 and CAP-H2). The two complexes appear to alternate along the axis of metaphase chromatids but are both present at the centromere, albeit in distinct regions (Ono, 2004). Depletion of these complexes in Xenopus and HeLa cells produced distinct defects in chromosome morphology, suggesting that the complexes may contribute differently to mitotic chromosome architecture (Ono, 2003). Furthermore, the centromere/kinetochore regions were structurally disorganised from the depletion of either condensin I or II subunits, suggesting a specific role for both complexes in centromere organisation (Ono, 2004; Savvidou, 2005 and references therein).
DNA topoisomerase II (topo II) has long been implicated in chromosome structure and dynamics, since its discovery as a chromosome 'scaffold' protein and its localization at the base of chromatin loops. SMC2 has also been shown to be a component of the chromosome 'scaffold' fraction. Yeast mutants in topo II exhibit segregation defects similar to those observed in condensin mutants. The decatenation activity of topo II facilitates the resolution of sister chromatids and the complete separation of chromosomes in anaphase. Topo II has also been shown to be required for chromosome condensation but its role in this process has been controversial since disruption of its function resulted in different chromosome morphologies. Recently, an unexpected role for topo II in chromosome arm congression emerged, after RNAi depletion in Drosophila cells (Chang, 2003). Localisation studies revealed that Topo II was axially distributed in the chromosome arms with some concentration at the centromeres, a distribution that was also observed for condensin subunits (Maeshima, 2003; Steffensen, 2001). The localisation pattern and the phenotype after the depletion of either the condensin complex or topo II suggested that these two components of the chromosome scaffold may collaborate to bring about chromosome compaction and organisation. Biochemical and localisation studies in Drosophila showed that CAP-H/Barren associates with topo II throughout mitosis, while SMC4 appeares to be responsible for the axial localisation of topo II on the chromosomes and for its decatenation activity (Bhat, 1996; Coelho, 2003). However, in S. pombe, the localisation but not the activity of topo II was affected in condensin mutants, while topo II localization to well-defined axial structures in Xenopus is independent of condensin. Given that chromosomes experience different constraints depending on the cell, organism, or time in development, it is perhaps not surprising that a 'unified' hypothesis regarding these two components has not emerged (Savvidou, 2005 and references therein).
This study, demonstrates that a condensin I complex exists in Drosophila. The cell cycle localization and role of the Drosophila CAP-D2 subunit in chromosome dynamics was examined during mitosis. Depletion of CAP-D2 by RNAi in Drosophila cultured cells results in a compromised mitotic chromosome architecture with defective sister chromatid resolution and subsequent chromosome bridges in anaphase. The fate of other members of the condensin complex and other chromosomal proteins essential for chromosome dynamics during mitosis is altered when a condensin subunit is disrupted either by depletion or mutation. Drosophila mutations of condensin subunits were examined to assess the stability of the complex in the context of the organism. Mitotic coordination with regard to chromosome passengers is compromised when condensin function is affected. Finally it was demonstrated that when both condensin and topo II are disrupted by RNAi, chromosomes are surprisingly still able to compact, intimating the existence of additional, currently unknown, factors essential to the assembly of mitotic chromosomes (Savvidou, 2005).
Barren, the Drosophila homolog of XCAP-H, is one of three non-SMC subunits of condensin, a conserved 13S multiprotein complex required for chromosome condensation. Mutations in barren (barr) were originally shown to affect sister-chromatid separation during mitosis 16 of the Drosophila embryo, whereas condensation defects were not detected. In contrast, mutations in yeast homologs of barren result in defective mitotic chromosome condensation as well as irregular chromatid separation. This study used double-stranded RNA-mediated interference (RNAi) to deplete Barren in Drosophila S2 cells. The analyses indicate that inactivation of barr leads to extensive chromosome condensation and disrupts chromatid segregation (Somma, 2003).
Double-stranded RNA-mediated interference (RNAi) was used to deplete barr function in Drosophila S2 cultured cells. The addition of 15 microg of barr double-stranded RNA (dsRNA) into S2 culture cells completely ablated the endogenous mRNA after 72 hr as shown by RT-PCR. Cytological analysis of treated metaphases revealed that chromosome condensation was highly defective upon depletion of the barr gene product. In 98% of metaphases scored, condensing chromatin appeared fuzzy and loose. Sister-chromatid morphology was largely disorganized and chromosomes were not distinguishable from one another. This chromatin undercondensation pattern was also detectable during prophase and by using different DNA dyes. To assess whether heterochromatin was also influenced by this global decondensation effect, fluorescence in situ hybridization (FISH) was performed on metaphase spreads using the dodecasatellite probe. The dodecasatellite probe specifically hybridizes to pericentromeric heterometaphases chromatin of chromosome 2. In all barr metaphases analyzed the fluorescent signals appeared larger and more diffuse with respect to untreated control metaphases (Somma, 2003).
The area of each fluorescent dot was measured and it was, on average, 1.8 times larger in barr cells than in controls. This suggests that some degree of chromatin decondensation may also occur at pericentromeric regions. This observation is consistent with Barren localization in the centromeric region (Steffensen, 2001) where it is thought to be required for the proper function of centromeres. In addition, studies in yeast have shown that brn-1 may be necessary for the formation of functional mitotic kinetochores (Somma, 2003).
To determine whether the phenotype associated with RNAi of barr reflected a general chromatin undercondensation phenomenon or whether it could be attributed directly to depletion of barr, S2 cells were treated with gluon dsRNA. gluon encodes the Drosophila SMC4 homolog and has been shown to be required for chromosome condensation and sister-chromatid resolution (Steffensen, 2001). It was found that the endogenous gluon mRNA was completely depleted after 72 hr treatment and that its depletion resulted in disruption of chromatin condensation. Interestingly, the chromatin decondensation pattern in gluon dsRNA-treated cells is distinct from that observed in barr, but very similar to that described for gluon mutant neuroblast chromosomes (Steffensen, 2001). Chromosomes in glu (RNAi) cells appear swollen and the chromatin downy. In contrast, barr chromatin seems unrolled and diffuse and appears to branch off the main chromosome axis, clearly visible in the Giemsa-stained spreads. Moreover, using version 1.6 of the National Institutes of Health (NIH) image tool for MacIntosh (http://rsb.info.nih.gov/nih-image), both chromosome length and width were measured in untreated and treated cells to ask whether axial condensation or completion of the chromatin loops, or both, was affected. For this purpose the analysis focussed on different stages of mitosis where both longitudinal and transversal axes were identifiable despite the irregular condensation phenotype. It was observed that in barr cells the average chromosome length and width did not significantly differ from that of the control whereas gluon chromosomes were longer than the control. Collectively, these data suggest that inactivation of these two different subunits (one SMC and one non-SMC) of the condensin complex has diverse effects on chromosome condensation and chromatin loop organization. Furthermore, these observations support the view that components of the condensin complex have distinct, specialized functions (Aono, 2002; Somma, 2003 and references therein).
Chromatin from cells treated with barr dsRNA appeared decondensed throughout anaphase and telophase. An extremely high proportion of these figures exhibited chromatin bridges and laggards), very likely a consequence of disorganized chromatin fibers. It is speculated that the extensive chromosome decondensation may give rise to chromosomes disentangling and failure of chromatid resolution (Somma, 2003).
It was next asked whether these chromatid segregation abnormalities were accompanied by spindle defects. Treated cells were immunostained with the anti-α-tubulin antibody and it was observed that spindle components were not affected by depletion of barr and appeared normal during all stages of mitosis. Moreover, mitotic progression of treated S2 cells was not influenced, as normally occurs when a high proportion of cells polyploid. The FACS analysis, however, revealed a general reduction of the 2C peak and an increase of the area between the 2C and 4C peaks. It is believed that the expanded area between 2C and 4C peaks represents 2C cells that, despite chromosome condensation defects, complete mitosis and linked to the centromere enter the subsequent cell cycle with an aberrant DNA content. To further characterize the phenotype of barr mutant cells, an analysis was carried out to see whether the localization of DNA topo isomerase II, which has previously been shown to interact with Barr, was affected in Barr-depleted cells. By immunostaining both mutant and control cells with an antitopoisomerase II antibody, it was observed the localization of this enzyme throughout the different stages of mitosis. It was found that topoisomerase II was associated with chromatin during prophase, metaphase, anaphase, and telophase in both barr and control cells, suggesting that its localization is independent of barr. Moreover, this analysis indicates that chromosome decondensation is a direct effect of Barr depletion and not a consequence of DNA topoisomerase II misbehavior (Somma, 2003).
To date, it is not understood why defects in chromosome condensation are present in Barr-depleted S2 cells and not in mutant embryos. Chromosome compaction was found to be normal in a null mutation of barren, so this discrepancy cannot be explained by allele-specific defects. It is possible that barr mutant embryos still retain maternal Barr product, which enables chromosomes to condense properly. Alternatively, it is conceivable that the organization of chromatin in embryonic nuclei differs from that of S2 cells, as the former support frequent and rapid mitotic divisions. In this highly dynamic scenario, Barren may not be strictly required for chromatin compaction, or its function may be redundant. One can also argue that the chromatin disorganization observed in S2 cells is a peculiarity of this cell type. It is believed that this is unlikely, however, since it was shown that RNAi of gluon causes failure in chromosome condensation, a phenotype similar to that elicited by gluon mutant neuroblast cells, thus indicating that S2-treated cells do mimic Drosophila mutations. In addition, the observation that dsRNAs of different condensin subunits have different effects on chromosome condensation makes RNA inter ference a useful tool to molecularly dissect this important aspect of chromosome dynamics (Somma, 2003).
Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin DmSMC4, a subunit of the complex, was depleted from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. These results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution (Coelho, 2003).
This study has shown that dsRNAi can be used to severely deplete DmSMC4 in tissue culture cells resulting in mitotic phenotypes that are very similar to those previously described for dmSmc4 mutant Drosophila cells (Steffensen, 2001). Already 24 hours after RNAi treatment some mitotic cells show abnormal resolution of sister chromatids and later, cells in anaphase or telophase begin to show chromatin bridges indicating that the frequency with which these phenotypes are observed depends on the level of depletion of DmSMC4. Loss of DmSMC4 causes the formation of short mitotic chromosomes with poorly defined sister chromatids. These chromosomes are unlikely to contain other proteins of the condensin complex since immunofluorescence and mitotic chromatin immunoprecipitation shows that binding of Barren to condensing chromosomes is dependent on DmSMC4. These observations are in agreement with previous findings indicating that non-SMC condensins can only bind DNA in the presence of the entire condensin complex. Also, it was shown in S. pombe and S. cerevisiae that all members of the regulatory subcomplex are essential for chromatin association of yeast condensin in vivo. Together, these results strongly suggest that, in Drosophila, the assembly of the condensin complex to mitotic chromatin requires all protein subunits. Moreover, the results demonstrate that certain aspects of chromosome condensation, namely shortening of the longitudinal axis of sister chromatids, can occur in the absence of condensins (Coelho, 2003).
Although, chromosomes do not condense normally in DmSMC4-depleted cells, genetic studies in Drosophila showed that loss of DmSMC4 (Steffensen, 2001) or Barren (Bhat, 1996) does not prevent cells from entering anaphase and attempting sister chromatid segregation. However, recently it has been suggested that the condensin complex might contribute to ensure proper function of the centromere. In S. cerevisiae, BRN1 the homologue of Barren, has been implicated in the formation of functional mitotic kinetochores (Ouspenski, 2000) and in C. elegans condensin activity is required for the normal orientation of the centromere towards the mitotic spindle (Hagstrom, 2002). This study has shown that depletion of DmSMC4 does not affect the localisation of centromere or kinetochore proteins and that microtubules associate with kinetochores. Furthermore, it was observed that at early stages of mitosis, kinetochores associated with spindle microtubules appear to stretch poleward, sometimes well beyond the chromatin. However, when microtubules are depolymerised by colchicine, kinetochores localise only over mitotic chromatin, suggesting that stretching of kinetochores is microtubule dependent. Similar observations were reported after expressing GFP-tagged centromeres in a BRN1 mutant background (Ouspenski, 2000). These results suggest that condensin is not required for the formation of functional kinetochores and that at metaphase-anaphase transition sister centromeres disjoin normally and segregate but sister chromatid arms remain attached causing stretching of the centromeres (Coelho, 2003).
Immunofluorescence and biochemical studies have suggested that condensed chromatids contain a central axial structure. Topoisomerase II and condensin have been identified at this elusive structure. Previously it was shown that in Drosophila S2 cells condensins associate with chromatin at prophase localising to the axis of sister chromatids throughout mitosis (Steffensen, 2001). This study shows that Topo II also localises to the axis of sister chromatids throughout mitosis. However, in prometaphase chromosomes it is clear that condensin and Topo II show only partial co-localisation. DmSMC4 and Topo II localise to discrete sites that alternate along the chromatid axis. Although similar patterns of localisation has been recently described for hBarren and Topo IIα in HeLa cells, hBarren appears to bind chromatin only during prometaphase while Topo IIα is present from prophase (Maeshima, 2003). This discrepancy in the kinetics of condensin accumulation at early stages of mitosis probably represents cell type-specific differences since it is unlikely that SMC and non-SMC subunits bind chromatin independently. Furthermore, this study shows that depletion of DmSMC4 abolishes the localisation of Topo II to a well-defined axial structure even though there is no significant reduction in the level of chromatin-associated Topo II. Similarly, in yeast, localisation of Topo II to mitotic chromatin has been shown to depend upon condensin function (Bhalla, 2002). However, more recent data has suggested that in chromatin assembled in Xenopus extracts, Topo II localisation to an axial structure of chromatids occurs independently of condensin (Cuvier, 2003). These apparently contradictory results could be explained if condensin was not completely depleted in the Xenopus extracts, allowing partial accumulation of Topo II to an axial chromatin structure. Since RNAi depletion studies no DmSMC4 was found either by immunoflourescence or western blotting, the results suggest that condensin plays an essential role in the organisation of the chromatin so that Topo II can localise to the chromatid axis. This structure is likely to be highly dynamic since recent live imaging and FRAP analysis in mammalian cells shows that Topo IIα exchanges rapidly between a cytoplasmic pool and that bound to chromosomes and centromeres. In vivo analysis of condensin accumulation to the axis of sister chromatids should provide valuable insights on the dynamics of this 'ill-defined' structure (Coelho, 2003).
The abnormal distribution of Topo II to mitotic chromatin resulting from depletion of DmSMC4 prompted a determination of whether its DNA decatenation activity was also compromised. This study showed, using an in vitro assay, that DNA decatenation activity of the endogenous Topo II is significantly reduced when DmSMC4 is depleted. Although these results are compatible with a direct interaction between DmSMC4 and Topo II, no co-immunoprecipitation was detected indicating that the interaction might be indirect. Nevertheless, the results suggest that proper activity of the enzyme requires condensin. A more direct interaction was reported previously since it was shown that Barren interacts in a yeast two-hybrid assay with Topo II and promotes its decatenating activity (Bhat, 1996). However, it has been shown that BRN1, the yeast homologue, is not required for Topo II activity in vivo (Lavoie, 2000). Furthermore, it is unlikely that depletion of DmSMC4 completely abolishes Topo II activity since mutation or inhibition of its activity has been shown to cause arrest at the metaphase-anaphase transition, a phenotype not produced by DmSMC4 depletion. Accordingly, it is believed that the chromatin bridges observed after depletion of DmSMC4 are due to inappropriate activity of Topo II resulting in the maintenance of catenated DNA between sister chromatids (Coelho, 2003).
Previous reports have suggested a possible mechanistic interaction between cohesins and condensins. However, this study shows that depletion of DmSMC4 does not alter the localisation or removal of cohesins from mitotic chromatin in Drosophila S2 cells. Similarly, in S. cerevisiae it has been shown that although sister chromatid separation does not occur normally in Ycs4 mutants, MCD1/SCC1 is released from chromosomes at the metaphase-anaphase transition. Conversely, depletion of cohesins in higher eukaryotes does not appear to affect chromosome condensation and in Xenopus extracts the release of cohesin during prophase is not required for chromatin compaction mediated by condensin. Taken together, these results indicate that the removal of cohesins during mitosis is independent of condensin activity (Coelho, 2003).
Depletion of cohesins causes premature sister chromatid separation and a significant prometaphase arrest. This prometaphase arrest could be due to the activity of the spindle checkpoint, which prevents exit from mitosis if proper chromosome orientation and organisation of a metaphase plate is not achieved. Strikingly, it was observed that simultaneous depletion of DmSMC4 and DRAD21 does not lead to premature sister chromatid separation or arrest during prometaphase but cells progress into anaphase and telophase showing extensive chromatin bridges. It is proposed that sister chromatids do not separate prematurely in the absence of cohesins because depletion of DmSMC4 prevents sister chromatid resolution by compromising Topo II activity. These cells then proceed into prometaphase, kinetochores can now bind spindle microtubules and chromosomes congress to the metaphase plate, allowing cells to satisfy the spindle checkpoint and initiate mitotic exit. Thus, in the absence of DmSMC4, abnormal decatenation of sister chromatids appears to provide an alternate mechanism to hold sisters together during early stages of mitosis (Coelho, 2003).
From these results it is proposed that condensin is essential to organise a clearly defined axial structure of sister chromatids where Topo II can localise. In the absence of this specific localisation, Topo II can still bind chromatin but its decatenation activity is not specifically directed and sister chromatids cannot resolve properly (Coelho, 2003).
Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. An investigation was carried out to see whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. Topoisomerase II (TopoII) and Barren (Barr) are shown to interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, the PcG protein Polyhomeotic (Ph) interacts physically with TopoII and Barr and Barr is required for Fab-7-regulated homeotic gene expression. Conversely, defects in chromosome segregation have been found associated with ph mutations. It is proposed that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression (Lupo, 2001).
PcG genes have been proposed to act as chromosomal components maintaining transcriptional repression by 'heterochromatinizing' their target sites. However, the molecular mechanisms underlying chromosomal silencing by the PcG, heterochromatin formation, and the transmission of the silenced state through mitosis are not known. It was reasoned that chromosome condensation machineries could provide an important functional link between the regulation of chromosome domain structure, gene silencing, and mitotic inheritance. Thus, the interaction of the PcG with the machinery involved in orchestrating chromosome dynamics has been investigated and in particular with those machines enabling mitotic chromosome condensation. The in vivo formaldehyde-fixed chromatin immunoprecipitation (X-ChIP) method was used to analyze the distribution in the BX-C locus of two proteins: TopoII, an enzyme involved in the regulation of DNA supercoiling, chromosome condensation, and segregation, and Barr. Barr is the homolog of the Xenopus XCAP-H and C. elegans DPY26 proteins, a TopoII-interacting protein associated with the SMC2/4 condensins complexes, known to be involved in mitotic chromosome condensation (Lupo, 2001).
A striking colocalization of TopoII and Barr with previously mapped PC binding sites was found, suggesting that the two groups of functions are at least acting on the same DNA regions. A clear colocalization was found at major PREs (Fab-7, Mcp, iab-3, bxd, and bx). In particular, the Fab-7 element appears to be a major TopoII/Barr binding site. Strong association of PC to Fab-7 is found. No Barr/TopoII binding site was found at the Fab-8 PRE, which might define the border between the repressed and active BX-C domains in SL-2 cells (Lupo, 2001).
In iab-2 and iab-3, large fragments (11.0 and 11.5 kb, respectively) have PRE activity. Here specific Barr and TopoII sites are also found. These sites do not match the PC/GAGA peaks previously described. Yet, since these regions show considerable levels of PC, it is suggested that minor PC binding sites adjacent to the reported 'peaks' may also be functionally relevant. Another important aspect of PcG function is the interaction with promoters; major PC binding sites include core promoters, and it is known that PREs perform better when combined with their natural target promoters. Interestingly, a striking colocalization of TopoII and Barr is also found at promoters (AbdB gamma, abdA II, and Ubx) (Lupo, 2001).
Based on the mitotic phenotype and previous immunolocalization data, a direct association of TopoII and Barr with chromosomes mostly at mitosis is expected. In this context, the colocalization of TopoII and Barr in regulatory regions of the BX-C is striking. Although asyncronous tissue culture cells were used, it is believed that the association of Barr and TopoII with the regulatory regions of the BX-C occurs not only at mitosis but also in interphase. In particular, in X-ChIP experiments, the number of mitotic cells at the time of formaldehyde fixation is around 5%, thus, if only mitotic cells contributed to the overall precipitated DNA, this approach would have been below the detectable limit. Hence, it is proposed that TopoII and Barr are associated with their target sites throughout the cell cycle (Lupo, 2001).
The short proximal isoform of Ph (Ph 140p) can be copurified from nuclear extracts with TopoII and Barr. This isoform is not found coimmunoprecipitated with Pc and Psc, and neither Barr nor TopoII copurified with Pc and Psc. The three PcG members Pc, Psc, and the long proximal product Ph 170p have been shown to coimmunopurify from nuclear extracts with antibodies against one of the three. Due to the absence of Ph 140p signals in the Pc/Psc immunoprecipitations, these results might be taken to indicate that there is no functional connection between the presumptive TopoII/Barr/Ph 140p complex and the Pc/Psc/Ph 170p complex. For three reasons this is thought to be unlikely. (1) Both the 170p and the 140p isoforms of Ph are derived from the same transcript by posttranscriptional regulation and differ by a 244 N-terminal stretch of amino acids present only in the 170p isoform. Functional domains of Ph (zinc finger, coiled-coil region, GTP binding site, serine/threonine-rich region, and SAM/SPM domain) are all contained in both isoforms, suggesting that both proteins can fulfill related functions. (2) X-ChIP data, obtained with the same Ph antibodies used in this study, show an extended overlap of Pc and Ph binding regions in the BX-C. Together with the finding of a colocalization of TopoII and Barr with PcG binding sites in regulative regions of the BX-C, this suggests that these proteins act on the same DNA regions. (3) The data show that a reduction of the amount of Barren protein in barren heterozygotes parallels PcG-negative effects on the silencing function of the Fab-7 PRE (Lupo, 2001).
An additional finding supports the conclusion that Ph protein(s) are involved both in PcG function and mitotic chromosome condensation. ph null embryos show defects in chromosome segregation, the same phenotype observed for barren mutant embryos. Conversely, the results of Barren haplo-insufficiency on Fab-7 silencing are suggestive of a role for Barr in early embryogenesis. Since in early embryogenesis Ph 140p is the only Ph product made, these defects are diagnostic of a specific role of Ph 140p in mitosis. These results with regard to Barren protein and Fab-7 silencing are reminiscent of another previously documented role for SMCs in gene regulation. In C. elegans, the DPY27 protein, a homolog of the Xenopus XCAP-C (SMC4), has been shown to bind the X chromosome in females, whereas its absence results in lethality due to abnormally high gene expression levels from the X chromosome. Thus, it is concluded that Ph 140p shares an important role with the Barr/TopoII condensin complexes in mitosis and cell memory processes (Lupo, 2001).
In order to further study interactions between barren and the PcG the null alleles ph502 and ph602 were used for genetic analysis, and strains heterozygous for ph and barren mutations were crossed. Surprisingly, no effect was found. PcG genes, in contrast, show dosage effects, suggesting that the interaction between PcG and Barr/TopoII may imply a different, more dosage-insensitive regulation. However, it has been shown that barren mutations affect PRE silencing in the same way as mutations in PcG genes do. Taken together, these results may indicate a nonstoichiometric relationship between PcG and Barr/TopoII protein complexes. It is proposed that major PcG and condensin proteins belong to distinct protein complexes, but that they nevertheless cooperate at PREs and promoters to maintain the silenced state of homeotic genes. From the SMC standpoint, these results are intriguing because they show that proteins involved in chromosome condensation and segregation processes bind to regulatory elements in chromosomal domains responsible for the inheritance of transcription states. This would suggest that the 'structural maintenance of chromosome' function could also affect epigenetic control of gene expression (Lupo, 2001).
These data reveal novel molecular aspects of BX-C regulation. The distribution of PC and TopoII/Barr sites in the BX-C appears as a reiterated array suggestive of heterochromatic hallmarks, perhaps providing in cis information for higher-order organization of the BX-C chromosomal domain. In particular, TopoII oligomerizes in a DNA-dependent manner. Similar interactions in trans are proposed to occur between PcG proteins in vivo. According to this ability, spaced molecules at distant sites on the DNA could come into contact, giving rise to more condensed domains. A model has been proposed to explain how condensin proteins and Topoisomerases may act together in condensation. In this model, the size of the condensin complex (perhaps 1000 Å) could introduce (+) supercoils by affecting the global writhe of DNA, thus creating a more condensed state. In this study, Barr is found only at discrete sites, whereas PC and other PcG proteins are associated also with large chromosomal regions. Possibly, one aspect of PcG protein function and binding to chromatin in interphase is to stabilize and expand the condensed state by topological effects (Lupo, 2001 and references therein).
The positioning of TopoII at complex regulatory regions (e.g., abx/bx and iab-3-iab-8) may indicate the existence of minidomains providing tight control on the chromatin structure of intervening regulatory DNA sequences by localized changes of DNA superhelicity. The activity of TopoII could be locally regulated by the association with other proteins like Barr and perhaps some PcG and trxG members [e.g., Ph 140p, CCF, E(z), and Gaga]. Interestingly, Barr has been found to stimulate TopoII activity. It has to be pointed out that these data show, in a direct way, where in vivo TopoII binds to single-copy genes but they cannot tell if these sites correspond to TopoII cutting sites. However, it is likely that a tight association with DNA corresponds to enzymatic activity. Thus, it is proposed that in vivo TopoII activity may be enhanced at specific sites, whereas at others it could be reduced, resulting in local differences in chromatin condensation states controlled by DNA topology (Lupo, 2001).
The presence of multiple Barr and TopoII sites within the BX-C could thus provide a powerful way to fine-tune the structure of each of the parasegment-specific chromosomal subdomains. As a direct consequence of controlled condensation of specific parts of the BX-C, determined states could be fixed by enabling or not enabling specific interactions between cis elements. The mechanism by which Fab-7 regulates the AbdB promoters is, in fact, not known. It has been proposed that a combination of 'chromatin effects' and insulating activity may regulate enhancer-promoter interactions. It is proposed that the homeotic loss-of-function phenotypes observed in Fab-7 or Mcp deletions could be due to a change in local DNA topology altering the communication of segment-specific enhancers with the AbdB promoters. In this way, local differences in chromosome domain topology may contribute to stabilize or interfere with correct phasing between regulatory elements and promoters. If topological effects are at least part of Fab-7 function, this may also help to explain distance-dependent effects on enhancer-promoter interactions. Interestingly, in Drosophila, mutations in the Nipped-B gene facilitate enhancer-promoter interactions by overcoming the action of ectopic insulator elements in the Ubx domain. Nipped-B is the homolog of the yeast SMC-associated protein Scc2 (sister chromatid cohesion 2), suggesting that adherins may have a broader role in chromosomal domain organization and gene regulation. It is proposed that chromatin condensation proteins may be involved in a pathway acting also in interphase that regulates chromosome domain structure by DNA topology and is essential for maintenance of gene expression (Lupo, 2001).
The segregation of chromosomes with high fidelity requires exquisite coordination of cellular processes. The mechanisms that coordinate the cycle of chromosome condensation and decondensation with the assembly, function, and subsequent disassembly of the mitotic spindle are poorly understood. Highly conserved genes essential for chromosome condensation have been found through genetic screens in yeasts and Drosophila. For example, five members of a protein complex known as condensin, have been identified that are functionally and structurally conserved. Mutants exhibit incomplete chromosome condensation associated with failure of segregation and the stretching of chromatin upon the spindle. Biochemical approaches also identified the protein complex in Xenopus and showed that it can promote chromatin condensation by directing the supercoiling of the DNA in an ATP-dependent manner. Chromosome condensation is also accompanied by phosphorylation of histones H1 and H3. Indeed, mutation of the mitotic phosphorylation site of histone H3 of Tetrahymena leads to both chromosome condensation and segregation defects. A direct link between histone H3 phosphorylation and condensin recruitment onto chromosomes has recently been suggested by the colocalization of members of the condensin complex with phosphorylated histone H3 during the early stages of mitotic chromosome condensation. However, the generality of the requirement for the phosphorylation of histone H3 for chromosome condensation and segregation must be questioned by the finding that budding yeast cells in which serine 10 of histone H3 is replaced with alanine show no apparent defects in cell cycle progression or chromosome transmission. Nevertheless, maximal chromosome condensation in meiosis does correlate with maximal levels of phospho-histone H3 in wild-type cells. The enzyme required for histone H3 phosphorylation in Saccharomyces cerevisiae is the aurora-related protein kinase Ipl1p. Moreover, one of its two counterparts from Caenorhabditis elegans, the air-2 protein kinase, has been shown to have the same function (Giet, 2001 and references therein).
The Aurora- and Ipl1-like protein kinases form a conserved family of enzymes, the founding members of which are encoded by the S. cerevisiae and Drosophila genes, IPL1 and aurora, respectively. While the yeast genome encodes only one such kinase required for accurate chromosome segregation, metazoan genomes have at least two subfamilies of aurora-like kinases. One is associated with centrosomes and is activated in early mitosis, and a second is associated with chromosomes and the spindle midbody and is activated later. These families are referred to as Aurora-like kinases A and B, respectively. The precise effects of loss of function of either of these enzymes varies a little between different organisms and cell types. Broadly speaking, however, the A-type enzymes are required to maintain the separation of centrosomes to give normal bipolar spindle structure. This is shown, for example, in Drosophila from the phenotype of aurora mutants (termed here aurora A); or in Xenopus, where the corresponding pEg2 kinase can be eliminated using antibodies or inactive mutants. In contrast, the B-type Aurora-like kinases appear to be required for cytokinesis, as shown, for example, by transfection of an inactive kinase mutant into cultured mammalian cells. An affect on cytokinesis has also been reported in mutants of the gene for the C. elegans B-type enzyme, Air-2, or after RNA interference. The air-2 encoded kinase is required for the positioning of Zen-4, a kinesin-like protein required at the midzone of the late central spindle for cytokinesis. Abnormal chromosome segregation is also observed after reduction of air-2 function (Giet, 2001 and references therein).
The dynamics of the localization of the Aurora B class of enzymes can be partially explained by recent findings showing they exist in a complex with an inner centromere protein (Incenp). Incenps are one example of so-called 'passenger proteins' that localize to the centromeric regions of chromosomes at metaphase and are then redistributed to the central spindle during cytokinesis. Defects in Incenp function lead to failure of chromosome congression and cytokinesis defects. These findings, and the fact that B-type Aurora kinase becomes incorrectly localized in human cells expressing mutant Incenps that fail to localize, has led to the idea that Incenp functions to target the B-type kinases, first to chromosomes and then to the spindle midzone. A physical interaction is also seen between the Air-2 kinase and the counterpart of Incenp in C. elegans, ICP-1. Moreover, the disruption of icp-1 function by RNAi leads to the same phenotype as air-2 RNAi. This direct functional interaction between the Aurora-like kinases and Incenp occurs not only in metazoan cells, but also in budding yeast where the counterpart of Incenp, Sli15p, was identified through a screen for genes that interact with Ipl1 (Giet, 2001 and references therein).
Although a B-type Aurora kinase gene has been identified in Drosophila, the lack of mutants at this locus has prevented any analysis of its potential mitotic function. Levels of the Aurora B kinase can be reduced by RNAi in cultured Drosophila S2 cells. This leads to cytokinesis failure, together with chromosome condensation and segregation defects strikingly similar to those that have been described for mutations in the condensin gene barren (Bhat, 1996). The segregation defects are accompanied by aberrant chromatin condensation, a reduction in the phosphorylated form of histone H3, and a failure to recruit the Barren protein onto condensed chromosomes (Giet, 2001 and references therein).
Using double stranded RNA interference it has been shown that the Aurora B kinase is required for mitotic chromosome condensation and segregation, and subsequently for cytokinesis. The Aurora B enzyme becomes perfectly positioned to execute these processes as mitosis proceeds. It is distributed throughout the chromatin as it condenses at prophase, then becomes concentrated around the centromeric regions of the condensed chromosomes at metaphase, and finally leaves for the company of the central spindle region during anaphase. As such it behaves as a so-called passenger protein. It appears from recent studies to be in an intimate relationship with a travelling companion Incenp. The interaction of Incenp, or its yeast counterpart Sli5p, with Aurora-like kinases in yeast, C. elegans, and Xenopus suggests that this interaction is universal. The dynamic association of Incenp with chromosome arms at prometaphase, the centromeric region at metaphase, and then the spindle midzone at anaphase makes it an attractive candidate for targeting the Aurora B kinase to these regions. Indeed, dominant mutants of Incenp in human cells disrupt the localization of the Aurora B-like kinase AIL2. The finding of abnormal chromosome segregation and cytokinesis after depletion of either the C. elegans Incenp, Icp-1, or its Aurora B-like kinase, Air-2, suggests the two passengers perform similar functions (Giet, 2001).
One striking effect of aurB RNAi is to permit progression through mitosis with improperly condensed chromosomes. It was possible to account for these condensation defects by a diminution of the phosphorylation of serine 10 of histone H3 and a failure to localize condensin on the chromosomes. The former finding is consistent with several studies that now implicate a requirement for the phosphorylation of the NH2-terminal region of histone H3 at this residue for chromosome condensation. Not only does the formation of mitotic chromosomes in a Xenopus cell-free extract by a nucleosome-associated kinase correlate with histone H3 phosphorylation, but when the serine 10 residue is mutated to alanine it results in abnormal segregation and chromosome loss during mitosis and meiosis in Tetrahymena. One enzyme credited with the ability to phosphorylate histone H3 at mitosis is the NIMA kinase of Aspergillus. However, the finding that levels of histone H3 phosphorylation are reduced after aurB RNAi in Drosophila cells is more in keeping with the report that the Aurora-like kinase homologs, Ipl1 of yeast and Air-2 (but not Air-1) of C. elegans, are required for histone H3 phosphorylation in these organisms. The finding of some residual histone H3 phosphorylation either could reflect the incomplete elimination of Aurora B by RNAi, or could indicate that an alternative kinase has this capability, offering an explanation of the partial chromosome condensation seen in the RNAi-treated cells. The current data are important in emphasizing the importance of histone H3 phosphorylation for chromosome transmission and as such are in line with the findings in Tetrahymena. This differs from the effects seen in budding yeast cells that continue through division cycles in the absence of histone H3 phosphorylation without showing defects in chromosome transmission. As an explanation, it has been suggested that other histones could be phosphorylated in addition to the histone H3 in the yeast cell and that such phosphorylation events could be sufficient to ensure normal chromosome dynamics. A major role of the yeast enzyme Ipl1p is to regulate the function of the kinetochore-associated protein Ndc10p through its phosphorylation. Therefore, the increase in ploidy reported in ipl1 mutant cells has been attributed more to inappropriate kinetochore function, and consequently the effects of Air-2 depletion upon chromosome condensation in C. elegans have been a little overshadowed. It seems likely that the abnormal chromosome segregation in Drosophila cells after aurB RNAi is due to incomplete condensation, since a similar phenotype is seen in mutants of the condensin subunit Barren (Bhat, 1996). Of course, this does not exclude the possibility that defects in the organization of the centromeric regions and kinetochores arise directly as a result of aurB RNAi or as either a direct or indirect consequence of condensation defects. The increase in ploidy seen after aurora B RNAi is reminiscent of the Ipl1 phenotype in budding yeast, but differs in that it arises from both chromosome segregation and cytokinesis defects (Giet, 2001).
The resemblance of the mitotic phenotype of cells after RNAi with aurB to that previously reported for Drosophila barren mutants (Bhat, 1996) can be further explained by the failure of Barren protein to be recruited to the mitotic chromosomes after aurB RNAi. Originally recognized through this mutant defect, it was later realized that Barren is the fly homolog of a member of the pentameric complex, condensin, first shown to be required for mitotic chromosome condensation in Xenopus. It is possible that Barren or other members of the condensin complex could themselves be directly phosphorylated by Aurora B during chromosome condensation. However, the process seems likely to involve a plethora of phosphorylation events: the nuclear A-kinase anchoring protein (AKAP95) appears to target the human hCAP-D2 condensin to chromosomes and phosphorylation of condensin subunits by cdk1 has been associated both with their nuclear accumulation and activation. It has been proposed that phosphorylation of the NH2 terminus of histone H3 leads to the recruitment or the activation of the condensin complex to the chromosome, where it can modify DNA topology. The data presented here indicate that phosphorylation of histone H3 by the Aurora B kinase and the localization of Barren onto chromosomes are associated events in mitosis. They support and extend a recent observation that human condensin proteins hCAP-E, hCAP-C, and hCAP-D2 colocalize with phosphorylated histone H3 in clusters in partially condensed regions of chromosomes in early prophase. The similarity of the effects seen on chromosome condensation resulting from loss of either aurora B or barren function is striking and points to the value of studying these processes in a single model organism amenable to both genetic manipulation and RNAi. It is perhaps surprising that in both cases partial chromosome condensation is achieved and that there can be some degree of segregation of chromatin to the poles (Giet, 2001 and references therein).
The second major mitotic abnormality observed after aurB RNAi in Drosophila cells is a failure of cytokinesis. Thus, like its mammalian and nematode counterparts AIM-1 and AIR-2, the enzyme encoded by aurora B appears essential for this process. Two proteins that play a role in cytokinesis have recently been shown to associate with the Aurora B-like kinases: Incenp, as discussed above, and the Zen-4 kinesin-like protein of C. elegans. The localization of the latter is disrupted after disruption of air-2 function using RNAi or conditional mutant alleles. Zen-4 is the C. elegans homolog of the Pavarotti KLP of Drosophila, which likewise is mislocalized on the central spindle from anaphase onwards after aurB RNAi. Pav-KLP also cooperates with Polo kinase to achieve its localization and function in Drosophila, suggesting that multiple mitotic kinases may be required to coordinate central spindle formation before cytokinesis, just as several kinases appear to be required for centrosome maturation and separation and chromosome condensation (Giet, 2001).
It is striking that aurB RNAi cells are not arrested by a mitotic checkpoint, given the abnormalities that they show in chromosome alignment at metaphase and the subsequent disorganization of the later mitotic spindle. However, the treated cells do undergo multiple cell cycles, as is clearly demonstrated in this cell culture system in which one can monitor the shift in ploidy by FACS analysis and the increase in chromosome and centrosome complements by immunocytology. It is possible that these abnormalities arise too late in the mitotic cycle to trigger checkpoint arrest, although this seems unlikely for the chromosome segregation defect. Although it is possible that Aurora B is itself required for checkpoint functions, it could also be that the kinetochore regions of chromosomes are insufficiently well organized after aurB RNAi to promote the checkpoint activity of the complex of Bub/Mad proteins that associate with unaligned centromeres. It is noteworthy that the C. elegans baculovirus inhibitor of apoptosis (IAP)-related repeat protein Bir-1 appears to be required for the localization of Air-2. Bir-1 localizes to chromosomes and then the spindle midzone and Air-2 fails to localize to these same sites in the absence of Bir-1. These IAP proteins, also known as survivin, are caspase inhibitors and as such counteract apoptosis. Is it possible that B-type Aurora kinases might play a role alongside survivin in an apoptotic checkpoint to promote mitosis (Giet, 2001)?
It is of considerable interest to know the multiple substrates of Aurora B kinase and to understand its mode of regulation in mitotic progression. It seems that subcellular localization of the enzyme could be one critical means of controlling access to its substrates. The enzyme localizes throughout condensing chromosomes when phosphorylation of histone H3 is required. Aurora B's subsequent concentration at centromeres could direct enzyme activity toward specific chromosomal proteins at these sites, but may be instrumental in its movement onto the central spindle at anaphase, thereby providing an effective way of removing the enzyme from the chromatin to facilitate chromosome decondensation at telophase. Understanding the intricacies of these processes will be a future challenge (Giet, 2001).
A Drosophila gene, barren (barr), is required for sister-chromatid segregation in mitosis. barr encodes a novel protein that is present in proliferating cells and has homologs in yeast and human. Mitotic defects in barr embryos become apparent during cycle 16, resulting in a loss of PNS and CNS neurons. Centromeres move apart at the metaphase-anaphase transition and Cyclin B is degraded, but sister chromatids remain connected, resulting in chromatin bridging. This phenotype is similar to that described in TOP2 mutants in yeast. Barren protein localizes to chromatin throughout mitosis. Colocalization and biochemical experiments indicate that Barren associates with Topoisomerase II throughout mitosis and alters the activity of Topoisomerase II. It is proposed that this association is required for proper chromosomal segregation by facilitating the decatenation of chromatids at anaphase (Bhat, 1996).
Search PubMed for articles about Drosophila Barren
Aono, N., et al. (2002). Cnd2 has dual roles in mitotic condensation and interphase. Nature 417: 197-202. PubMed ID: 12000964
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: 632-645. PubMed ID: 11854418
Bhat, M. A., et al. (1996). Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 87: 1103-1114. 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: 4715-4726. PubMed ID: 14600258
Cobbe, N. and Heck, M. M. S. (2004). The evolution of SMC proteins: phylogenetic analysis and structural implications. Mol. Biol. Evol. 21: 332-347. PubMed ID: 14660695
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. PubMed ID: 14600262
Cuvier, O. and Hirano, T. (2003). A role of Topoisomerase II in linking DNA replication to chromosome condensation. J. Cell Biol. 160: 645-655. PubMed ID: 12604590
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: 895-906. PubMed ID: 15514062
Giet, R. and Glover, D. M. (2001). Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152(4): 669-82. PubMed ID: 11266459
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: 729-742. PubMed ID: 11914278
Hagstrom, K. A. and Meyer, B. J. (2003). Condensin and cohesin: more than chromosome compactor and glue. Nat. Rev. Genet. 4: 520-534. PubMed ID: 12838344
Hudson, D. F., et al. (2003). Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev. Cell 5: 323-336. PubMed ID: 12919682
Jager, H., M. Rauch, and S. Heidmann. 2005. The Drosophila melanogaster condensin subunit Cap-G interacts with the centromere-specific histone H3 variant CID. Chromosoma 113: 350-361. PubMed ID: 15592865
Kimura, K. and Hirano, T. (2000). Dual roles of the 11S regulatory subcomplex in condensin functions. Proc. Natl. Acad. Sci. 97: 11972-11977. PubMed ID: 11027308
Lavoie B. D., et al. (2000). Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren. Mol. Biol. Cell 11: 1293-1304. 10749930
Lupo, R., et al. (2001). Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and maintain Fab-7 PRE silencing. Mol. Cell 7(1): 127-136. 11172718
Maeshima, K. and Laemmli, U. K. (2003). A two-step scaffolding model for mitotic chromosome assembly. Dev. Cell 4: 467-480. PubMed ID: 12689587
Oliveira, R. A., Coelho, P. A. and Sunkel, C. E. (2005). The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis. Mol. Cell Biol. 25(20): 8971-84. PubMed ID: 16199875
Oliveira, R. A., Heidmann, S. and Sunkel, C. E. (2007). Condensin I binds chromatin early in prophase and displays a highly dynamic association with Drosophila mitotic chromosomes. Chromosoma 116(3): 259-74. PubMed ID: 17318635
Ono, T., Losada, A., Hirano, M., Myers, M. P., Neuwald, A. F. and Hirano, T. (2003). Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115: 109-121. PubMed ID: 14532007
Ono, T., Fang, Y., Spector, D. L. and Hirano, T. (2004). Spatial and temporal regulation of condensins I and II in mitotic chromosome assembly in human cells. Mol. Cell. Biol. 15: 3296-3308. PubMed ID: 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: 1305-1313. 10749931
Pek, J. W. and Kai, T. (2010). A role for Vasa in regulating mitotic chromosome condensation in Drosophila. Curr, Biol. 21: 39-44. PubMed ID: 21185189
Pek, J. W. and Kai, T. (2011). DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation. Proc Natl Acad Sci U S A 108(29): 12007-12012. PubMed ID: 21730191
Savvidou, E., Cobbe, N., Steffensen, S., Cotterill, S. and Heck, M. M. (2005). Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids. J. Cell Sci. 118(Pt 11): 2529-43. PubMed ID: 15923665
Somma, M. P., Fasulo, B., Siriaco, G. and Cenci, G. (2003). Chromosome condensation defects in barren RNA-interfered Drosophila cells. Genetics 165(3): 1607-11. PubMed ID: 14668407
Steffensen, S., Coelho, P. A., Cobbe, N., Vass, S., Costa, M., Hassan, B., Prokopenko, S. N., Bellen, H., Heck, M. M. S. and Sunkel, C. E. (2001). A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis. Curr. Biol. 11: 295-307. PubMed ID: 11267866
date revised: 15 October 2011
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