Function of Barren, a regulatory component of the Gluon containing condensin complex

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).

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).

Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis

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 Rad21, 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 ).

Thus 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. 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, fully supporting the previous results (Coelho, 2003).

Although, chromosomes do not condense normally in DmSMC4-depleted cells, genetic studies in Drosophila show that loss of DmSMC4 or Barren 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 and in C. elegans condensin activity is required for the normal orientation of the centromere towards the mitotic spindle. Depletion of DmSMC4 has been demonstrated in this study to not affect the localisation of centromere nor kinetochore proteins and microtubules were shown to associate with kinetochores. Furthermore, 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. 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. In Drosophila S2 cells condensins associate with chromatin at prophase localising to the axis of sister chromatids throughout mitosis. 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 IIalpah in HeLa cells, hBarren appears to bind chromatin only during prometaphase while Topo IIalpha is present from prophase. 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, 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. However, in chromatin assembled in Xenopus extracts, Topo II localisation to an axial structure of chromatids occurs independently of condensin. 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 in the current RNAi depletion studies DmSMC4 was not 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 IIalpha 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 an examination of whether its DNA decatenation activity was also compromised. DNA decatenation activity of the endogenous Topo II was shown to be significantly reduced when DmSMC4 is depleted. Although these results are compatible with a direct interaction between DmSMC4 and Topo II, it was not possible to detect co-immunoprecipitation indicating that the interaction might be indirect. Nevertheless, the results suggest that proper activity of the enzyme requires condensin. A more direct interaction has been seen, since it was shown that Barren interacts in a yeast two-hybrid assay with Topo II and promotes its decatenating activity. However, it has been shown that BRN1, the yeast homologue, is not required for Topo II activity in vivo. 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, 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, 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).

gluon: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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