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Address correspondence to Tatsuya Hirano, Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: (516) 367-8370. Fax: (516) 367-8815. E-mail: hirano{at}cshl.org
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Abstract |
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Key Words: Xenopus egg extracts; condensin; decatenation; compaction; SMC
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Introduction |
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Topoisomerase II (topo II), which catalyzes a transient breakage and reunion of double-stranded DNA, was the first protein shown to be essential for mitotic chromosome condensation (Uemura et al., 1987). A role for topo II in the structural maintenance of mitotic chromosomes has been suggested on the basis of the finding that topo II is a major constituent of the chromosome scaffold (Earnshaw et al., 1985; Gasser et al., 1986). A study using a Xenopus egg cell-free extract demonstrated a stoichiometric contribution of topo II to chromosome assembly (Adachi et al., 1991). However, the exact role of topo II in chromosome organization remains controversial because different approaches failed to detect a stable association of topo II with mitotic chromosomes (Hirano and Mitchison, 1993; Swedlow et al., 1993). Moreover, recent studies have shown that the association of topo II with chromosomes in living cells appears to be more dynamic than predicted before (Christensen et al., 2002; Tavormina et al., 2002).
One prominent phenotype of condensin mutants is a defect in chromosome segregation in anaphase (Saka et al., 1994; Strunnikov et al., 1995; Sutani et al., 1999; Steffensen et al., 2001; Bhalla et al., 2002; Hagstrom et al., 2002). This is reminiscent of (if not identical to) the phenotype observed in topo II mutants. On the basis of these observations and other mechanistic considerations, it has been proposed that one of the important roles of chromosome condensation is to assist topo IImediated decatenation of sister chromatids (Koshland and Strunnikov, 1996; Hirano, 2000; Holmes and Cozzarelli, 2000). Consistent with this notion, it was reported that a regulatory subunit of condensin (Barren) interacts directly with topo II in Drosophila (Bhat et al., 1996), and that a different subunit (YCS4p) is required for the binding of topo II to chromatin in Saccharomyces cerevisiae (Bhalla et al., 2002). However, other studies did not detect a direct interaction between the two proteins or their interdependent loading onto chromosomes (Hirano et al., 1997; Lavoie et al., 2000). Thus, it remains to be established exactly how condensin and topo II might cooperate to support chromosome condensation and segregation.
In this work, we have used Xenopus egg extracts to study the functional interactions between condensin and topo II in mitotic chromosome condensation. We find that the presence or absence of DNA replication affects the modes of action of topo II in chromosome condensation during subsequent mitosis. Only when mitosis is induced after DNA replication, topo II becomes concentrated on an axial structure that can be visualized in the absence of condensin. This subchromosomal structure can then be converted into mitotic chromosomes by subsequent action of condensin. We propose that topo II has a previously uncharacterized role in linking DNA replication to mitotic chromosome condensation.
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Results |
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To test whether condensin might affect topo II activities in the extracts, we used two different assays. In the first one, the topo IIspecific inhibitor VM-26 was added to measure topo IImediated cleavage of plasmid DNA (Fig. 1 D, left), and in the second, kinetoplast DNA was used to measure the decatenation activity of topo II (Fig. 1 D, middle). We found that neither of the two activities was affected in the absence of condensin, although they were barely detectable when topo II was depleted from the extracts. Topo IImediated DNA cleavage was also tested in the context of sperm chromatin (Fig. 1 D, right). Again, it was not affected by depletion of condensin. These results show that condensin and topo II have distinct roles in chromosome assembly in the cell-free extracts.
Topo II and condensin must function simultaneously when chromosome assembly is induced from unreplicated substrates
Next, we tested whether condensin and topo II must function simultaneously in mitotic chromosome assembly or whether their actions are temporarily separable. To this end, we set up a stepwise protocol for chromosome assembly. Sperm chromatin was first incubated with condensin-depleted interphase HSS, and then with mitotic HSS that had also been depleted of condensin. Finally, condensin was supplemented by adding topo IIdepleted mitotic HSS into the assembly mixture (Fig. 2
A, protocol 1). Rod-shaped chromosomes were successfully assembled under this condition (Fig. 2 B, ac). Because HSS supports no DNA replication (Fig. 2 B, d), the assembled chromosomes were composed of single chromatids. In contrast, when the topo II inhibitor VM-26 was added before allowing condensin to function (Fig. 2 A, protocol 2), chromosome assembly was severely impaired and no individual chromosome was observed (Fig. 2 B, eg). These results suggest that topo II and condensin must work simultaneously to assemble mitotic chromosomes under this condition, although the two proteins play distinct roles in this process.
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Active condensin converts the topo IIcontaining axes into chromosome-like structures
We reasoned that the topo IIcontaining axes might function as templates for the subsequent action of condensin in LSS. To investigate the functional interaction between topo II and condensin more closely, we took advantage of the use of AMP-PNP, a nonhydrolyzable analogue of ATP that allows condensin to bind to DNA, but prevents its DNA supercoiling/knotting activity in vitro (Kimura and Hirano, 1997; Kimura et al., 1999; Bazzett-Jones et al., 2002). The standard chromosome assembly protocol was used in LSS, except that AMP-PNP was added together with condensin at the final step (Fig. 5
A, protocol 2). The diffuse localization of topo II in interphase chromatin (Fig. 5 B b, protocol 1) was converted into axial structures on entry into mitosis. The topo IIcontaining structure remained intact after addition of VM-26 and condensin in the presence of AMP-PNP (Fig. 5 B, f). Under this condition, no individual chromosomes were assembled (Fig. 5 B, e), most likely because AMP-PNP inhibited the action of condensin. Interestingly, condensin was found to largely colocalize with topo II within the uncondensed chromatin (Fig. 5 B, g and h), suggesting that condensin may have high affinity for the topo IIenriched axes. Close inspection of the structure showed that DNA extended out of the topo IIcontaining axes in the presence of AMP-PNP (Fig. 5 C, d). When AMP-PNP was replaced with ATP at the final stage of incubation (Fig. 5 A, protocol 3), the fuzzy chromatin mass was converted into well-individualized chromosomes (Fig. 5 C, e). The topo II signal was converted from thin axes to chromosome-like structures (Fig. 5 C, f), and largely overlapped with the DAPI-stained areas (Fig. 5 C, g and h). These results suggest that, once the topo IIcontaining axes are formed in a DNA replicationdependent manner, subsequent addition of condensin is sufficient to assemble chromosomes in the absence of active topo II.
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Discussion |
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Previous studies showed that topo II is tightly associated with metaphase chromosomes isolated from somatic cells, leading to the proposal that topo II plays a role in the structural maintenance of chromosomes as an integral component of the scaffold (Earnshaw et al., 1985; Gasser et al., 1986). In striking contrast, topo II was found to be easily extracted from single-chromatid chromosomes assembled in HSS without changing their overall structure (Hirano and Mitchison, 1993). The current results show that the presence or absence of preceding DNA replication affects the distribution and salt-extractability of topo II on chromosomes, providing at least a partial explanation for the discrepancy between the previous studies. Our conclusion is further supported by the observation that preventing DNA replication with aphidicolin in LSS abolishes the tight association of topo II with the chromosomes. Because topo II displays highly dynamic behavior in vivo during both interphase and mitosis (Swedlow et al., 1993; Christensen et al., 2002; Tavormina et al., 2002), we favor the idea that topo II function as part of a complex molecular assembly that undergoes a dynamic rearrangement during the cell cycle and links interphase chromatin organization to mitotic chromosome architecture. This is not necessarily inconsistent with the model that topo II may play a role in forming chromosome loops by binding to scaffold-attachment regions in an early stage of chromosome assembly (for review see Hart and Laemmli, 1998). However, our current results do not address the question of whether topo II function may continuously be required for the structural maintenance of chromosomes after their assembly is complete.
Topo II links DNA replication to chromosome condensation
Chromosome condensation is not a simple compaction process of a linear DNA molecule, and it requires the relief of a number of physical constraints that would otherwise impede compaction (Hirano, 2000). It has been proposed that the DNA decatenation activity of topo II is required to untangle catenations between different chromosomes (nonreplicative catenanes) and between sister chromatids (replicative catenanes) before the completion of mitosis (Downes et al., 1994; Gimenez-Abian et al., 2000). Although topo II activity is not required for DNA replication, per se, it acts behind the replication fork and unlinks replicating DNA by removing precatenates in Xenopus egg extracts (Lucas et al., 2001). This unlinking process does not require entry into mitosis, at least in the case of small circular DNA substrates. It is tempting to speculate that the close association of topo II with the fork movement may prime the assembly of a subchromosomal structure during S-phase, which can only be visualized on mitotic entry. Such a replication-dependent reorganization of topo II distribution might also facilitate the removal of nonreplicative catenanes. Our results show that topo II plays a crucial role in linking DNA replication to mitotic chromosome condensation. Several recent studies also start to reveal this previously understated connection between the two important chromosomal events (for review see Pflumm, 2002). For example, several origin recognition complex mutants in Drosophila produce shorter and thicker chromosomes (Pflumm and Botchan, 2001), or irregularly condensed chromosomes with the severest defect in late-replicating regions (Loupart et al., 2000). It has been hypothesized that the correct timing of DNA replication or the density of functional replication origin might affect the assembly of chromosomes during subsequent mitosis. It would be interesting to test whether mutations in replication factors may perturb proper distribution of topo II during DNA replication and thereby lead to condensation defects.
The interplay between topo II and condensin
How do topo II and condensin functionally interact with each other to assemble chromosomes? Unlike topo II, which binds to chromatin throughout the cell cycle, the binding of condensin to chromosomes is mitosis-specific in the cell-free extracts (Hirano et al., 1997; Losada et al., 1998). Our current data clearly show that topo II and condensin are both required for chromosome assembly in the presence or absence of DNA replication. However, after DNA replication, topo IIcontaining axial structure is observed in the absence of condensin, and this structure can be converted into a metaphase chromosome-like structure by subsequent addition of condensin. One potential mechanism might be that the targeted condensin drives DNA supercoiling, which in turn facilitates topo IImediated decantation of the nonreplicative and replicative catenanes, and thereby allows compaction of each chromatid. It should be noted that the actions of the two proteins in chromosome assembly are tightly linked under normal conditions. The topo IIcontaining axes reported in the current paper would not be visualized when condensin is present.
A number of genetic studies from different model organisms clearly show that condensin function is required for proper chromosome condensation and segregation in vivo (Saka et al., 1994; Strunnikov et al., 1995; Sutani et al., 1999; Ouspenski et al., 2000; Bhalla et al., 2002; Hagstrom et al., 2002). However, it remains to be determined exactly how condensin contributes to the assembly of metaphase chromosomes at a mechanistic level. For example, the Drosophila SMC4 mutants produce very fat chromosomes with unresolved sister chromatids, suggesting that condensin function may be required for sister chromatid resolution, but not for axial compaction (Steffensen et al., 2001). When SMC-4 is depleted by RNA interference in Caenorhabditis elegans embryos, the most drastic condensation defect is observed in prometaphase, but not in metaphase (as judged by metaphase plate formation; Hagstrom et al., 2002; Kaitna et al., 2002). Additional factors, including topo II, are likely to contribute to a certain level of compaction. It would be informative to carefully compare and contrast the defective phenotypes associated with condensin and topo II mutants in different organisms. It would also be important to note that the relative contribution of condensin and other factors to chromosome architecture might vary depending on the size of chromosomes, assembly pathways (with or without preceding DNA replication), or developmental stages. For instance, in organisms that contain short chromosomes, like S. cerevisiae, chromosome compaction requires condensin, but apparently not topo II (Lavoie et al., 2002). Future studies should integrate information from biochemistry, genetics, and structural analysis of chromosomes to fully understand the interplay of condensin and topo II in chromosome assembly at a mechanistic level.
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Materials and methods |
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Immunodepletions
Immunodepletions of topo II and condensin were performed as described previously (Hirano and Mitchison, 1993; Hirano et al., 1997), with minor modifications. For condensin depletion, a mixture of affinity-purified anti-XCAP-C, -E, and -G (6 µg each) and anti-XCAP-D2 (12 µg) was incubated with 30 µl Affi-Prep® Protein A support (Bio-Rad Laboratories) for 1 h. For topo II depletion, 30 µl of anti-topo II serum was loaded onto 30 µl beads. For mock depletion, 30 µg control IgG or 30 µl nonimmune rabbit antiserum was used. After washing the antibody-coupled beads with XBE2 (10 mM K-Hepes, pH 7.7, 100 mM KCl, 2 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, and 50 mM sucrose), 100 µl HSS or LSS supplemented with an ATP-regenerating system (Hirano and Mitchison, 1993) was added and incubated on ice for 1 h with occasional mixing. The supernatants were recovered by two rounds of brief spins and used as freshly depleted extracts. For immunodepletion of topo II, two successive rounds of incubation were performed.
Chromosome assembly in Xenopus egg extracts
Chromosomes were assembled from sperm chromatin in HSS or LSS as described previously (Hirano et al., 1997; Losada et al., 1998), except that topo II- or condensin-depleted extracts were used as indicated. To monitor the efficiency of DNA replication, biotin-16-dUTP (Boehringer) was added into interphase extracts at a final concentration of 10 µM. For DNA replication inhibition, 100 µg/ml aphidicolin or 1.5 µg/ml of nondegradable recombinant geminin (McGarry and Kirschner, 1998) was added to the extracts. Mitosis was induced by adding 1/2 volume of mitotic extracts or purified recombinant cyclin B90 (Solomon et al., 1990). When necessary, VM-26 (4'-demethylepipodophyllotoxin thenylidene-ß-D-glucoside; a gift from Brystol-Myers Squibb Company) was added at a final concentration of 10 µM. AMP-PNP or ATP was added to 1 mM when indicated.
Analyses of chromosomes assembled in vitro
Chromosome assembly reactions were performed as described previously (Hirano et al., 1997; Losada et al., 1998), by incubating sperm chromatin with HSS or LSS at 22°C for 2 h. Salt extraction of chromosomes was done as described previously (Hirano and Mitchison, 1993), by adding 1/10 reaction volume of buffer containing 10x NaCl into assembly reactions. For biochemical analysis of chromosomal proteins (Fig. 1 A), the reaction mixtures were loaded onto a 30% sucrose cushion and spun at 10,000 rpm for 15 min (Hirano and Mitchison, 1994). For morphological analysis (Figs. 16), the assembled chromosomes were fixed and recovered onto coverslips by centrifugation through a 30% glycerol cushion, as described previously (Hirano and Mitchison, 1991), with minor modifications. 10 µl assembly mixture was fixed by the addition of 50 µl 0.8% formaldehyde and incubated at RT for 10 min. For sedimentation of salt-treated chromosomes or chromatin assembled in condensin-depleted extracts, a 10% (instead of 30%) glycerol cushion was used. The coverslips were washed with TBS containing 0.1% Triton X-100 (TBST) for 10 min, and were blocked with TBS containing 2% BSA (TBS-BSA) at 22°C for 1 h in a humid chamber. Samples were then incubated with primary antibodies diluted in TBS-BSA at 4°C overnight. The antibodies used were anti-topo II (Hirano and Mitchison, 1993), anti-cohesin (XSMC3; Losada et al., 1998), and anti-condensin (Hirano et al., 1997). For double immunostaining, a combination of mouse anti-condensin (XCAP-G) and rabbit anti-topo II antibodies was used. After washing with TBST, fluorescein- or rhodamine-labeled goat antirabbit IgG or donkey antimouse antibodies (Jackson ImmunoResearch Laboratories) were applied at 1:100 dilution for 1 h at 22°C, followed by TBST washes and incubation with 10 µg/ml fluorescein-conjugated avidin D for 30 min. After washing with TBST and DAPI counterstaining, samples were mounted in medium (Vectashield®) for microscopy.
Other assays
Topo IImediated DNA cleavage and kinetoplast DNA decatenation assays were done as described previously (Hirano and Mitchison, 1991, 1993).
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Footnotes |
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health (to T. Hirano) and by fellowships from the Cold Spring Harbor Laboratory Association and the Human Frontier Science Program (to O. Cuvier).
Submitted: 5 September 2002
Revised: 15 January 2003
Accepted: 15 January 2003
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