Article |
2 Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution of Washington, Baltimore, MD 21210
Address correspondence to Brigitte D. Lavoie, Department of Medical Genetics and Microbiology, University of Toronto, Medical Sciences Building, Room 4278, Toronto, ON M5S 1A8, Canada. Tel.: (416) 978-6123. Fax: (416) 978-6885. E-mail: brigitte.lavoie{at}utoronto.ca
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Abstract |
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Key Words: sister chromatid cohesion; mitotic chromosome structure; SMC proteins; chromosome dynamics; MCD1/SCC1
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Introduction |
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In addition to condensin, other proteins have been implicated in mitotic chromosome condensation; in particular phosphohistone H3 (phospho-H3), topoisomerase II (topo II), and proteins involved in mediating sister chromatid cohesion (Hirano, 2000). Whether they are required for condensation in all eukaryotes and their exact roles in this process remain unclear. For example, the posttranslational modification of histone H3 by phosphorylation at serine 10 has long been correlated with the onset of mitosis, and mutants in this residue show condensation defects in Tetrahymena (Wei et al., 1999). In contrast, a recent report suggests a poor correlation between H3 phosphorylation and levels of condensation (Adams et al., 2001). Similarly, in both budding yeast and Sordaria, chromosome condensation requires the cohesion machinery, which mediates pairing between sister chromatids from their replication until their separation in anaphase (Hirano, 2000; Koshland and Guacci, 2000; Nasmyth et al., 2000; Skibbens, 2000). Mutations in cohesion factors such as MCD1/SCC1, TRF4, PDS5/SPO76, and CTF18 not only perturb sister chromatid cohesion but also fail to establish and/or maintain chromosome condensation (Castano et al., 1996; Guacci et al., 1997; Hartman et al., 2000; Hanna et al., 2001). To account for these data, it has been proposed that cohesins bound to chromosomes could modulate the extent of chromosome compaction according to the density of cohesin binding sites (Guacci et al., 1997). Although the current structural data on cohesins correlate well with these models, several issues remain to be resolved. Direct interactions between the cohesins and condensins have not been described, and the two machineries neither colocalize on chromosomes nor show interdependent chromatin binding in any system (Losada et al., 1998; Toth et al., 1999). Furthermore, cohesin-depleted extracts support condensation in vitro (Losada et al., 1998; Sonoda et al., 2001). Thus the question arises, are the in vivo roles of cohesins and condensins in condensation mechanistically linked?
As a first step toward understanding the in vivo mechanism of mitotic chromosome folding, we have used a comparative approach to probe known and putative components of the condensation apparatus using conditional alleles that allow rapid protein inactivation and therefore minimize secondary consequences of chromosome transmission defects. We define essential parameters of in vivo condensation in Saccharomyces cerevisiae and discuss the implications of our in vivo data in relation to in vitro models.
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Results |
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Protein requirements for chromosome condensation
To ascertain whether YCG1 and YCS4 are required for the establishment of chromosome condensation, fluorescence in situ hybridization (FISH) was performed. Previous studies have shown that in G1 cells, euchromatic probes are maximally separated and the rDNA adopts a disordered, puffed morphology (Fig. 2 A, cdc28-1; Guacci et al., 1994). In contrast, in early M phase cells (Nz arrest), the distance between euchromatic probes is minimized, and the rDNA adopts a distinct looped or line-like structure (Fig. 2 A, Nz; Guacci et al., 1994). The rDNA serves as an excellent reporter for the condensation state of chromosomes because it reflects the action of condensin at both repetitive and unique sites (Guacci et al., 1994, 1997; Strunnikov et al., 1995; Freeman et al., 2000; Hartman et al., 2000; Lavoie et al., 2000). To inactivate Ycg1p and Ycs4p function over a window of the cell cycle when chromosome condensation is established and maintained, ycg1-1, ycg1-2, and ycs4-2 strains were synchronized in G1, shifted to 37°C, released into Nz-containing medium to rearrest in mitosis, and then processed for FISH. Under these conditions, all three mutants exhibited G1-like rDNA rather than the looped structures characteristic of M phase (Fig. 2). Thus, Ycs4p and Ycg1p, like Brn1p and Smc2p, are required for the establishment of condensation.
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As chromatin spreads have not been extensively used to assess condensin binding, we initially characterized Smc4HA binding in WT cells. Smc4p staining was detected on chromosomes at all times during the cell cycle (Fig. 4 A), and in >95% of asynchronously growing cells (unpublished data). In addition, the Smc4p signal appeared concentrated over the rDNA locus, which stains poorly by DAPI (Fig. 4 A). This localization likely reflects that of the condensin complex, because a similar pattern is observed for both Ycs4MYC12p and Ycg1HA3p (unpublished data), and the complex is known to exist in the nucleus throughout the cell cycle (Freeman et al., 2000). In addition, recent in vivo studies have shown a nucleolar enrichment of GFP-tagged condensin (Freeman et al., 2000).
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Chromosome condensation is reversible in a single mitosis
The transient inactivation of the non-SMCs just before chromosome segregation leads to a loss of condensin chromatin binding and chromosome decondensation. However, when the cells are returned to the permissive temperature, cell viability remains high (Fig. 5 A). This apparent contradiction can be resolved if condensins rapidly regain activity upon return to the permissive temperature and recondense their chromosomes before chromosome segregation ensues. To test this possibility, BRN1 and brn1-9 cultures were synchronized in M phase at 23°C to allow condensation to occur. As shown in Fig. 5 B, both BRN1 and brn1-9 cells show similar levels of condensation at the permissive temperature. Upon a shift to 37°C, condensation was disrupted in the brn1-9 mutant and, significantly, was efficiently regained within 1 h of returning to the permissive temperature. This recondensation must be biologically relevant, because the viability of cells after transient inactivation in M phase is high. Furthermore, the fact that condensin can promote a second round of condensation suggests that neither the chromosomes nor the condensins are irreversibly modified (or inactivated) during condensation. Thus, the activity of condensin is maintained at least until the metaphase checkpoint.
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Although these data argue against an activator role for cohesin, they are consistent with a regulatory function. In this scenario, the inability of chromosomes to properly condense in cohesin mutants results from the malfunction of condensin. To demonstrate that the irreversible state observed in the mcd1-1 strain was dependent on condensin activity, we generated double mutants. Both condensin and cohesin function were inactivated from G1 to M phase in brn1-9 mcd1-1 and ycg1-2 mcd1-1 strains. Upon return to the permissive temperature in M phase, the double mutants showed a dramatic restoration of condensation, indicating that chromosomes remain good substrates for folding when both condensin and cohesin are inactivated (Fig. 8 A). Furthermore, in contrast to that seen in the mcd1-1 strain, the expression of functional Mcd1p before the temperature downshift stimulated the level of condensation in the brn1-9 mcd1-1 strain, suggesting that cohesin action must precede that of condensin, as would normally occur in vivo. One explanation for the restoration of condensation in the double mutants versus the mcd1-1 mutant is that condensin mutants suppress sister chromatid cohesion defects. To assess this directly, we performed FISH using a CEN16 proximal probe to monitor the extent of precocious separation of sisters in both brn1-9 mcd1-1 and ycg1-2 mcd1-1 cells, after the temperature downshift (Fig. 8 B). In both cases, two spots were detected in >50% of the nuclei, indicating a precocious dissociation of sister chromatids that was not restored after the return to the permissive temperature. Thus, the failure of chromosome condensation in an mcd1-1 mutant is unlikely to result from the irreversible loss of sister chromatid cohesion, because sister chromatid pairing per se does not appear essential for chromosome condensation.
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Discussion |
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The characterization of multiple condensin subunits provided a basis to analyze the contribution of other potential condensation factors such as phospho-H3 and topo II. In fact, in budding yeast, neither of these proteins had been directly tested for a role in condensation, and we now show that in contrast to condensins, neither phospho-H3 nor topo II activity are required for condensation (this study). These results are consistent with the robust viability of histone H3 mutants in budding yeast (Hsu et al., 2000). In addition, studies in Drosophila found a poor correlation between H3 phosphorylation and chromosome condensation, suggesting that the link between phospho-H3 and condensation is not absolute (Adams et al., 2001). Similarly, we show that topo II activity is not required for condensation in budding yeast, however this contrasts with previous work in S. pombe (Uemura et al., 1987). One possible explanation for this difference is that condensation of the rDNA fundamentally differs from that at euchromatic sites. This seems unlikely because our work, and that of others, has shown that condensin function is closely mirrored between unique and repetitive DNA regions. In addition, the rDNA is a bona fide in vivo substrate of condensin, because both its morphology and transmission are condensin dependent (this study; Freeman et al., 2000; Lavoie et al., 2000; Ouspenski et al., 2000). Alternatively, the observed rDNA condensation in the topo II mutant could reflect the relatively small size of budding yeast chromosomes. Although a precise role for topo II in higher order chromosome folding remains controversial (Adachi et al., 1991; Hirano and Mitchinson, 1993), it is noteworthy that our results concur with recent in vivo studies of mammalian chromosome condensation in the presence of topo II inhibitors (Andreassen et al., 1997). Taken together, these data fail to provide compelling evidence that either phospho-H3 or topo II activity are ubiquitous components of the in vivo condensation machinery, and indicate that condensin remains the only functionally conserved component known to date.
Reversibility of chromosome condensation in mitosis
Using a reversible condensin mutant, we demonstrate that condensation can occur twice in a single mitosis. Therefore, the process of condensation itself does not lead to an irreversible change in either chromosome structure or the condensin complex. This recondensation is not specific to the brn1-9 allele, because the ycg1-2 mutant also supports efficient chromosome refolding. Furthermore, the restoration of viability indicates that this recondensation is both morphologically and functionally correct. The condensation state of chromosomes can be modulated in mitosis through the activity of the non-SMC subunits, but, could the cell exploit this potential in a more physiological context? One possibility exists in vertebrate cells that initiate the decondensation of chromosomes and a reversal of the cell cycle after either irradiation in early prophase (Rieder and Cole, 1998) or a treatment with microtubule-depolymerizing drugs (Rieder and Cole, 2000). Our data suggest that the mechanism by which this decondensation and subsequent refolding occurs could involve changes in the activity of the non-SMCs. Consistent with this, cdc2 kinase is down-regulated as early prophase nuclei revert into interphase nuclei (Rieder and Cole, 1998), and this kinase controls the chromatin association of Xenopus condensin through phosphorylation of the non-SMC components (Kimura et al., 1998, 2001). It will therefore prove interesting to determine the phosphorylation state of condensins in irradiated prophase cells.
Role of cohesins in condensation
In striking contrast to the reversibility of condensation in condensin mutants, an irreversible uncondensed rDNA state is produced in the absence of cohesin. This uncondensed rDNA can be distinguished both structurally and functionally from that produced in condensin mutants; in the absence of functional Mcd1p, condensin binds chromatin yet fails to promote condensation. We infer that condensin is active and misregulated because the irreversible, presumably misfolded, chromosome structure generated in the absence of cohesin is condensin dependent.
These data provide the first evidence of a mechanistic link between condensins and cohesins; therefore, these proteins are not acting in independent pathways. How then, do cohesins regulate chromosome condensation? As higher order chromosome structure is highly reproducible, it is likely to be orchestrated through cis determinants. These determinants could in principle be provided by site-specific binding of condensins. In fact, such sites have been suggested by a previous study (Freeman et al., 2000), however, we have been unable to reproduce these results using multiple tagged subunits (unpublished data). Alternatively, cohesin-defined domains could dictate condensin distribution along chromosomes (Guacci et al., 1997; Hartman et al., 2000). Cohesins are known to bind chromosomes regularly, roughly every 10 kb (Blat and Kleckner, 1999; Megee et al., 1999; Tanaka et al., 1999; Laloraya et al., 2000), and this coincides with biochemical estimates of condensin (1 complex per 810 kb in S. pombe; Sutani and Yanagida, 1997). Cohesins could then act in a manner reminiscent of boundary elements, restricting condensin activity to defined domains and consequently imposing a regular array of loops. Indeed, the cohesin subunit Smc1 has been implicated in boundary element activity at the MAT mating type locus (Donze et al., 1999). In the absence of such "stops," condensin function would not be confined to regions of chromatin, and this deregulation would preclude the normal higher order chromosome structure (Fig. 9).
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A second attractive feature of this model is the prediction that condensin function is regulated by cis determinants on chromosomes. Whether cohesins delimit domains of condensin action in all eukaryotes remains to be demonstrated. Current in vitro and in vivo studies suggest that in higher eukaryotes, the loss of cohesins does not abrogate chromosome condensation (Losada et al., 1998; Sonoda et al., 2001). Consistent with this, and in contrast to budding yeast, bulk cohesins are removed from chromosomes in prophase (Losada et al., 1998; 2000; Waizenegger et al., 2000), suggesting that low levels of cohesins are sufficient to promote condensation and/or additional regulatory factors are required in these systems. Indeed, the demarcation of condensation domains could be provided by other boundary-like elements such as AT-rich sequences, which have been proposed to act as cis determinants for higher order chromosome structure (Hart and Laemmli, 1998). Alternatively, condensin could modulate only a portion of the total mitotic chromosome compaction observed in higher eukaryotes, such that its loss or deregulation in the absence of cohesins would not eliminate condensation. This idea is consistent with in vivo data where a loss of condensin in flies causes partial defects in condensation (Bhat et al., 1996; Steffensen et al., 2001), and could explain why significant chromosome condensation was observed in Mcd1/Scc1depleted TD40 cells (Sonoda et al., 2001). It does not, however, account for the dramatic loss of chromosome condensation in condensin-depleted extracts in vitro (Hirano et al., 1997), and further experiments will be required to resolve these issues. Indeed, because direct comparisons of condensin versus cohesin knockouts have as yet only been done in vivo in budding yeast, the extension of this approach to other organisms should prove helpful in determining the roles of cohesins and condensins in higher order chromosome dynamics.
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Materials and methods |
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Yeast strains and plasmids
Yeast strains are in Table I and were constructed using standard techniques (Guthrie and Fink, 1991). YCG1 and YCS4 genes were cloned in pRS316 (Sikorski and Hieter, 1989) or YCplac33 (Gietz and Sugino, 1988) by gap repair from YPH501-generating pBL235 and pZF1. Because of a sequence error in Saccharomyces Genome Database (missing G at position 1117105), the initiating methionine for Ycg1p is misidentified, predicting a protein that has a 16amino acid NH2-terminal extension. The actual start codon is at 1117118. Both ycg1::KAN and ycs4::KAN knockouts were generated by single step gene replacement in YPH501, selecting for growth on 150 µg/ml G418. Diploids were sporulated and 20 tetrads were dissected. 2:2 segregation confirmed that both YCG1 and YCS4 are essential (no surviving spores were G418R).
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To generate SMC4HA3, single step gene replacement used the S. pombe his5+ gene to complement ura3 deficiency in YPH501. The 3xHAhis5+ was amplified from p473 with 5' and 3' sequences corresponding to SMC4. Integration into the correct site was verified by colony PCR and Western blotting. After sporulation and dissection, 18/20 tetrads showed 4:0 viability and 2:2 segregation of the tagged allele. Galactose-inducible Mcd1p strains were generated by integration of NcoI-linearized pPCM87 (URA3: GAL1-MCD1-HA6; a gift from Paul C. Megee, University of Colorado, Denver, CO) into 985-7c and BLY22. The JHY90, JHY91, and JHY93 strains were gifts from J.Y.A. Hsu and M.M. Smith (University of Virginia, Charlottesville, VA).
Cell growth and viability
Permissive and restrictive temperatures were 23°C and 37°C, respectively. For viability assays, strains were grown in YPD at 23°C, synchronized in M phase with 20 µg/ml Nz for 3 h, shifted to the restrictive temperature for 1 h, sonicated, counted, and plated. Percent viability was scored as the number of colony forming units divided by the number of cells plated (x100). For the M/G1 viability experiment, an aliquot from the M phasearrested culture was taken and released from the Nz block by washing the cells three times in warmed YPD. The cells were then resuspended in their original volume of warmed YPD, containing -factor (10-8 M), and incubated for 1 h at 37°C until >85% of the cells had a schmoo morphology. The cells were then sonicated, counted, and plated.
Cell cycle synchronization
Cultures were first synchronized in G1 (10-8 M -factor for bar1 strains vs. 3 x 10-5 M for BAR1) for 2.5 h at 23°C (8595% schmoos), shifted to 37°C for 0.5 h, released by washing the cells three times with 37°C YPD containing 0.1 mg/ml pronase E (Sigma-Aldrich), and incubated for 2.5 h in 20 µg/ml Nz (37°C), as described in Lavoie et al. (2000). For indirect immunofluorescence (IIF) or FISH, cells were fixed with 0.36% formaldehyde for 1.5 h. Arrests were verified microscopically by cell and nuclear morphology and confirmed by FACS®. Rescues of condensation experiments were performed similarly except that cells were shifted back to 23°C for 1 h after only 2 h at 37°C, to maintain the arrest. Maintenance experiments were performed by synchronizing cells in G1, releasing into Nz at 23°C for 2 h, and then shifting to 37°C for 1 h. All mutants possessed >85% rDNA loops at 23°C. S phase arrests were performed by adding HU to cells (0.2 M final concentration) for 3 h at 23°C.
IIF, chromatin spreads, and FISH
IIF of tubulin was performed using 1:500 Yol1/34 rat mAb-tubulin (Serotec) and 1:500 antirat-FITC (Sigma-Aldrich) as described in Lavoie et al. (2000). After G1 arrest, cultures were shifted to 37°C, released, and then followed microscopically until a maximal level of anaphase cells was reached (80100 min after release). Ycg1-1, ycg1-2, and ycs4-2 cells were then fixed and processed for IIF of tubulin and DAPI staining of chromosomes. Chromatin spreads were performed as previously described (Michaelis et al., 1997). Both mouse anti-HA and goat antimouse-FITC antibodies used for spreads were at 1:500. DNA was stained with DAPI. Images were obtained using a ZEISS epifluorescence microscope, and were recorded digitally with the use of a Princeton Scientific Instruments charge-coupled device with Scanalytics processing software, which allows image superimposition. To allow direct comparisons of signal between different chromatin spreads, identical exposures (1 s) were taken and background levels of the images were adjusted to the same range using the Scanalytics software. Images shown reflect what was observed directly through the microscope. Longer exposures (3 s) enhanced background signal in all of the samples including the untagged control (unpublished data). FISH was performed as previously described (Guacci et al., 1994; Lavoie et al., 2000) using digoxigenin-labeled rDNA or CEN16 proximal probes and FITC-conjugated secondary and tertiary antibodies. Chromosomes were counterstained with propidium iodide in antifade mounting medium (Intergen).
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Footnotes |
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Acknowledgments |
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D. Koshland is funded by the Howard Hughes Medical Institute.
Submitted: 18 September 2001
Revised: 18 January 2002
Accepted: 20 January 2002
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References |
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