Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
Author for correspondence (e-mail: hirano{at}cshl.edu)
Accepted 1 March 2005
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Summary |
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Key words: Sister chromatid cohesion, Chromosome assembly, RNA interference, Xenopus
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Introduction |
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Genetic screens in yeast and fungi have identified several classes of cohesion factors (Uhlmann, 2003). Among them is Pds5/BimD/Spo76, a protein conserved from yeast to human (Denison et al., 1993
; Geck et al., 1999
; van Heemst et al., 1999
). Pds5 and cohesin have been shown to interact both genetically and physically (Holt and May, 1996
; Hartman et al., 2000
; Panizza et al., 2000
; Sumara et al., 2000
; Tanaka et al., 2001
; Wang et al., 2002
). In S. cerevisiae, Pds5 is essential for viability and is required to maintain cohesion (Panizza et al., 2000
; Stead et al., 2003
) and condensation (Hartman et al., 2000
). In contrast, Pds5 is non-essential in Schizosaccharomyces pombe, and a
pds5 mutant shows cohesion defects only after prolonged arrest in G2/M (Tanaka et al., 2001
; Wang et al., 2002
). In Sordaria and Aspergillus, Spo76/BimD is important for both cohesion and condensation in mitosis and meiosis, but to a variable extent (van Heemst et al., 1999
; van Heemst et al., 2001
). Cohesion defects are also observed during meiosis in Caenorhabditis elegans elv-14/pds5 mutants whereas the mitotic phenotype is far less severe (Wang et al., 2003
). Thus, different organisms seem to have substantially different requirements for Pds5 function, and the exact nature of the observed interaction between Pds5 and cohesin remains to be determined. Moreover, very little is known about the role of Pds5 proteins in vertebrates.
Here we report the biochemical and functional characterization of two Pds5 proteins, Pds5A and Pds5B, in human tissue culture cells and Xenopus egg cell-free extracts. In HeLa cells, a reduction in the levels of Pds5A or Pds5B by RNA interference (RNAi) leads to the assembly of abnormal mitotic chromosomes with partial defects in sister chromatid cohesion. In Xenopus egg extracts depleted of Pds5A and Pds5B, cohesion along chromosome arms appears normal but the altered localization of the inner centromere protein INCENP suggests that centromeric cohesion may be loosened. Unexpectedly, these chromosomes have an unusually high level of cohesin. In the light of these results, we propose that Pds5 participates in both stabilization and destabilization of cohesin-mediated cohesion.
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Materials and Methods |
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Preparation of antibodies
Rabbit polyclonal antisera were raised against synthetic peptides corresponding to the C-terminal sequence of Xenopus Pds5A (TAKKTAQRQIDLHR), Xenopus Pds5B (VAPSTGRLRSAKKR) and human Pds5B (VSTVNVRRRSAKRERR). The antibody against Xenopus Pds5A also recognizes human Pds5A. For a detailed list of all the other antibodies used in this study, see supplementary material.
HeLa cell nuclear extracts
HeLa cell nuclear extracts were prepared in buffer B (20 mM K-HEPES, pH 8.0, 100 mM KCl, 2 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 1 mM ß-mercaptoethanol, 20% glycerol) as described previously (Losada et al., 2000). For immunoprecipitations, 2 µg affinity-purified antibody were added to 70 µl extract and incubated on ice for 2 hours. Immunoprecipitates were recovered on protein A-agarose beads (Invitrogen) and they were washed several times with buffer B before being analyzed by silver staining and immunoblotting.
siRNA-mediated depletion of cohesin and Pds5 in HeLa cells
Exponentially growing HeLa cells were transfected twice with 100 nM oligo RNA duplexes (Dharmacon) using Oligofectamine (Invitrogen) at 0 and 24 hours (Elbashir et al., 2001). Control cells were transfected with a mixture containing no siRNA. Cells were seeded on wells containing poly-L-lysine-coated coverslips at 48 hours and they were processed for immunofluorescence at 72 hours. Total cell extracts were also prepared at this time to check the extent of depletion by immunoblotting. The sequences of the sense strand of the siRNA duplexes were: Scc1-1 5'-AUACCUUCUUGCAGACUGUdTdT-3'; Scc1-2: 5'-GCACUACUACUUCUAACCUdTdT-3'; Pds5A-1, 5'-UUCUUCCUCAGGAACCCCAdTdT-3'; Pds5A-2, 5'-GCUCCAUAUACUUCCCAUGdTdT-3'; Pds5B-1, 5'-GCUCCUUACACAUCCCCUGdTdT-3'; Pds5B-2, 5'-GAGACGACUCUGAUCUUGUdTdT-3'. Although a similar set of defects was observed after transfection with Scc1-1 or Scc1-2, their frequency was slightly higher with Scc1-1, consistent with a more efficient depletion of Scc1 as judged by immunoblotting. The two oligonucleotides used for Pds5A and Pds5B depletion produced essentially similar results.
Immunofluorescence
HeLa cells grown on coverslips were fixed with 2% paraformaldehyde in PBS pH 7.4, for 15 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes at room temperature. For the experiments shown in Fig. 3B,C, cells were pre-extracted with 0.5% Triton X-100 in CSK buffer (10 mM PIPES, pH 7.0, 100 mM NaCl, 3 mM MgCl2 and 300 mM sucrose) for 5 minutes before fixation. For the staining shown in Fig. 4A, cells were incubated in 60 mM KCl at room temperature for 30 minutes before fixation. Metaphase chromosome spreads (Fig. 4C) were prepared as described (Ono et al., 2003), except that colcemid was added to the culture medium 2 hours before harvesting cells. Immunolabeling was carried out as described (Losada et al., 1998
). Double labeling with anti-CAP-H2 and Alexa 488-conjugated anti-CAP-G (Fig. 7A) or with anti-CENP-E and Alexa 488-conjugated anti-INCENP (Fig. 7C) was done as described (Ono et al., 2003
).
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Assembly of Xenopus chromatin and chromosomes
Xenopus sperm chromatin was added to interphase HSS (3000 nuclei/µl extract) or LSS (1000 nuclei/µl). After incubation at 22°C for 2 hours, the extract was converted into a mitotic state by adding half a volume of cytostatic factor (CSF)-arrested extracts. For biochemical analysis, the reaction mixtures were diluted fivefold (in the case of HSS) or tenfold (in the case of LSS) with XBE2 containing 0.25% Triton X-100, and chromatin fractions were isolated by centrifugation as described previously (Losada et al., 1998). For morphological analyses, 10 µl of the reaction mixtures were diluted with 90 µl of 2% formaldehyde in XBE2 containing 0.25% Triton X-100, spun over coverslips and processed for immunofluorescence as described above.
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Results |
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Association of Pds5A with chromatin requires functional cohesin in HeLa cells
To test the functional significance of the interactions detected between Pds5 and cohesin, we designed small interfering RNAs (siRNAs) specific to Pds5A and Pds5B, as well as the cohesin subunit Scc1. Transfection of HeLa cells with each of the siRNA duplexes substantially decreased the level of the target protein without affecting the other two, as judged by immunoblotting of whole cell extracts (Fig. 3A, three upper panels). None of the treatments affected the levels of Smc1, Smc3 or an unrelated protein such as Orc2, a subunit of the origin recognition complex (Fig. 3A, two lower panels). To examine the association of the cohesin complex with chromatin after siRNA treatment, the cells were pre-extracted with detergent, fixed and analyzed by immunofluorescence with anti-Smc3. We found that the level of chromatin-bound Smc3 was drastically reduced in Scc1 siRNA-treated cells compared with control cells. Such a reduction was not observed in cells transfected with Pds5A or Pds5B siRNAs (Fig. 3B). This result suggests that Scc1, but neither Pds5A nor Pds5B, is required for the stable association of Smc3 with chromatin. Immunofluorescent labeling of HeLa cells showed that Pds5A, like cohesin, localized to the interphase nucleus (Fig. S1 in supplementary material). As expected, the nuclear signal of Pds5A was greatly reduced in Pds5A siRNA-treated cells compared with control cells, but it was unchanged in cells treated with Pds5B siRNA (Fig. 3C). Importantly, we found that the chromatin-bound population of Pds5A was reduced in cells treated with Scc1 siRNA, indicating that a functional cohesin complex is required for stable association of Pds5A with chromatin. Although the quality of the currently available anti-Pds5B did not allow us to determine whether the association of Pds5B with chromatin also depends on cohesin, this is indeed the case in Xenopus egg extracts (see below).
Cohesion defects in HeLa cells with reduced levels of cohesin and Pds5
We next examined the morphology of mitotic chromosomes assembled in the cells treated with siRNAs specific to Scc1, Pds5A and Pds5B. Cells were subject to a hypotonic treatment, fixed and labeled with antibodies against the condensin subunit CAP-E/Smc2 and the mitotic protein kinase aurora B. In control metaphase cells, the axes of the sister chromatids, labeled with anti-CAP-E, were paired along the their entire length, and aurora B localized to a single dot at the primary constriction of each chromosome (Fig. 4A, control). In cells transfected with siRNAs, we observed abnormal phenotypes that could be classified into three groups (Fig. 4A, types I to III). The `type I' phenotype was the most striking, and consisted of chromosomes in which cohesion between the two sister chromatids was lost. The resulting single chromatids were usually shorter and thicker, and presented a wavy axis. The aurora B signal was no longer concentrated at the primary constriction but spread along the chromatid arm. In the `type II' phenotype, chromosomes exhibited loosely paired sister chromatids, and the aurora B signal was split into two dots at the primary constriction, suggesting a defect in centromeric cohesion. In the chromosomes categorized as `type III', centromeric cohesion was apparently normal, but sister chromatids were shorter and more separated and displayed a wavy axis. More than half of the metaphase cells transfected with Scc1 siRNA showed the type I phenotype (Fig. 4B). The type II and type III phenotypes were more prevalent among the cells transfected with Pds5B and Pds5A siRNAs respectively, and only small fraction of cells showing the type I phenotype were found in these cultures. However, when metaphase chromosome spreads were prepared from colcemid-arrested cells, the fraction of cells with single chromatids (type I) increased in Scc1 siRNA- and Pds5B siRNA-treated cells (Fig. 4C). Taken together, these results suggest that the human Pds5 proteins contribute to the proper assembly of metaphase chromosomes with tightly paired sister chromatids, and further imply that there may be some division of labor between Pds5A and Pds5B in arm and centromeric cohesion, respectively.
Chromosome alignment defects in mitotic cells with reduced levels of cohesin and Pds5 proteins
Consistent with the observed defects in chromosome assembly, we also found a number of defects in chromosome alignment and segregation in Scc1-, Pds5A- and Pds5B-deficient cells (Fig. 5). In most control metaphase cells, all chromosomes become bi-oriented at the spindle equator, forming a tight metaphase plate (Fig. 5a). In contrast, a large proportion of the mitotic cell population in the Scc1 siRNA-treated samples corresponded to prometaphase cells with striking chromosome alignment defects and multipolar spindles (Fig. 5b) or pseudo-anaphase cells with elongated spindles and prematurely separated sister chromatids (Fig. 5c). A two- to threefold increase of the mitotic index was observed in these samples. HeLa cells treated with Pds5A or Pds5B siRNAs showed similar defects, albeit to a lesser extent. The most commonly observed abnormality was a metaphase cell with a bipolar spindle in which a number of chromosomes (from one to five) had failed to congress to the metaphase plate (Fig. 5d). In addition, more severely disorganized mitotic figures with abnormally condensed chromosomes were found after Pds5 depletion (Fig. 5e). Whether these abnormal chromosomes are the primary consequence of the lack of Pds5 function or a secondary effect of a prolonged metaphase arrest remains to be determined. No major variation of the mitotic index was observed in Pds5A or Pds5B siRNA-treated cell cultures compared to control.
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To examine the chromatin binding properties of Pds5 proteins in the cell-free extracts, chromatin was first assembled in an interphase extract, and then converted to mitotic chromosomes. Chromatin fractions were isolated in the course of the reaction and analyzed by immunoblotting. We observed progressive loading of Pds5B on interphase chromatin and substantial dissociation upon entry into mitosis, coincident with the association of condensin. This behavior was essentially identical to that of the cohesin subunit Scc1 (Fig. 6B, three bottom panels). Unlike Pds5B, only a small population of Pds5A was recovered on chromatin during interphase and no clear dissociation was observed in mitosis (Fig. 6B, top panel).
To test the interdependency of Pds5B and cohesin in their association with chromatin, interphase chromatin was assembled in extracts that had been mock-depleted or depleted of Pds5B or cohesin and was isolated and analyzed by immunoblotting (Fig. 6C). The loading of Pds5B on chromatin was severely compromised in the cohesin-depleted extract whereas a normal level of cohesin bound to chromatin in the absence of Pds5B. Similarly, depletion of Pds5A or simultaneous depletion of Pds5A and Pds5B had little effect in the binding of cohesin to interphase chromatin (Fig. S2 in supplementary material and data not shown). Conflicting observations reported in two previous studies made uncertain whether this was the case in S. cerevisiae (Hartman et al., 2000; Panizza et al., 2000
). Our results in Xenopus egg extracts and HeLa cells showing that cohesin loading does not depend on Pds5 are consistent with those from Hartman et al. (Hartman et al., 2000
) as well as with more recent results from studies in S. pombe (Tanaka et al., 2001
; Wang et al., 2002
).
Depletion of Pds5 proteins disturbs cohesin regulation in Xenopus egg extracts
To test whether Pds5 proteins are important for sister chromatid cohesion in Xenopus egg extracts, we performed a cohesion assay (Losada et al., 1998). Metaphase-like chromosomes were assembled in extracts that had been mock depleted, depleted of cohesin or depleted of both Pds5A and Pds5B. The efficiency of each depletion was higher than 95% in all cases (Fig. S2A in supplementary material and data not shown). The chromosomes were isolated from each extract and labeled with antibodies against condensins I and II (Ono et al., 2003
). Although many of the chromosomes assembled in a cohesin-depleted extract showed dramatic defects in sister chromatid cohesion both along the arms and at the centromeric region (Fig. 7A, cohesin-dep), no apparent defects were observed in those assembled in the extract depleted of Pds5A and Pds5B (Fig. 7A, Pds5-dep). A subpopulation of condensin II was enriched at the centromeric region of the in vitro assembled chromosomes, as has been recently shown in HeLa cell chromosomes (Ono et al., 2004
), and this enrichment was not compromised in the absence of cohesin or Pds5 (Fig. 7A, two lower rows). Despite their apparently normal morphology, chromosomes assembled in the Pds5-depleted extract retained an abnormally high level of cohesin, as judged both by immunolabeling with anti-SA1 (Fig. 7B) and by immunoblotting with anti-Scc1 (Fig. S2B in supplementary material).
Previous reports have shown that the distribution of the chromosome passenger protein INCENP changes upon depletion of functional cohesin in chicken and Drosophila cells (Sonoda et al., 2001; Vass et al., 2003
). We wished to test whether this might also be the case in Xenopus egg extracts. In the chromosomes with tightly paired chromatids assembled in mock-depleted extracts, INCENP staining was detected between the sister chromatids throughout the pericentromeric region, flanked by the signals of kinetochore protein CENP-E (Fig. 7C, left). In chromosomes assembled in cohesin-depleted extracts, however, the INCENP signal was no longer present in the inner pericentromeric region, being instead restricted to the kinetochores. This was the case even in a population of cohesin-depleted chromosomes that showed a relatively mild cohesion defect (Fig. 7C, center). The same localization of INCENP was observed in many of the chromosomes isolated from extracts depleted of Pds5A and Pds5B (Fig. 7C, right). Thus, the Pds5-depleted chromosomes may have a mild defect in centromeric cohesion that is not readily detectable with our standard cohesion assay.
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Discussion |
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The cell cycle-regulated localization of Pds5A and Pds5B in HeLa cells is essentially identical to that of cohesin (see Fig. S1 in supplementary material) (Losada et al., 2000; Sumara et al., 2000
). In nuclear extracts, a specific interaction between each Pds5 protein and cohesin is readily detectable by co-immunoprecipitation. We also show here that association of Pds5A, and most likely Pds5B, with chromatin is dependent on functional cohesin in HeLa cells. Although cohesin can be loaded on chromatin in cells treated with Pds5A or Pds5B siRNA, the morphology of mitotic chromosomes in these cells is compromised. Some chromosomes display loosened cohesion at both the centromeric and arm regions, whereas others show separated arms with wavy chromatid axes but no major impairment of centromeric cohesion. A similar, yet more severe phenotype was observed in cells depleted of the cohesin subunit Scc1. In this case, more than half of the mitotic cells show completely unpaired chromatids, a phenotype also found in human cells expressing a dominant-negative version of Scc1 (Hoque and Ishikawa, 2002
) and in Drosophila cells depleted of Scc1/Rad21 by RNAi (Vass et al., 2003
). The milder phenotypes found in Pds5A- or Pds5B-deficient cells could be due to incomplete depletion of each of the target proteins by RNAi treatment. Alternatively, Pds5A and Pds5B may have redundant functions, and one may be able to substitute for the other. As we have been unable to find experimental conditions that allow the simultaneous, efficient reduction of the two Pds5 proteins in HeLa cells, this possibility remains open.
By quantitative immunoprecipitation analysis, we estimate the relative abundance of Pds5A or Pds5B to cohesin to be 1:20 in Xenopus egg extracts, a value substantially smaller than the
1:3 or 1:4 ratio estimated in HeLa cell nuclear extracts. Furthermore, only a minor population of Pds5 proteins is found associated with cohesin in the egg extracts. Although other interpretations are possible, these two observations suggest that the function of Pds5 proteins may be less important in the embryo than in somatic cells. Consistently, in the embryonic extracts, no prominent defect in arm cohesion is observed in the absence of Pds5A and Pds5B. It is nevertheless important to emphasize that the association of Pds5B with chromatin depends on cohesin in Xenopus egg extracts, and that centromeric cohesion appears to be loosened in chromosomes from Pds5-depleted extracts, as judged by delocalization of INCENP. The finding of an abnormal level of cohesin on these chromosomes was somewhat unexpected. Given the proposed role of Pds5 in cohesion maintenance (e.g. Stead et al., 2003
), one would anticipate that the association of cohesin with chromatin would be destabilized rather than stabilized in the absence of Pds5 function. We speculate that Pds5 may facilitate conformational changes of cohesin and thereby regulate its behavior both positively and negatively. Such dual functions of Pds5 have been hinted at by a genetic study in S. pombe showing that Pds5 not only contributes to the maintenance of cohesion, but also acts as an inhibitor of its establishment (Tanaka et al., 2001
). In Xenopus egg extracts, Pds5B is phosphorylated in a mitosis-specific manner (see Fig. S3 in supplementary material). This modification could render cohesin accessible to the dissociation machinery or prevent re-association of the cohesin complexes that had just been removed from chromatin (Losada et al., 2000
). Recent results in yeast suggest that Pds5 is regulated by sumoylation (Stead et al., 2003
). So far we have been unable to show convincing sumoylation of either of the two Pds5 proteins in HeLa or Xenopus extracts. However, it is conceivable that sumoylation of vertebrate Pds5 is restricted to a very small population, for instance present at centromeres of mitotic chromosomes, making its detection very difficult.
How might Pds5 proteins regulate the action of cohesin at a mechanistic level? It is interesting to note that this class of proteins contains HEAT repeats (Neuwald and Hirano, 2000; Panizza et al., 2000
), which are also shared by the cohesin loading factor Scc2 and some of the non-SMC subunits of condensins (Ono et al., 2003
). It is possible that the HEAT motifs act as regulators of the SMC ATPase (Kimura and Hirano, 2000
) and thereby modulate the opening and closing of the ring- or V-shaped SMC molecules (Arumugam et al., 2003
; Weitzer et al., 2003
). Alternatively, a superhelical scaffolding structure formed by HEAT-repeat domains (Kobe and Kajava, 2000
) may promote protein-protein interactions between neighboring cohesin complexes. It has recently been suggested that, after being loaded onto chromatin, cohesin relocates and accumulates at sites of convergent transcription (Glynn et al., 2004
; Lengronne et al., 2004
). In this scenario, Pds5 could facilitate the sliding of cohesin rings along the chromatids and/or stabilize their clustering. The clusters would contribute to the establishment of robust cohesion, and also to its efficient dissolution in subsequent mitosis. Pds5-mediated stabilization of cohesion is probably more important for organisms with small chromosomes and little non-coding DNA (e.g. S. cerevisiae) in order to achieve strong arm cohesion despite active transcription throughout the cell cycle. In vertebrate cells, stable cohesion in the large blocks of centromeric heterochromatin could be facilitated by a Pds5-independent mechanism, such as that involving the RNAi machinery (Fukagawa et al., 2004
). Although purely speculative, this model may help us understand the diverse requirements for Pds5 function in different organisms as well as devise new strategies to dissect the molecular mechanism of action of this puzzling class of cohesion factor.
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Acknowledgments |
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Footnotes |
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* Present address: Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, Madrid 28029, Spain
Present address: SUNY Upstate Medical University, Syracuse, NY13210, USA
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References |
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