Shugoshin regulates cohesion by driving relocalization of PP2A in Xenopus extracts
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Sister chromatid cohesion is mediated by cohesin. At the onset of mitosis, most cohesin dissociates from chromatin with the exception of a small population, present along chromosome arms and enriched at centromeres. A protein known as shugoshin (Sgo) is essential to maintain arm and centromeric cohesion until the onset of anaphase in transformed human cells, but not in other organisms like Drosophila or mouse. We have used Xenopus egg extracts to further explore this issue. Chromosomes assembled in extracts depleted of Sgo have little or no cohesin at centromeres and display centromeric cohesion defects. Unlike transformed human cells, however, arm cohesion is maintained in the absence of Sgo. Furthermore, Sgo depletion impairs the prophase dissociation of cohesin. This phenotype can be rescued by inhibition of PP2A. The protein phosphatase interacts with Sgo and accumulates at centromeres in mitosis in a Sgo-dependent manner. We propose that Sgo drives relocalization of PP2A from arms to centromeres and, in this way, coordinates release of arm cohesin with protection of centromeric cohesin in mitosis.
Sister chromatid cohesion is essential for proper chromosome segregation. It entails the creation and maintenance of a physical linkage between the two copies of a chromosome that arise from the duplication process until their separation at the metaphase to anaphase transition. Cohesin, a ring-shaped multiprotein complex conserved from yeast to human, is a major mediator of this linkage (Losada and Hirano 2005; Nasmyth and Haering 2005). It consists of a heterodimer of SMC1 and SMC3 and two regulatory subunits, Scc1/Rad21 and Scc3/SA. In vertebrate cells, cohesin is loaded on chromatin in early G1 phase and establishes cohesion during S phase. Most cohesin is released from chromatin during prophase, and only a small population remains between the sister chromatids enriched in the pericentric region (Losada et al. 1998; Waizenegger et al. 2000). This population is essential in preventing precocious separation of the sister chromatids and allowing proper chromosome alignment (Vass et al. 2003; Vagnarelli et al. 2004; Losada et al. 2005). The dissociation of cohesin in prophase requires Polo and Aurora B kinases as well as a number of additional factors that include Wapl (Losada et al. 2002; Sumara et al. 2002; Hauf et al. 2005; Gandhi et al. 2006; Kueng et al. 2006). At the onset of anaphase, cleavage of Scc1/Rad21 by separase drives dissociation of the remaining cohesin and sister chromatid separation ensues (Hauf et al. 2001).
Sequential loss of cohesion is essential also in meiosis (Marston and Amon 2004). Arm cohesion is released in anaphase I to allow resolution of chiasmata and segregation of homologous chromosomes. Centromeric cohesion persists until anaphase II, when it is dissolved to allow segregation of the sister chromatids. In both anaphase I and anaphase II, loss of cohesion involves separase-mediated cleavage of cohesin (Terret et al. 2003; Brar et al. 2006; Kudo et al. 2006). A family of proteins known as shugoshins (Sgo), orthologues of Drosophila melanogaster MEI-S332, protect centromeric cohesin from separase cleavage in meiosis I (Kerrebrock et al. 1995; Kitajima et al. 2004; Rabitsch et al. 2004). One or two orthologues of this protein can be found in most eukaryotes, and they function both in meiosis and mitosis. It has been proposed that one specific variant of the protein phosphatase PP2A associates with Sgo and protects centromeric cohesin in meiosis I by counteracting Rec8 phosphorylation by Polo (Riedel et al. 2006). A second report showed that PP2A associates with Sgo proteins also in HeLa cells and most likely counteracts SA2 phosphorylation by Polo (Kitajima et al. 2006). In Saccharomyces cerevisiae, an organism in which there is no prophase release of cohesin, Sgo1 is not required for cohesion but for sensing tension between sister chromatids (Indjeian et al. 2005). Human Sgo2 also acts as a sensor of tension (Huang et al. 2007; Lee et al. 2008), whereas a number of studies in HeLa cells show that knock down of Sgo1 by small interfering RNA (siRNA) leads to premature separation of the sister chromatids and prometaphase arrest (Salic et al. 2004; McGuinness et al. 2005). Therefore, in these transformed human cells, Sgo1 appears to protect a small population of cohesin both at arms and at centromeres from the prophase pathway (Nakajima et al. 2007). In contrast, the pioneer studies of D. melanogaster mei-S332 mutants indicated the absence of cohesion defects in mitotic cells (Kerrebrock et al. 1995). After removing arm cohesion by treatment with colchicine, sister chromatid separation was observed in 18% of the mutant cells compared with 9% of the wild-type cells, suggesting that shugoshin MEI-S332 was specifically involved in centromeric cohesion (LeBlanc et al. 1999). A similar conclusion can be drawn from the study of Bub1-null mouse embryonic fibroblasts (MEFs; Perera et al. 2007). Bub1 is a component of the spindle assembly checkpoint, and it is also required for proper localization of Sgo at centromeres (Tang et al. 2004; Kitajima et al. 2005; Vaur et al. 2005; Boyarchuk et al. 2007). In Bub1-null MEFs treated with the APC/C inhibitor MG132 to prevent anaphase entry, there is no precocious separation of sister chromatids even though there is no Sgo1 at centromeres. Thus, while the role of Sgo in the protection of centromeric cohesion in meiosis is conserved in most species, its role in regulation of mitotic cohesion is less clear.
We have used Xenopus laevis egg cell-free extracts to further understand how Sgo regulates the behavior of cohesin in mitosis. To date, a single Sgo protein has been identified in X. laevis (Salic et al. 2004). We report that chromosomes assembled in extracts without Sgo show increased distance between sister centromeres, consistent with a centromeric cohesion defect caused by the lack of cohesin enrichment at this region. However, these chromosomes have paired chromosome arms. In fact, cohesin release from chromosome arms is partially defective in extracts without Sgo, most likely due to mislocalization of PP2A.
Materials and methods
Two fragments of Xenopus Bub1 (p1, aminoacids 1–331, and p2, aminoacids 568–828, a gift from J. Maller, University of Colorado) were expressed and purified from bacteria as 6× His-tagged fusion proteins and used to produce rabbit polyclonal and mouse monoclonal antibodies, respectively. Mouse monoclonal and rabbit polyclonal antibodies were also raised against a maltose binding protein fusion of full-length Xenopus Sgo protein (MBP-XSgo, a gift from A. Salic). Rabbit polyclonal sera against Xenopus CENP-A were obtained using a synthetic peptide as immunogen (MRPGSTPPSRRKSRPPRRVC). Other antibodies used in this study were as follows: anti-SMC1, anti-SMC3, and anti-Scc1 (Losada et al. 1998); anti-SA1 (Losada et al. 2000); anti-XCAP-G (Hirano et al. 1997); anti-XINCENP and anti-XAurora B (MacCallum et al. 2002); and anti-Plx1 (Losada et al. 2002). A mouse monoclonal antibody against the catalytic subunit of PP2A (clone 1D6) was purchased from Upstate.
Preparation and depletion of Xenopus egg extracts
Cytostatic factor (CSF)-arrested, low-speed supernatants (LSS) of Xenopus eggs were prepared in XBE2 buffer [10 mM K-Hepes (pH 7.7), 0.1 M KCl, 2 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, and 50 mM sucrose] as described (Losada et al. 1998). Interphase LSS were generated by addition of 100 μg/ml cycloheximide and 0.4 mM CaCl2 to CSF-arrested LSS followed by incubation at 22°C for 30 min. To deplete 50 μl of extract, the following antibodies were bound to 25 μl of Dynabeads protein A (Dynal): for Bub1 depletion, 8 μg of rabbit polyclonal anti-Xenopus Bub1 (40 min); for cohesin depletion, 4 μg of anti-SMC3 plus 2.5 μg of anti-SMC1 (two rounds of 40 min); for Aurora B depletion, 3.5 μg of anti-XINCENP plus 3.5 μg of anti-XAurora B (two rounds of 40 min); and for Polo depletion, 8 μg of anti-Polo (two rounds of 40 min). For the depletion of Sgo, 50 μl of rabbit polyclonal serum was bound to 25 μl of Affi-prep protein A Support (BIO-RAD). In all cases, mock depletions were performed using beads coated with pre-immune rabbit IgG (Sigma).
Chromosome assembly and analysis
Sperm nuclei (800–1,000 nuclei per microliter) were incubated at 22°C for 2 h with freshly depleted interphase LSS supplemented with energy mix (1 mM MgATP, 10 mM creatine phosphate, and 50 μg/ml creatine kinase). The extracts were driven into mitosis by addition of an equal volume of CSF-arrested extract and incubated at 22°C for another 2 h. For biochemical analysis of chromatin-bound proteins, samples were diluted tenfold with XBE2 buffer containing 0.25% Triton X-100. The chromatin fractions were isolated by centrifugation through a sucrose cushion (30% sucrose in XBE2) in an Eppendorf 5417R centrifuge at 10,000×g for 15 min. For immunofluorescence, the assembly mixtures were fixed with ten volumes of 2% paraformaldehyde in XBE2 containing 0.5% Triton X-100 for 10 min and centrifuged onto coverslips. Primary antibodies were used at 1–2 μg/ml, whereas Cy3 or fluorescein isothiocyanate-conjugated donkey anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch) were used at 1:200. DNA was counter-stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). In some cases, double immunostaining with two rabbit polyclonal antibodies was performed with one of the antibodies directly labeled with a fluorophore, as described previously (Ono et al. 2004). A Leica DM6000 microscope was used to obtain grayscale images, which were later pseudo-colored and merged using Adobe Photoshop.
Soluble cohesin was purified from HeLa cell nuclear extracts with anti-SMC3 as described (Losada et al. 2000) and was first incubated with a Polo kinase fraction (or an Aurora B kinase fraction as control) immunoprecipitated from a CSF extract on Dynabeads protein A. After 20 min, the Polo-incubated cohesin was incubated with a Sgo fraction immunoprecipitated from a CSF extract in the presence or absence of 1 μM okadaic acid. To detect phosphorylation/dephosphorylation of cohesin, the mobility of the SA1 and SA2 subunits were analyzed by immunoblotting.
Xenopus Sgo binds throughout chromatin in interphase but accumulates at centromeres in mitosis
Centromeric cohesion is loosened in chromosomes assembled in the absence of Sgo
Arm cohesion is not affected by depletion of Sgo
Cohesin distribution is affected by Sgo depletion
Anomalous PP2A distribution in the absence of Sgo impairs cohesin release
The role of Sgo in the regulation of mitotic cohesion has been addressed in a number of recent studies, mainly in yeast and human cells (Salic et al. 2004; McGuinness et al. 2005; Vaur et al. 2005; Kitajima et al. 2006; Tang et al. 2006; Kawashima et al. 2007; Vanoosthuyse et al. 2007; Lee et al. 2008). However, this is the first time that the Xenopus egg cell free system, in which vertebrate cohesin was initially characterized, is used. Our results show that Sgo is a protector of cohesin around centromeres in chromosomes assembled in these extracts, as we see a perfect correlation between increased distance between sister centromeres and decreased cohesin staining in the absence of Sgo or its targeting kinase Bub1. This is important because it corroborates the previously proposed role of Sgo as a protector of centromeric cohesin from the prophase dissociation pathway in a “clean” system (Xenopus extracts) without interference of the spindle assembly checkpoint in which Sgo also participates (Indjeian et al. 2005; Huang et al. 2007). In contrast, the role of Sgo as a protector of cohesin along chromosome arms, proposed for human cells, seems not to be relevant in this system (Nakajima et al. 2007). It is possible that mechanistic differences in the regulation of cohesion exist between species or even between primary and transformed cells. These differences could affect cohesin per se or some other aspect of cohesion, e.g., the contribution of catenation. However, since arm cohesion is defective in the absence of cohesin in Xenopus egg extracts (Losada et al. 1998), it is clear that catenation alone cannot account for arm cohesion in the absence of Sgo, and cohesin must play a major role in keeping the sister chromatids together along the entire length of the chromosome. Regarding regulation of cohesin, the prophase dissociation pathway could be less efficient in Xenopus extracts compared with transformed human cells, perhaps due to increased amount and/or activity of the phosphatase PP2A. It is even conceivable that a fraction of PP2A is present along chromosome arms independently of Sgo and contributes to maintain arm cohesion in the chromosomes assembled in these extracts.
It has been previously shown that Aurora B is required for relocalization of Sgo from arms to centromeres (Resnick et al. 2006; Boyarchuk et al. 2007). Our results suggest that conversely, Sgo also contributes to the proper accumulation of the CPC at the inner centromere region. Both in S. cerevisiae and Schizosaccharomyces pombe, Sgo proteins have been reported to regulate at least some CPC components (Kawashima et al. 2007; Vanoosthuyse et al. 2007; Yu and Koshland 2007). The interdependence of Aurora B and Sgo localization at centromeres remains to be investigated in detail, but it is likely to underlie the role of both proteins as sensors of tension across the sister kinetochores (Goulding and Earnshaw 2005). This task appears to be more specific of Sgo2 in human and fission yeast (Kawashima et al. 2007). We suspect that a single Sgo protein in Xenopus may carry out the tasks assumed by Sgo1 and Sgo2 in other organisms.
On the other hand, we unexpectedly found increased amounts of cohesin on chromosome arms after Sgo depletion. This could be due to a problem in cohesin dissociation or could result from redistribution of cohesin from centromeres to arms in the absence of centromeric Sgo. We favor the first possibility. No report to date has shown that cohesin is enriched at centromeres prior to mitosis in metazoa, although this is the case in yeast (e.g., Lengronne et al. 2004). Thus, centromeric cohesin enrichment may be the result of preferential dissociation of cohesin from chromosome arms and protection at centromeres. In this scenario, redistribution is unlikely. In addition, depletion of Bub1 does not result in a similar phenotype of increased arm cohesin, even though Sgo is absent from centromeres also in this case. A fraction of PP2A left along the arms of chromosomes assembled in the absence of Sgo most likely counteracts cohesin phosphorylation by Polo and thereby prevents its release. Consistent with this interpretation, co-depletion of both Polo and Sgo increases only modestly the amount of cohesin left on chromosomes with respect to the single depletion of the kinase. In the case of Aurora B, Sgo appears not to be the only relevant target of the kinase in this prophase pathway. Other possible targets could be condensin, heterochromatin protein 1 (HP1), or some other protein yet to be identified (Fischle et al. 2005; Hirota et al. 2005; Lipp et al. 2007).
We thank A. Salic (Harvard Med. School) for Xenopus Sgo cDNA and the antibody used in our initial studies, J. Maller (University of Colorado Med. School) for Bub1 cDNAs, M. Rodríguez for excellent technical help, and J. Méndez (CNIO) for critically reading the manuscript. We are funded by the Spanish Ministry of Science and Education (grants BFU2007-66627 and CSD-Inesgen and a Ramón y Cajal Contract for A.L.), the Fundación Caja Madrid and the EU (Epigenome NoE and MIRG-31126).
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