The condensin complex is a key determinant of higher-ordered chromosome structure. We show here that the complex is also important for the correct alignment of chromosomes on the meiosis I spindle. Unlike during mitosis and meiosis II, when sister chromatids attach to microtubules emanating from opposite spindle poles (biorientation), accurate meiosis I chromosome segregation requires that sister chromatids attach to microtubules emanating from the same spindle pole (coorientation). The monopolin complex, consisting of Lrs4, Csm1, and the meiosis-specific component Mam1, brings about meiosis I coorientation. We find that in the absence of functional condensin complexes, a fraction of sister kinetochores biorient on the meiosis I spindle and association of the monopolin complex subunit Mam1 with kinetochores is decreased. Our studies uncover a new locus-specific effect of the condensin complex.
MEIOSIS is a cellular division consisting of a single DNA synthesis phase followed by two chromosome segregation phases and is employed in the generation of gametes. During the first meiotic division, homologous chromosomes segregate, requiring that each pair of sister chromatids cosegregates toward one pole (coorientation); during the second meiotic division, sister chromatids separate toward opposite poles (biorientation). In budding yeast, the monopolin complex brings about the coorientation of sister chromatid kinetochores to allow only one microtubule attachment per pair of sisters (Winey et al. 2005; reviewed in Marston and Amon 2004). The monopolin complex is composed of four components: Mam1, expressed only during meiosis, which localizes to kinetochores from late pachytene until metaphase I (Toth et al. 2000); Lrs4 and Csm1, two nucleolar components that are released from the nucleolus during prophase I and targeted to kinetochores by the polo-like kinase Cdc5 (Clyne et al. 2003; Lee and Amon 2003; Rabitsch et al. 2003); and Hrr25, a casein kinase (Petronczki et al. 2006). The monopolin complex is thought to clamp sister kinetochores together through a cohesin-independent mechanism and fuse the two sister kinetochores into a single microtubule attachment site to facilitate coorientation (Monje-Casas et al. 2007). Deletion of genes encoding monopolin complex subunits results in the biorientation of sister chromatids during meiosis I (Toth et al. 2000; Lee and Amon 2003; Rabitsch et al. 2003; Petronczki et al. 2006).
The condensin complex is a conserved pentameric complex. In budding yeast, it is composed of two coiled-coil structural maintenance of chromosomes (SMC) subunits, Smc2 and Smc4 (Freeman et al. 2000) that form a heterodimer, and a globular head made up of Ycs4 (Bhalla et al. 2002), Ycg1, and Brn1 (Ouspenski et al. 2000). Condensin is best known for its role in chromosomal compaction during mitosis (reviewed in Hirano 2005) and meiosis (Yu and Koshland 2003; Chan et al. 2004). During meiosis, the complex is also required for the repair of double-strand breaks and chromosome axis morphogenesis, a prerequisite for recombinatorial repair and homolog synapsis as well as resolution of recombination-dependent linkages between homologs (Yu and Koshland 2003, 2005).
At the rDNA, the site of rRNA synthesis, condensins regulate rDNA silencing and prevent unequal sister chromatid exchange by presumably joining sister chromatid rDNA repeats (Freeman et al. 2000; Lavoie et al. 2002; Huang et al. 2006; Waples et al. 2009). Two components of the monopolin complex, Lrs4 and Csm1, share this role with the condensin complex. Both the Lrs4-Csm1 and the condensin complexes reside in the nucleolus (Bhalla et al. 2002; Rabitsch et al. 2003; D'Amours et al. 2004; Huang et al. 2006). There, Lrs4 and Csm1 are part of the regulator of nucleolar silencing and telophase exit (RENT) complex that binds to the replication fork barrier site within the nontranscribed spacer region NTS1 in the ribosomal DNA repeats (Huang et al. 2006; Waples et al. 2009). Lrs4 and Csm1 regulate rDNA functions by recruiting condensins to the rDNA (Johzuka and Horiuchi 2009). Not only do condensins and Lrs4-Csm1 share functions at the rDNA, but also their localization at the rDNA appears to be coregulated. During late anaphase, the mitotic exit network, a signaling pathway that triggers exit from mitosis by promoting the release of the protein phosphatase Cdc14 from the nucleolus, also promotes the dissociation of both the Lrs4-Csm1 complex and condensins from the rDNA (Huang et al. 2006; Varela et al. 2009; I. L. Brito, unpublished observations).
Lrs4-Csm1 and condensins colocalize at the rDNA where they regulate rDNA stability. During meiosis, Lrs4 and Csm1 associate with kinetochores. Condensins also accumulate at kinetochores in budding (Wang et al. 2004; D'Ambrosio et al. 2008) and fission yeast (Nakazawa et al. 2008). These observations raise the possibility that the two protein complexes also regulate the same process at kinetochores. Our findings lend support to this idea. We find that condensins, like the Lrs4-Csm1 complex, are required for full sister kinetochore coorientation during meiosis I by promoting the localization of Mam1 to kinetochores. We propose that condensin helps to establish a pericentromeric architecture required for Mam1 binding.
MATERIALS AND METHODS
Strains and growth conditions:
Derivatives of W303 are described in supporting information, Table S1; derivatives of SK1 strains are in Table S2. Proteins were tagged using the PCR-based method described in Longtine et al. (1998). GFP dots were constructed by integrating an array of bacterial TET operator sites 2 kb from the centromere on CENIV in the W303 strains or 1.4 kb from the centromere of one homolog of chromosome V in the diploid SK1 strains (Toth et al. 2000). Conditions for arrest with α-factor and release from the arrest are as described in Amon (2002). α-Factor was readded to all cultures 90 min after release from the G1 arrest to prevent cells from entering the next cell cycle. Growth conditions for individual experiments are described in the figure legends.
Cells were grown to saturation in YEP + 2% glucose (YPD) for 24 hr, diluted into YEP + 2% KAc (YPA) at OD600 = 0.3, and grown overnight. Cells were then washed with water and resuspended in SPO medium [0.3% KAc (pH = 7.0)] at OD600 = 1.9 at 30° to induce sporulation. Cells carrying temperature-sensitive alleles of condensin subunits were induced to sporulate at 25° for 1 hr and then shifted to 34°.
Indirect in situ immunofluorescence was carried out as described in Visintin et al. (1999) for tubulin-, HA-, and MYC-tagged proteins. CEN GFP dot visualization was performed as described in (Monje-Casas et al. 2007). Two hundred cells were counted for each time point. Chromosomes were spread as described in Nairz and Klein (1997). HA-tagged proteins were detected with a mouse α-HA.11 antibody (Covance) at a 1:500 dilution. MYC-tagged proteins were detected with a mouse anti-MYC 9E10 antibody (Babco) at a 1:500 dilution. Both were followed by a secondary anti-mouse CY3 antibody (Jackson ImmunoResearch) at a 1:1000 dilution. Endogenous luminescence was sufficient for visualizing Ndc80-GFP on chromosome spreads. In spreads of meiotic cells expressing Ndc10-6HA and Mam1-9MYC, mouse anti-HA and rabbit anti-MYC were used at 1:500 dilution, followed by anti-mouse FITC antibodies and anti-rabbit CY3 antibodies, also used at 1:500 dilution. In each experiment, at least 50 cells were counted per strain.
Western blot analysis:
Cells were harvested, incubated in 5% trichloroacetic acid (TCA), and lysed as described in Moll et al. (1991). Immunoblots were performed as described in Cohen-Fix et al. (1996). HA-tagged proteins were detected with a mouse α-HA.11 antibody (Covance) at a 1:500 dilution. MYC-tagged proteins were detected with a mouse anti-MYC 9E10 antibody (Babco) at a 1:1000 dilution. vATPase was detected using a mouse anti-vATPase antibody (Molecular Probes, Eugene, OR) at a 1:2000 dilution. The secondary antibody used was a goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch) at a 1:2000 dilution. Cdc28 was detected using a rabbit anti-Cdc28 antibody at a 1:1000 dilution. The secondary antibody used was a donkey anti-rabbit antibody conjugated to HRP (Jackson ImmunoResearch) at a 1:2000 dilution.
Condensins are required for sister kinetochore coorientation induced by CDC5 and MAM1:
The observation that the monopolin and condensin complexes function together at the rDNA and are capable of binding to each other (Johzuka and Horiuchi 2009) prompted us to test the possibility that these proteins function together to maintain genomic integrity at other chromosomal locations. During meiosis, the main function of Lrs4 and Csm1 is to coorient sister kinetochores (Rabitsch et al. 2003). To determine whether condensins are necessary for this process, we first tested the requirement for the protein complex in a system developed to induce sister kinetochore coorientation during mitosis. We previously showed that overexpression of the meiosis-specific coorientation factor Mam1 and the Polo kinase Cdc5 was sufficient to induce sister kinetochore coorientation during mitosis, leading to cosegregation of sister chromatids during anaphase (Monje-Casas et al. 2007). To follow the segregation of a single pair of sister chromatids, a tandem array of tetO sequences was integrated proximal to the centromere of chromosome IV and a tetR-GFP fusion protein, which binds the tetO sequence, was expressed to visualize the repeats (Michaelis et al. 1997). Overexpression of CDC5 and MAM1 led to cosegregation of sister chromatids during anaphase in 22% of cells at 34° (Figure 1A) (Monje-Casas et al. 2007). Deletion of LRS4 or CSM1 reduces cosegregation by ∼50%, whereas deletion of both mitotic components of the monopolin complex almost completely suppressed the cosegregation of sister chromatids induced by high levels of Cdc5 and Mam1 (Figure 1A) (Monje-Casas et al. 2007). Inactivation of YCS4 or BRN1 reduced Cdc5- and Mam1-induced sister kinetochore cosegregation by ∼50% (Figure 1A), signifying a role for the condensin complex in establishing sister kinetochore orientation.
The 50% reduction in sister chromatid cosegregation caused by loss of YCS4 and BRN1 function is likely to be an underestimation of the effect of condensin on this process. First, cosegregation of sister chromatids was analyzed only in cells with fully divided nuclei to ensure that these cells had reached anaphase. Cells with stretched or nondivided nuclei, which is indicative of a more complete inactivation of condensin function, were not included in this analysis as cosegregation of sister chromatids cannot be unambiguously determined in such cells. Second, due to low levels of sister chromatid cosegregation already seen in the ycs4-1 and brn1-60 single mutants (Bhalla et al. 2002), the extent of the effects of inactivating YCS4 and BRN1 in reducing Cdc5/Mam1-induced sister coorientation is underrealized (Figure 1A; note that the low-level cosegregation of sister chromatids observed in the condensin single mutants is probably not due to sister kinetochore coorientation but the result of failed chromosome segregation caused by defects in the decatenation of sister chromatids) (Ouspenski et al. 2000; Bhalla et al. 2002). Finally, because the GAL1-10 promoter does not function as well at 37° compared to 25°, we performed the experiment at 34° when the temperature-sensitive condensin alleles may not be completely inactivated. Indeed, at 34°, both ycs4-1 and brn1-60 mutants exhibited intermediate phenotypes with respect to chromosome segregation during mitosis (Ouspenski et al. 2000; Bhalla et al. 2002). Nevertheless, our results indicate that condensins are required for full sister kinetochore coorientation induced by Cdc5 and Mam1 overproduction. This loss of cosegregation in the absence of condensin was not due to reduced levels of Cdc5 and Mam1 produced in condensin mutants (Figure S1). We conclude that condensin is required for the function of coorientation factors rather than their production.
To determine how condensins affect sister kinetochore coorientation, we analyzed the ability of overexpressed MAM1 to associate with kinetochores in condensin mutants by chromosome spreads. In cells overexpressing CDC5 and MAM1 during mitosis, Mam1 colocalizes with centromeric GFP dots in ∼70% of cells with divided nuclei (Figure 1, B and C). In contrast, cells carrying the temperature-sensitive condensin allele ycs4-1 or brn1-60 exhibited reduced Mam1-9MYC localization to kinetochores. Only 25% of anaphase ycs4-1 cells and 37% of anaphase brn1-60 cells were able to target Mam1 to kinetochores at 34° (Figure 1, B and C). Our results show that condensins are required for full Cdc5/Mam1-induced coorientation of sister chromatids during mitosis.
Condensins are required for full sister kinetochore coorientation during meiosis I:
Examination of the role of condensin in coorienting sister chromatids during meiosis I is confounded by its other meiotic functions. Condensin is required during prophase I for processing of double-strand breaks and resolving recombination-dependent chromosome linkages (Yu and Koshland 2003). To isolate the effect of condensin on coorientation, we analyzed cells arrested in metaphase I, by depleting the anaphase promoting complex activator Cdc20. In this arrest, sister kinetochores are tightly associated. When the centromere of one homolog is GFP tagged (heterozygous GFP dots), the pair appears as one focus in the arrest (Lee and Amon 2003). By contrast, when coorientation is disrupted, as occurs when MAM1 is deleted, sister chromatids biorient in metaphase I and tension exerted by the meiosis I spindle allows two GFP dots to become visible (Lee and Amon 2003). To examine the consequences of inactivating condensins on sister kinetochore coorientation, we analyzed the separation of heterozygous GFP dots in cells carrying temperature-sensitive alleles of YCG1 and YCS4, two genes encoding condensin subunits. Cells were transferred into sporulation-inducing medium; 1 hr later, they were shifted to 34°. Sixty percent of Cdc20-depleted cells lacking MAM1 arrested in metaphase and approximately half of these cells exhibited separated CENV GFP dots (Figure 2) (Lee and Amon 2003). In Cdc20-depleted strains carrying either the ycg1-2 or the ycs4-2 alleles only 40% of cells reached metaphase, yet, similar to Cdc20-depleted cells lacking MAM1, approximately half of the cells showed CENV GFP dot separation (Figure 2). We conclude from this experiment that condensin activity is required to achieve wild-type levels of coorientation during meiosis I. Thus, the condensin complex is required for monopolin complex activity or functions in parallel to the complex to promote sister kinetochore coorientation.
We also examined sister kinetochore coorientation in cells depleted of condensins. We generated a depletion allele of the condensin component Brn1 by placing the gene encoding it under the control of the mitosis-specific CLB2 promoter. Epitope-tagged Brn1 produced from the native promoter can be detected throughout meiosis. In contrast, when placed under the CLB2 promoter, Brn1 levels are undetectable after transfer to sporulation medium (Figure 3A). In cells depleted of Brn1, sister kinetochore coorientation was impaired but the defect was not as severe as that observed in temperature-sensitive condensin mutants. Forty percent of cells reached metaphase I and 15% showed CENV GFP dot separation (Figure 3B). Similar results were obtained in cells depleted of the condensin subunit Ycs4 (data not shown). The difference in penetrance between temperature-sensitive condensin alleles and the depletion allele could be due to incomplete depletion of Brn1 in CLB2-BRN1 cells and/or due to high temperature exaggerating the coorientation defect of condensin mutants.
A fraction of cells depleted of Brn1 exhibited stretching of the tetO array. This is evident as lines of tetR-GFP signal (Figure 3C) and has been observed in bioriented cells lacking structural integrity at the kinetochores (He et al. 2000; Oliveira et al. 2005; Gerlich et al. 2006; Warsi et al. 2008). These observations suggest a requirement for condensin in sister kinetochore coorientation and in establishing structural integrity at kinetochores during meiosis.
Condensins are required for Mam1 localization to kinetochores in meiosis I:
On the basis of the evidence that condensins were required to localize Mam1 in mitotic cells overexpressing CDC5 and MAM1, we asked whether condensin was also needed for Mam1 association with kinetochores during meiosis I. Mam1 protein accumulation was not affected by the inactivation of condensin (Figure 4A). However, Mam1 association with kinetochores was. Mononucleate cells were viewed 6 hr after transfer into sporulation medium when ∼30% of wild-type and 20% of Brn1-depleted cells were in metaphase I (Figure 4B). Fifty-two percent of wild-type cells showed strong colocalization between tagged versions of Mam1 and Ndc10, as expected (Toth et al. 2000). In these cells, the majority of Ndc10 foci (>50%) were spatially associated with Mam1 foci. Another 27% of cells showed weak colocalization, as defined by a minority of Ndc10 foci displaying Mam1 colocalization (Figure 4, C and D). In cells depleted of the condensin component Brn1, only 18% of cells showed Mam1 colocalization with over half of the Ndc10 foci and only an additional 17% showed colocalization with a small fraction (<50%) of Ndc10 foci (Figure 4, C and D). Results obtained with cells depleted of the condensin component Ycs4 were similar (data not shown). As in cells lacking LRS4, Mam1 associated with chromatin in Brn1-depleted cells (Figure S2, data not shown), but not with kinetochores. We conclude that condensin is required for full association of Mam1 with kinetochores and to bring about coorientation during meiosis I.
The condensin complex and Lrs4-Csm1 associate with kinetochores through independent mechanisms:
To determine whether the association of the Lrs4-Csm1 complex with meiosis I kinetochores required condensins, we examined Lrs4-6HA localization in chromosome spreads of wild-type and Brn1-depleted cells 6 hr post-transfer into sporulation medium when ∼20% of cells were in metaphase I (Figure 5A). We were unable to test the localization using temperature-sensitive condensin alleles because they are lethal in the presence of tagged versions of the monopolin complex components (data not shown; Waples et al. 2009.) Lrs4-13MYC was released from the nucleolus (Figure 5A) and colocalized with Ndc10-6HA foci to the same extent in cells depleted for Brn1 as in wild-type cells (Figure 5, B and C). In agreement with previous reports that demonstrate that Lrs4 and Csm1 function in a single complex (Rabitsch et al. 2003; Huang et al. 2006), we found Lrs4 and Csm1 localization to be interdependent (Figure S3, A–C) and Lrs4 protein accumulation to require CSM1 (Figure S3 D). Our results indicate that condensin is required for Mam1 to associate with kinetochores but not Lrs4 and Csm1, raising the interesting possibility of sequential recruitment of coorientation factors to kinetochores during meiosis I.
To test whether condensins required the monopolin complex to localize to kinetochores, we used mitotic anaphase-arrested cdc14-3 cells because the congregation of kinetochores during this point of the cell cycle facilitates the analysis of kinetochore proteins (Guacci et al. 1997) and because Lrs4 and Csm1 are enriched at kinetochores in this mutant (I. L. Brito, unpublished observations). We found condensins to be enriched at kinetochores during anaphase (Figure 6), which is consistent with previous results in budding and fission yeast (Wang et al. 2004; D'Ambrosio et al. 2008; Nakazawa et al. 2008). MYC-tagged versions of the condensin subunits Ycs4 (Figure 6, A and B) or Smc4 (Figure 6, C and D) were found to colocalize with a GFP-tagged version of the kinetochore component Ndc80 in ∼80% of cells. Deletion of LRS4 (Figure 6, A and B) or CSM1 (Figure 6, C and D) did not affect this kinetochore association. Together, our findings indicate that although the Lrs4-Csm1 complex is needed for recruitment of the condensin complex to the rDNA (Johzuka and Horiuchi 2009), the two complexes associate with kinetochores independently of each other.
Our studies and those of others indicate that members of the condensin complex and the mitotic components of the monopolin complex, Lrs4 and Csm1, bind at specialized genomic sites where they function to link sister chromatids. We show here that Lrs4-Csm1 and the condensin complex bind to sister kinetochores independently of each other, but that during meiosis, both of these complexes are required to recruit Mam1 (Figure 7, A and B). At the rDNA, Lrs4 and Csm1 recruit condensins to inhibit the unequal exchange between sister chromatids (Huang et al. 2006; Johzuka and Horiuchi 2009), a function most simply explained by a role in linking sister chromatids so that movement of the repeats is restricted with respect to one another (Figure 7C). Together, the two complexes thus provide cohesive properties to sister chromatids and impose steric constraints, to prevent recombination at the rDNA, and at kinetochores, to facilitate their attachment to microtubules emanating from the same spindle pole.
Our results indicate that condensins are required for wild-type levels of association of Mam1 with kinetochores to promote the coorientation of sister chromatids during meiosis I. How could condensin bring this about? It is unlikely that Mam1 fails to associate with kinetochores due to a loss of overall centromeric and pericentromeric structure in condensin mutants. First, kinetochore assembly does not appear to be affected in condensin mutants, as is evident by the sister chromatids' ability to separate in metaphase I-arrested cells in condensin mutants. This activity requires kinetochores to have captured a microtubule and be under tension. Second, inactivation of the cohesin complex, another SMC chromosome structure complex, does not affect binding of the monopolin complex to kinetochores (Monje-Casas et al., 2007). Finally, inactivation of condensin does not appear to interfere with the association of Lrs4 with kinetochores. We propose that the condensin complex creates a higher-ordered chromatin structure at the kinetochore, which not only provides a scaffold for the recruitment of Mam1 but also contributes to the coorientation process (Figure 7B). Our results also raise the interesting possibility that assembly of the monopolin complex is stepwise, with Lrs4-Csm1 associating with kinetochores independently of Mam1. There they, together with condensins, establish a Mam1 binding platform. We speculate that Lrs4-Csm1 and condensin provide rigidity and hence steric constraints to the centromeric regions and facilitate the linking of microtubule binding sites. This favors orientation of the two sister kinetochores toward one spindle pole. Mam1 could be needed to stabilize these linkages.
Condensins do not appear to be involved in meiosis I sister kinetochore coorientation in other eukaryotes. In Drosophila melanogaster hypomorphic alleles in the gene encoding the non-SMC condensin subunit DCAP-G (homologous to Saccharomyces cerevisiae Ycg1) do not show defects in coorientation (Resnick et al. 2009). Caenorhabditis elegans mutants in hcp-6, a non-SMC condensin II subunit, show defects in chromosome segregation during meiosis I (Chan et al. 2004) but whether sister kinetochore coorientation is affected in this mutant is not clear. It appears that in most species, the other SMC-containing chromosome structure complex, the cohesin complex, facilitates sister kinetochore coorientation. In fission yeast, meiotic cohesin complexes associate with the core centromere where they, together with the coorientation factor Moa1, facilitate a kinetochore geometry that favors sister kinetochore coorientation (Yokobayashi and Watanabe 2005; Sakuno et al. 2009). In maize and Arabidopsis, cohesins are also essential for sister kinetochore coorientation (Yu and Dawe 2000; Chelysheva et al. 2005). Clearly, SMC protein-containing complexes play critical roles in establishing coorientation but a role for condensin has thus far only been conclusively demonstrated in yeast. We speculate that employing different SMC complexes in promoting sister kinetochore coorientation reflects differences in kinetochore architecture. Organisms in which sister kinetochore coorientation requires cohesins have regional centromeres with large pericentromeres that are heterochromatic in nature. In contrast, budding yeast centromeres are only 147 bp in length and lack pericentromeric heterochromatin. Structural rigidity appears necessary for sister kinetochore coorientation (Sakuno et al. 2009). At centromeres surrounded by heterochromatin, cohesin could be sufficient to bring about this rigidity. At centromeres that lack a heterochromatic pericentromere, additional rigidity factors such as condensins could be important. Consistent with a role of condensins in providing chromatin firmness in the pericentromere is our observation that tetO arrays located next to the centromere appear stretched in condensin mutants. By structuring pericentromeric regions and linking them to centromeric proteins, namely Lrs4 and Csm1, condensins could promote sister kinetochore coorientation.
Our data further add to the growing body of evidence to suggest that condensins not only establish gross chromosomal architecture (reviewed in Hirano 2005) but also play a role in creating locus-specific chromosome structures. At the rDNA, condensin binds distinct rDNA repeats to prevent recombination between mismatched rDNA repeats (Huang et al. 2006; Johzuka and Horiuchi 2009). Clustering of tRNA genes by condensin is used to modulate their expression levels (Haesler et al. 2008; D'Ambrosio et al. 2008). Our data suggest that condensin plays a role in coorientation at centromeric regions. The generation of specialized local chromatin structure may utilize condensin's ability to aggregate DNA in a locus-specific manner.
We thank Frank Solomon, Iain Cheeseman, and members of the Amon Lab for their comments on the manuscript. We also thank Hui Jin of the Yu lab for her technical assistance. H.G.Y. is supported by the National Science Foundation (NSF-MCB0718384). This research was supported by National Institutes of Health grant GM62207 to A.A. A.A. is also an Investigator of the Howard Hughes Medical Institute.
Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.115139/DC1.
Communicating editor: S. E. Bickel
- Received February 3, 2010.
- Accepted February 26, 2010.
- Copyright © 2010 by the Genetics Society of America