A Genome-Wide Screen Identifies Genes Required for Centromeric Cohesion

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Science  27 Feb 2004:
Vol. 303, Issue 5662, pp. 1367-1370
DOI: 10.1126/science.1094220


During meiosis, two chromosome segregation phases follow a single round of DNA replication. We identified factors required to establish this specialized cell cycle by examining meiotic chromosome segregation in a collection of yeast strains lacking all nonessential genes. This analysis revealed Sgo1, Chl4, and Iml3 to be important for retaining centromeric cohesin until the onset of anaphase II. Consistent with this role, Sgo1 localizes to centromeric regions but dissociates at the onset of anaphase II. The screen described here provides a comprehensive analysis of the genes required for the meiotic cell cycle and identifies three factors important for the stepwise loss of sister chromatid cohesion.

In eukaryotes, the meiotic cell cycle allows for the formation of haploid gametes. This cycle is characterized by two rounds of chromosome segregation after a single DNA replication phase. During the first segregation phase, meiosis I, homologous chromosomes are separated. During meiosis II, sister chromatids separate. Three events must take place for meiosis to succeed [reviewed in (1)]: (i) One reciprocal recombination event (crossover) must occur between homologous chromosomes to physically link them, which is essential for accurate segregation during anaphase I. (ii) The kinetochores of sister chromatids must attach to microtubules emanating from the same spindle pole (co-orientation) during meiosis I to ensure sister chromatid cosegregation, but must attach to microtubules from opposite spindle poles (bi-orientation) for sister chromatids to separate during meiosis II (2). (iii) Cohesion between sister chromatids must be lost in a stepwise manner. Cohesion loss from chromosome arms is necessary for the resolution of chiasmata, the physical manifestation of crossover events, which is a prerequisite for homolog disjunction during meiosis I [reviewed in (1)]. Cohesion at centromeres, however, must be maintained during meiosis I for the faithful segregation of sister chromatids during meiosis II.

To identify genes required for meiotic cell cycle progression, we used a collection of Saccharomyces cerevisiae strains in which individual genes were deleted (3). Diploids were constructed that were homozygous for a particular deletion and carried green fluorescent protein arrays (GFP dots) on both copies of chromosome III (fig. S1) (4). We then analyzed two meiotic events: sporulation efficiency and the segregation pattern of the GFP-marked chromosome III. Using this systematic genome-wide approach, we assembled a comprehensive list of genes required for meiosis (table S1) (5). Mutants were first classified on the basis of their sporulation phenotypes. Mutants that failed to form spores efficiently or that formed two-spored asci at an increased frequency are listed in tables S2 and S3, respectively (5). The second classification was based on the pattern of chromosome missegregation. Meiosis I nondisjunction results in an increase in tetrads where only two spores contain GFP dots (Fig. 1, A and B). Strains producing asci with this GFP dot distribution at a high frequency are listed in table S4 (5). Meiosis II nondisjunction or premature sister chromatid separation is characterized by an increase in tetrads containing GFP dots in just three of the four spores. Mutants that fail to maintain cohesion around centromeres during meiosis I are expected to fall into this class (Fig. 1C), because the Drosophila mutant mei-S332, in which centromeric cohesion is lost prematurely, exhibits random meiosis II chromosome segregation (6, 7). We identified several mutants in which the fraction of tetrads with GFP dots in three of the four spores was elevated, although it never reached 50% (table S5). The segregation pattern of GFP dots in asci carrying a deletion of YOR073w (also known as SGO1) (8) was most similar to that predicted for mutants that prematurely lose centromeric cohesion. Deletion of the kinetochore components IML3 and CHL4 also led to a similar, although less pronounced, pattern of chromosome missegregation (table S5).

Fig. 1.

Chromosome segregation pattern of cells exhibiting random meiosis I and meiosis II chromosome segregation. A pair of homologs (bivalent) is shown, one homolog in red and the other in blue. The green dot denotes a GFPdot located on the chromosomes. (A) During a wild-type meiosis, homologs are segregated during meiosis I and sister chromatids are segregated during meiosis II, leading to the formation of tetrads with a sister chromatid and hence one GFPdot in each spore. (B) Homologs segregate randomly during meiosis I (shown here for a recombination mutant). After the first meiotic division, 50% of binucleate cells contain one homolog in each nucleus and 50% contain both homologs in one of the two nuclei. Meiosis II is normal, leading to 50% of tetrads containing one sister chromatid in each nucleus and 50% of tetrads with two spores containing two sister chromatids and two spores lacking a copy of this chromosome. (C) Meiosis I chromosome segregation is normal but random during meiosis II. Hence, 25% of tetrads will contain four spores with a GFPdot each, 25% of tetrads will contain spores with GFP dots in two of the four spores, and 50% of tetrads will contain spores with GFP dots in three of the four spores.

Because of the importance of retaining centromeric cohesion during meiosis I, we chose SGO1, IML3, and CHL4, whose gene products have been shown to localize to kinetochores during mitosis (912), for a more detailed characterization. Deletion of these genes in a SK1 background led to a chromosome missegregation pattern similar to that observed in the screen (Fig. 2A). iml3Δ chl4Δ double mutants exhibited a missegregation frequency similar to that of the single mutants (13); this finding indicates that the two genes function in the same pathway, which is consistent with the observation that the two proteins form a complex (911).

Fig. 2.

Centromeric cohesion is lost prematurely in iml3Δ, chl4Δ, and sgo1Δ mutants. (A and B) Wild-type, iml3Δ, chl4Δ, and sgo1Δ cells in which one (B) or both (A) copies of chromosome V were marked with GFP dots at URA3 (∼35 kb or 7 cM from the centromere) were induced to sporulate. The distribution of GFP dots in tetranucleate (A) and binucleate (B) cells was determined. The category “other” indicates cells in which more than four GFP dots were observed. (C) Wild-type, sgo1Δ, iml3Δ, and chl4Δ cells were induced to sporulate. The percentages of binucleate (open symbols) and bi- and tetranucleate (solid symbols) cells were determined. (D and E) Wild type, sgo1Δ, iml3Δ, and chl4Δ cells carrying REC8-13MYC and NDC10-6HA fusions were induced to sporulate. The percentage of anaphase I and/or metaphase II cells positive for the centromere marker Ndc10-6HA and visible Rec8 at centromeres was determined on nuclear spreads (D). Examples of anaphase I wild-type and sgo1Δ cells are shown in (E); DNA is stained with DAPI (4′,6′-diamidino-2-phenylindole).

To determine whether meiosis I segregation was affected in the mutants, we constructed strains in which only one of the two homologs carried a GFP dot (heterozygous GFP dots). In a normal first meiotic division, sister chromatids cosegregate so that a dot is observed in just one nucleus after the first meiotic division (in binucleate cells). Normal meiosis I division was observed in iml3Δ, chl4Δ, and sgo1Δ mutants (Fig. 2B). However, whereas sister chromatids remained tightly associated in wild-type binucleate cells, two GFP dots within one nucleus were often observed in sgo1Δ mutants (Fig. 2B).

These results suggest that meiosis I chromosome segregation is normal in iml3Δ, chl4Δ, and sgo1Δ mutants. However, sister chromatids appear to separate prematurely before anaphase II and are segregated randomly during the second meiotic division in sgo1Δ mutants, which indicates that sister chromatid cohesion is lost prematurely in sgo1Δ cells. iml3Δ and chl4Δ mutants also exhibited an increase in meiosis II nondisjunction, but premature separation of sister chromatids was, for unknown reasons, not evident in binucleate cells (Fig. 2B).

Meiotic cell cycle progression revealed a delay in all three mutants (Fig. 2C). sgo1Δ mutants were delayed in a cell cycle stage prior to entry into metaphase I (13), whereas iml3Δ and chl4Δ mutants were delayed in progression through metaphase I (fig. S2). Furthermore, spore formation was reduced in the mutants. Wild-type cells produced 76% tetrads, whereas sgo1Δ mutant cells formed 4% tetrads and iml3Δ and chl4Δ mutants 37%.

To establish whether SGO1, IML3, and CHL4 were required for retaining cohesin at centromeres during anaphase I, we analyzed the localization of the cohesin subunit Rec8 (5). In wild-type cells, Rec8 was lost from chromosome arms during anaphase I, but centromeric Rec8 [as identified by colocalization with the kinetochore protein Ndc10 (14)] was maintained until the onset of anaphase II (15) (Fig. 2, D and E). In sgo1Δ, iml3Δ, and chl4Δ mutants, Rec8 localization on chromosomes was indistinguishable from that of wild-type cells during prophase and metaphase I. However, Rec8 was undetectable around centromeres in sgo1Δ mutants during anaphase I (Fig. 2, D and E). Rec8 was also not visible at centromeric regions in 71% and 49% of anaphase I iml3Δ and chl4Δ mutants, respectively. Moreover, Rec8 was less abundant around centromeres in iml3Δ and chl4Δ mutants that retained Rec8 (Fig. 2D).

Our results suggest that SGO1 prevents Rec8 from being removed from centromeric regions during meiosis I. IML3 and CHL4 may also be required for this process. It is also possible that the loss of Rec8 from centromeric regions in binucleate iml3Δ and chl4Δ mutants is due to a phenotype similar to that of FEAR network mutants, in which meiosis II events (such as loss of centromeric cohesion) continue to occur despite defects in chromosome segregation (1618). We favor the idea that IML3 and CHL4 are involved in retaining Rec8 around centromeres, because cells lacking other nonessential kinetochore components such as CTF3, MCM16, and MCM22 [reviewed in (19)] did not exhibit the missegregation pattern observed in iml3Δ and chl4Δ mutants (table S1).

We next examined the localization patterns of Sgo1, Iml3, and Chl4 during meiosis (5). Iml3 and Chl4 localized to kinetochores throughout meiosis (Fig. 3, B and C) (fig. S3), as they do during mitosis (911). Sgo1 was undetectable during G1, because Sgo1 protein levels were low during this cell cycle stage (Fig. 3A). Sgo1 localized to centromeric regions from prophase I until metaphase II but was not detected at centromeric regions in anaphase II cells, which is at least in part due to degradation of the protein (Fig. 3A) (fig. S3).

Fig. 3.

Iml3, Chl4, and Sgo1 localize to meiotic kinetochores. Cells carrying NDC10-6HA and SGO1-9MYC, IML3-9MYC, or CHL4-9MYC fusions were induced to sporulate, and samples were collected for analysis of spread nuclei and protein levels of Sgo1, Iml3, and Chl4 by Western blot (5). Representative images of Sgo1 (A), Iml3 (B), and Chl4 (C) localization are shown. Quantifications are shown in fig. S3. Sgo1, Iml3, and Chl4 are shown in green; Ndc10-6HA in red; and DNA in blue. Western blots are shown below. Pgk1 and Kar2 were used as loading controls in Western blots.

A role for IML3 and CHL4 in mitotic chromosome segregation is well established (911). Deletion of SGO1 also led to defects in progression through mitosis. sgo1Δ cells released from a pheromone-induced G1 arrest (5) were delayed in progression through metaphase, indicating that some aspect of chromosome segregation was defective in the mutant (Fig. 4, A and B). To determine whether defects in sister chromatid cohesion around centromeres could be responsible for this chromosome segregation defect, we examined whether GFP dots localized 35 kb away from the centromere on chromosome V were separating prematurely. In wild-type cells, GFP dot separation occurred concomitant with the onset of anaphase (Fig. 4A). Although 10% of sgo1Δ cells harbored more than one GFP dot, presumably because of chromosome instability, GFP dot separation occurred before anaphase onset in some cells (Fig. 4B). Consistent with a role in regulation of mitotic cohesion and/or kinetochore function, Sgo1 localized to centromeric regions during S phase and metaphase but was absent during anaphase (Fig. 4, C and D). Loss of Sgo1 from kinetochores was likely due to protein degradation, because Sgo1 levels declined as cells entered anaphase (Fig. 4E). Our results suggest that Sgo1 is important for preventing premature separation of sister chromatids and/or kinetochore function during mitosis.

Fig. 4.

Sgo1 is important for progression through mitosis. (A and B) Wild-type (A) and sgo1Δ (B) cells carrying GFP dots at URA3 (∼35 kb from the centromere) were arrested in G1 with the use of α-factor (3 μg/ml) for 180 min, followed by release from the block as described (5). The percentage of cells with metaphase and anaphase spindles was analyzed. Also shown are percentages of cells in which two separate GFP dots were visible up to the onset of anaphase. The ∼10% of cells with two GFP dots at the zero time point is presumably due to cells bearing two copies of chromosome V. (C to E) Wild-type cells carrying SGO1-6HA were released from α-factor–induced G1 arrest (5) and the localization of Sgo1 was examined as cells progressed through the cell cycle. (C) Representative images of Sgo1 localization in the indicated cell cycle stages. Sgo1-6HA is shown in red, mitotic spindles in green, and DNA in blue. (D) Analysis of Sgo1 localization during the cell cycle. Localization to the kinetochore was defined as Sgo1 staining around the spindle pole body in S phase and/or along the mitotic spindle in metaphase, which is a characteristic of kinetochore proteins (19). Protein levels were analyzed throughout the cell cycle by Western blot analysis (E). Clb2 cyclin levels were used to monitor progression through mitosis.

The screen described here represents a comprehensive functional analysis of meiotic chromosome segregation. As a proof of principle we characterized three mutants, iml3Δ, chl4Δ, and sgo1Δ, whose chromosome segregation pattern predicted an involvement in the maintenance of centromeric cohesion during meiosis I. This analysis showed that all three proteins (to different extents) were required to retain cohesin around centromeres during anaphase I. Consistent with this role, Sgo1, Iml3, and Chl4 localize to centromeric regions. The findings that Sgo1, like Mei-S332, is a coiled-coil protein and dissociates from centromeric regions at the onset of anaphase during mitosis and the onset of anaphase II during meiosis (6, 7, 20) further raise the possibility that SGO1 is the functional homolog of MEI-S332 in S. cerevisiae. How SGO1 prevents loss of centromeric Rec8 during meiosis I is not yet known. Rec8 is cleaved by a protease known as separase at the onset of anaphase I, which leads to the removal of cohesin from chromosome arms (21). Perhaps Sgo1 is required to prevent Rec8 cleavage around centromeres during anaphase I. Factors that function together with Sgo1 to prevent Rec8 removal around centromeres or regulate Sgo1 localization may be among the uncharacterized genes identified by our screen.

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Materials and Methods

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Figs. S1 to S3

Tables S1 to S5

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