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Distinct Cohesin Complexes Organize Meiotic Chromosome Domains

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Science  16 May 2003:
Vol. 300, Issue 5622, pp. 1152-1155
DOI: 10.1126/science.1083634

Abstract

Meiotic cohesin complexes at centromeres behave differently from those along chromosome arms, but the basis for these differences has remained elusive. The fission yeast cohesin molecule Rec8 largely replaces its mitotic counterpart, Rad21/Scc1, along the entire chromosome during meiosis. Here we show that Rec8 complexes along chromosome arms contain Rec11, whereas those in the vicinity of centromeres have a different partner subunit, Psc3. The armassociated Rec8-Rec11 complexes are critical for meiotic recombination. The Rec8-Psc3 complexes comprise two different types of assemblies. First, pericentromeric Rec8-Psc3 complexes depend on histone methylation-directed heterochromatin for their localization and are required for cohesion during meiosis II. Second, central core Rec8-Psc3 complexes form independently of heterochromatin and are presumably required for establishing monopolar attachment at meiosis I. These findings define distinct modes of assembly and functions for cohesin complexes at different regions along chromosomes.

Eukaryotic sister chromatid cohesion is established during S phase and is maintained throughout G2 until M phase of the cell cycle. This cohesion is mediated by cohesin, a multisubunit complex (14). The mitotic cohesin complex is composed of two SMC (structural maintenance of chromosome) family proteins, Smc1 and Smc3, and two accessory subunits, Scc1 and Scc3 [Psm1, Psm3, Rad21, and Psc3 in fission yeast, respectively (5)], all of which are essential for cohesion function and cell proliferation. In meiosis, Rad21/Scc1 is dispensable (6, 7) and is largely replaced by its meiotic counterpart, Rec8 (810). During meiotic prophase, Rec8 complexes play a central role in establishing sister chromatid cohesion and facilitating recombination. In addition, centromeric Rec8 is required for ensuring that each kinetochore within a sister chromatid pair attaches to the same spindle pole (monopolar attachment) in fission yeast (6, 9). At anaphase I, Rec8 is disrupted along the arms, whereas centromeric Rec8 persists so that recombined homologs separate but sister chromatids move together to the same spindle pole (7, 9, 11). At meiosis II, when sister chromatids separate, centromeric Rec8 is disrupted. Thus, Rec8 complexes at centromeres and along chromosome arms appear to play different roles and must be differentially regulated through meiosis.

Fission yeast have two Scc3-like proteins, Psc3 and Rec11 (12, 13). Psc3 plays an essential role in sister chromatid cohesion during mitosis by forming a complex with Rad21 (fig. S5) (5). In contrast, Rec11 is meiosis-specific, and its mutation reduces recombination, like Rec8 (14, 15). Thus, circumstantial evidence suggests that Rec11 may function together with Rec8 as a component of the meiosis-specific cohesin complex. To evaluate this possibility, we determined whether Rec11 is required for sister chromatid cohesion in meiosis by monitoring green fluorescent protein (GFP) fluorescence associated with the cut3 locus (cut3-GFP), which is located at the middle of the left arm of chromosome 2. During prophase of meiosis I, wild-type cells show two cut3-GFP dots (Fig. 1A), indicating stable associations between each pair of sister chromatids. However, rec11Δ cells often contain three or four cut3-GFP dots (Fig. 1A), representing dissociation in these regions, as observed in rec8Δ cells. This premature separation of sister cut3 sequences appears in rec11Δ cells at the end of premeiotic DNA replication (fig. S1) and is not an indirect effect of decreased recombination, as sister cohesion is unimpaired in cells lacking rec12 (the SPO11 homolog in fission yeast) and, thus, lacking meiotic recombination entirely (16). Identical results were obtained by monitoring ade3-GFP, which lies in the middle of the left arm of chromosome 1 (17). Hence, Rec11, like Rec8, plays a crucial role in sister chromatid cohesion along chromosome arms during meiotic prophase I.

Fig. 1.

Distinct roles of the cohesin subunits of Rec8, Rec11, and Psc3 during meiosis. (A) The cut3-GFP dots were monitored in meiotic cells arrested by mei4Δ at late prophase I (19). Examples of wild-type and rec11Δ cells are shown at bottom. (B) One of the homologs was marked with cen1-GFP, cen2-GFP, and cen3-GFP and monitored for segregation pattern during meiosis. The segregation patterns of cen-GFP during meiosis are illustrated with examples of rec11Δ and rec8Δ cells. The equational pattern of wild-type cells (*) is mostly caused by recombination (19). (C) The segregation pattern of cen3-GFP marked on both homologs was monitored after meiosis I by arresting cells with the mes1 mutation. Examples of rec11Δ cells are shown at bottom. (D) The indicated cells were grown on the yeast extract agar (YEA) plate. Note that rec11+ is not expressed during mitosis, therefore rad21Δ rec8+o.p. psc3+ rec11+ cells sustain viability by the Rec8-Psc3 pair. The same cells were induced to meiosis and monitored for the segregation pattern of cen2-GFP marked on one chromosome. Representative cells are shown at bottom.

To determine whether Rec11 is also required for centromere functions, we marked a centromere-linked sequence (cen-GFP) on only one of the two homologs in a zygote and monitored segregation of the GFP dots during meiosis (Fig. 1B). rec8Δ cells undergo predominantly equational (i.e., +/–, +/–) chromosome segregation at meiosis I (9). If Rec11 worked with Rec8 at centromeres, rec11Δ cells would show this equational segregation pattern at meiosis I. However, nearly all pairs of sister chromatids in rec11Δ cells move together to the same pole (reductional-like +/+, –/–pattern) (Fig. 1B). Recombination-deficient rec12Δ cells also show a +/+, –/–segregation pattern, and deletion of rec8+ from either rec11Δ or rec12Δ cells shifts their segregation pattern from reductional-like to equational. Therefore, monopolar spindle attachment of sister centromeres is independent of crossing over or chiasmata formation and is directly regulated by centromeric Rec8 in a Rec11-independent manner.

Despite the fact that sister chromatid movement appears normal, rec11Δ cells nevertheless exhibit low spore viability (<70% viable) (15, 17). Experiments in which cen3 were marked with GFP on both homologs revealed that >20% of rec11Δ cells exhibit homolog nondisjunction, in which both homolog pairs move to the same pole at meiosis I (Fig. 1C). Nonrandom segregation of homologs in rec11Δ cells (different from rec12Δ cells) can be explained by residual levels of recombination (15) and, presumably, residual cohesion as well. rec11Δ cells undergo faithful disjunction during meiosis II (fig. S2C), indicating that centromeric cohesion persists through meiosis I. Thus, the abnormal chromosome segregation and reduced spore viability of rec11Δ cells stem from defective arm cohesion and recombination with ensuing nondisjunction at meiosis I.

Disruption of monopolar attachment in rec8Δ cells but not in rec11Δ cells suggests that Rec11 is not the sole partner of Rec8. Though Rec11 is indeed meiosis-specific, Psc3 is expressed during both mitosis and meiosis (fig. S2A), suggesting some meiotic role for Psc3. Mitotic cells carrying a temperature-sensitive allele of psc3 (psc3-2T) (18) displayed extensive separation of cut3-GFP dots (fig. S2B), whereas arm cohesion of psc3-2T cells is completely intact during meiotic prophase (fig. S2C). The psc3-2T mutation slightly enhances the dissociation of cut3 sequences in a rec11Δ background (fig. S2C), suggesting that Psc3 may assist arm cohesion if Rec11 is absent in meiosis and that Psc3 may secure the residual meiosis I disjunction of rec11Δ cells. In contrast to the dramatic reduction of meiotic recombination in rec11Δ cells, wild-type levels of recombination occur in psc3-2T cells (fig. S2D). Moreover, the overexpression of psc3+ leads to partial recovery of the arm cohesion defect of rec11Δ, but the recombination defect remains unimproved (fig. S3). Thus, Psc3 may play a minor role in cohesion along meiotic chromosome arms, but it has no ability to promote recombination.

Because Rec11 is dispensable for the centromeric functions of meiotic cohesin, Psc3 might instead partner with Rec8 at centromeres. Indeed, psc3-2T cells show defects in sister chromatid segregation during meiosis (fig. S2E). To definitively illuminate the meiotic roles for Psc3, we exploited the fact that ectopic expression of the Rec8-Rec11 pair sustains viability of rad21Δ psc3Δ cells (Fig. 1D), thus allowing meiotic induction in the complete absence of Psc3. The control rec8+ overexpressing (o.p.) psc3+ rec11+ cells undergo proper meiotic chromosome segregation, both reductional (meiosis I) and equational (meiosis II). However, rec8+o.p. psc3Δ rec11+o.p. cells show defective sister chromatid movement at both meiotic divisions (Fig. 1D). Thus, Psc3 plays a crucial role in kinetochore regulation at both meiotic divisions, and this function of Psc3 cannot be replaced by overexpression of Rec11. In contrast, the Rec8-Rec11 pair sustains mitotic growth better than Rec8-Psc3 (Fig. 1D; fig. S4A), underscoring the meiosis specificity of kinetochore regulation by Rec8-Psc3.

The distinct functions of Rec11 and Psc3 should be reflected by differential localization along meiotic chromosomes. In situ immunofluorescence showed that both Rec11 and Rec8 appear only during meiosis, whereas Psc3 is detected during both mitosis and meiosis. During meiotic prophase, Rec8 first appears at the centromeres and later distributes throughout the chromosome (Fig. 2A) (9). Rec11 does not coexist with Rec8 at centromeres in early meiotic prophase, but later it colocalizes with the distal chromosomal signals of Rec8. Conversely, Psc3 almost exclusively colocalizes with centromeric Rec8 throughout prophase I (Fig. 2A). To more precisely delineate the localizations of these cohesin proteins, we used a chromatin immunoprecipitation (ChIP) assay (19) using primers that amplify the centromeric central core (cnt) or pericentromeric (dg) regions, a centromere-proximal arm region (lys1), and a middle arm region (mes1) (Fig. 2B). The data reveal that Rec8 associates more with centromere regions than with chromosome arms (20), whereas Rec11 associates with arm regions more than with the centromere. Although Rec11 is not fully excluded from centromeres, the centromere-enrichment ratio (cnt/mes1) of Rec11 is less than one-fifth that of Rec8. In contrast, Psc3 associates exclusively with the centromere, showing a centromere-enrichment ratio >4 times higher than Rec8 and >20 times higher than Rec11 (Fig. 2B). In rec11Δ cells, the association of Rec8 is reduced selectively at arm sites, as shown by both ChIP and immunofluorescence assays (Fig. 2, A and B, rec11Δ). Moreover, when Rec8-GFP is expressed ectopically during mitosis, it localizes to centromeres much more efficiently if Psc3 is coexpressed than if Rec11 is coexpressed (fig. S4). Furthermore, immunoprecipitation experiments demonstrated that Rec8 interacts with Psc3, Rec11, and Psm3/Smc3 in vivo (fig. S5). At anaphase of meiosis I, Rec11 signals become faint in the nucleus, but Psc3 persists, together with Rec8, at the clustered centromeres (Fig. 2C). The centromeric Rec8-Psc3 dots disappear at meiosis II. The foregoing results suggest a scheme in which arm-associated Rec8 cooperates primarily with Rec11 to promote recombination and maintain arm cohesion until the end of meiosis I, whereas centromeric Rec8 works together with Psc3 throughout meiosis I until meiosis II.

Fig. 2.

In meiosis, Psc3 and Rec11 locate at centromeres and along chromosome arms, respectively. (A) Rec11-HA or Psc3-HA was counterstained with Rec8-GFP and DAPI (4′,6′-diamidino-2-phenylindole) in meiotic prophase (19). In the merged images, Rec11-HA or Psc3-HA is represented by red and Rec8-GFP by green. Early and middle prophase nuclei were selected according to the Rec8-GFP pattern. In rec11Δ, these nuclei cannot be discriminated because of the decrease of arm Rec8 throughout prophase. (B) Schematic map of chromosome 1 and the primers used (cnt, dg, lys1, mes1). ChIP assays (19) with GFP antibodies were used to measure Rec8-GFP, Rec11-GFP, or Psc3-GFP levels in wild-type cells and Rec8-GFP levels in rec11Δ cells throughout the indicated chromosome sites. The ratio of cnt/mes1 in each cell is shown at the bottom. (C) The locations of Rec8-GFP, Rec11-GFP, Psc3-GFP, and Psm3-GFP were examined during the progression of meiosis.

How do the two types of Rec8 complexes associate with distinct regions of chromosomes? Recent studies from our laboratory and others have shown that Rad21-Psc3 complexes preferentially assemble at pericentromeric heterochromatin regions. This assembly depends on histone H3 methylation by Clr4/Suv39h-Rik1 and the consequent binding of the heterochromatin protein Swi6/HP1 (18, 21, 22). Psc3 interacts with the chromodomain of Swi6 in a two-hybrid assay (18), whereas Rec11 does not. Therefore, Rec8-Psc3 complexes could target the centromeric regions via the Psc3-heterochromatin interaction. To test this possibility, we assessed the effects of swi6 or clr4 deletion on Rec8 localization to centromeres. The level of Rec8 association with pericentromeric regions is markedly reduced in these mutants, whereas Rec8 is still enriched at the central core (Fig. 3A). These results suggest that preferential localization of Rec8-Psc3 at centromeres is promoted by two independent mechanisms, a Clr4-Swi6–dependent mechanism at pericentromeric regions and a Clr4-Swi6–independent mechanism at the central core. The replacement of histone H3 by its variant CENP-A at the central core (23) might provide a basis for the localization of Rec8-Psc3 there.

Fig. 3.

Pericentromeric localization of Rec8-Psc3 requires histone H3 methyl transferase and heterochromatin and is required for persisting cohesion throughout meiosis I. (A) Location of Rec8-GFP at the centromeres was examined by ChIP assays in the indicated strains arrested at meiotic prophase. Central core (cnt, imr) and pericentromeric regions (dg, dh) were examined. (B) One of the homologs marked with cen1-GFP was monitored for segregation pattern in tetra-nucleated cells. (C) Separation of sister cen2-GFP dots after meiosis I is evident in clr4Δ cells. (D) The Rec8 signal is nearly lost from centromeres in clr4Δ cells arrested after meiosis I by the mes1 mutation.

The restriction of histone modification-dependent localization of cohesin to the pericentromeric region prompted us to address the role of pericentromeric cohesin in normal meiotic chromosome segregation. In swi6Δ and clr4Δ cells marked on one pair of sister chromatids with cen1-GFP, sister chromatid pairs move together to the same nucleus during meiosis I, indicating that monopolar attachment is intact in these mutants (Fig. 3B). Moreover, homologous chromosomes undergo faithful disjunction at meiosis I in these mutants (Fig. 3C) (17). At meiosis II, however, sisters fail to segregate properly, undergoing nondisjunction in 20 to 40% of cells (Fig. 3B). The defect in meiosis II is more penetrating in clr4Δ cells than in swi6Δ cells, with a pattern approximating random segregation. This is reconcilable with the more thorough decrease in pericentromeric Rec8 in clr4Δ cells (Fig. 3A). Reinforcing the foregoing results, clr4Δ cells frequently display precocious separation of cen2-GFP signals (Fig. 3C) and a decreased level of Rec8 at the centromeres if arrested after meiosis I (Fig. 3D). Thus, Clr4-Swi6–dependent enrichment of Rec8-Psc3 at pericentromeric regions is required to preserve centromeric cohesion through meiosis I, thereby ensuring equational segregation in meiosis II (Fig. 4). The defects accompanying loss of pericentromeric heterochromatin in fission yeast resemble those of Drosophila lacking the MEI-S332 protein, which is a proposed guardian of centromeric cohesion in meiosis II (24). Although we do not know the precise mechanism for how the centromeric cohesin is protected from degradation until meiosis II, our results suggest that pericentromeric cohesin might be the major target of protection from degradation at anaphase I. Given that pericentromeric Rec8 is dispensable for the monopolar attachment, Rec8 bound to the centromeric central core could be responsible for this role at meiosis I (Fig. 4), as predicted in our previous model (6, 20).

Fig. 4.

A model for the action of meiotic cohesins. The arm-specific complex Rec8-Rec11 required for recombination and for holding homologs is disrupted during meiosis I. The heterochromatin-dependent location of Rec8-Psc3 at pericentromeric regions is required for persisting centromere cohesion until meiosis II. The central core Rec8-Psc3 is presumably required for establishing monopolar attachment of sister kinetochores for meiosis I.

Previous studies have established that the cohesin complex in meiosis is different from that in mitosis. Our findings indicate that the species of cohesin complex also varies with location along the chromosome, thereby organizing distinct chromosome domains. Mammals have at least three Scc3-like proteins, STAG1-3/SA1-3. STAG3 is meiosis-specific, localizing along chromosome arms during meiotic prophase I but becoming undetectable thereafter (25), indicating that STAG3 is most likely an ortholog of Rec11. Drosophila also has a meiosis-specific Scc3-like protein (26), suggesting the conservation of this principle. During mitosis, a substantial release of cohesin from chromosome arms accompanies the process of chromosome condensation, producing two recognizable chromatids (27). Nonetheless, important levels of cohesin remain, especially at centromeres, until the onset of anaphase. Cohesin enrichment at centromeres during mitosis is mediated through an interaction between cohesin and heterochromatin in fission yeast and also, presumably, in mammals (18, 28). This system might be insufficient for the more complex process of meiosis, as cohesion along chromosome arms plays a crucial role in promoting recombination and holding homologs tightly together to ensure disjunction at meiosis I. Thus, the meiosis-specific cohesin subunit Rec11/STAG3 might have evolved to strengthen or develop the arm function of cohesin. We have shown that defects in the arm-specific Rec11-associated cohesin cause meiosis I nondisjunction. This phenotype differs markedly from the more disruptive chromosome segregation seen in rec8Δ. Therefore, such arm-specific cohesion factors may be particularly important to consider with regard to human reproductive problems like Down syndrome, which arises mainly from nondisjunction in meiosis I.

Supporting Online Material

www.sciencemag.org/cgi/content/full/300/5622/1152/DC1

Materials and Methods

Figs. S1 to S5

References

  • * These authors contributed equally to this work.

References and Notes

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