Centromere mapping functions for aneuploid meiotic products: Analysis of rec8, rec10 and rec11 mutants of the fission yeast Schizosaccharomyces pombe.

Recent evidence suggests that the position of reciprocal recombination events (crossovers) is important for the segregation of homologous chromosomes during meiosis I and sister chromatids during meiosis II. We developed genetic mapping functions that permit the simultaneous analysis of centromere-proximal crossover recombination and the type of segregation error leading to aneuploidy. The mapping functions were tested in a study of the rec8, rec10, and rec11 mutants of fission yeast. In each mutant we monitored each of the three chromosome pairs. Between 38 and 100% of the chromosome segregation errors in the rec8 mutants were due to meiosis I nondisjunction of homologous chromosomes. The remaining segregation errors were likely the result of precocious separation of sister chromatids, a previously described defect in the rec8 mutants. Between 47 and 100% of segregation errors in the rec10 and rec11 mutants were due to nondisjunction of sister chromatids during meiosis II. In addition, centromere-proximal recombination was reduced as much as 14-fold or more on chromosomes that had experienced nondisjunction. These results demonstrate the utility of the new mapping functions and support models in which sister chromatid cohesion and crossover position are important determinants for proper chromosome segregation in each meiotic division.     

Molecular Mechanisms of Homologous Chromosome Pairing and Segregation in Plants

In most eukaryotic species, three basic steps of pairing, recombination and synapsis occur during prophase of meiosis I. Homologous chromosomal pairing and recombination are essential for accurate segregation of chromosomes. In contrast to the well-studied processes such as recombination and synapsis, many aspects of chromosome pairing are still obscure. Recent progress in several species indicates that the telomere bouquet formation can facilitate homologous chromosome pairing by bringing chromosome ends into close proximity, but the sole presence of telomere clustering is not sufficient for recognizing homologous pairs. On the other hand, accurate segregation of the genetic material from parent to offspring during meiosis is dependent on the segregation of homologs in the reductional meiotic division （MI） with sister kinetochores exhibiting mono-orientation from the same pole, and the segregation of sister chromatids during the equational meiotic division （MII） with kinetochores showing bi-orientation from the two poles. The underlying mechanism of orientation and segregation is still unclear. Here we focus on recent studies in plants and other species that provide insight into how chromosomes find their partners and mechanisms mediating chromosomal segregation.     

A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.     

A component of C. elegans meiotic chromosome axes at the interface of homolog alignment, synapsis, nuclear reorganization, and recombination

A universal feature of meiotic prophase is the pairing of homologous chromosomes, a fundamental prerequisite for the successful completion of all subsequent meiotic events. HIM-3 is a Caenorhabditis elegans meiosis-specific non-cohesin component of chromosome axes that is required for synapsis. Our characterization of new him-3 alleles reveals previously unknown functions for the protein. HIM-3 is required for the establishment of initial contacts between homologs, for the nuclear reorganization characteristic of early meiotic prophase, and for the coordination of these events with synaptonemal complex (SC) assembly. Despite the absence of homolog alignment, we find that recombination is initiated efficiently, indicating that initial pairing is not a prerequisite for early steps of the recombination pathway. Surprisingly, RAD-51-marked recombination intermediates disappear with apparent wild-type kinetics in him-3 null mutants in which homologs are spatially unavailable for recombination, raising the possibility that HIM-3's presence at chromosome axes inhibits the use of sister chromatids as templates for repair. We propose that HIM-3 is a molecular link between multiple landmark events of meiotic prophase; it is critical for establishing chromosome identity by configuring homologs to facilitate their recognition while simultaneously imposing structural constraints that later promote the formation of the crossover essential for proper segregation.     

The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesin's meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events.     

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes.     

Slk19p is necessary to prevent separation of sister chromatids in meiosis I

BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis. It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region. The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery. RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I. When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies. The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores). Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I. In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells. Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I. CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I.     

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis,Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.     

Cis-Acting Determinants Affecting Centromere Function, Sister-Chromatid Cohesion and Reciprocal Recombination during Meiosis in Saccharomyces Cerevisiae

We have employed a system that utilizes homologous pairs of human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to assess the specific role (s) of conserved centromere DNA elements (CDEI, CDEII and CDEIII) in meiotic chromosome disjunction fidelity. Thirteen different centromere (CEN) mutations were tested for their effects on meiotic centromere function. YACs containing a wild-type CEN DNA sequence segregate with high fidelity in meiosis I (99% normal segregation) and in meiosis II (96% normal segregation). YACs containing a 31-bp deletion mutation in centromere DNA element II (CDEIIδ31) in either a heterocentric (mutant/wild type), homocentric (mutant/mutant) or monosomic (mutant/--) YAC pair configuration exhibited high levels (16-28%) of precocious sister-chromatid segregation (PSS) and increased levels (1-6%) of nondisjunction meiosis I (NDI). YACs containing this mutation also exhibit high levels (21%) of meiosis II nondisjunction. Interestingly, significant alterations in homolog recombination frequency were observed in the exceptional PSS class of tetrads, suggesting unusual interactions between prematurely separated sister chromatids and their homologous nonsister chromatids. We also have assessed the meiotic segregation effects of rare gene conversion events occurring at sites located immediately adjacent to or distantly from the centromere region. Proximal gene conversion events were associated with extremely high levels (60%) of meiosis I segregation errors (including both PSS and NDI), whereas distal events had no apparent effect. Taken together, our results indicate a critical role for CDEII in meiosis and underscore the importance of maintaining sister-chromatid cohesion for proper recombination in meiotic prophase and for proper disjunction in meiosis I.     

Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions.     

Grades of chromatid organisation in mitotic and meiotic chromosomes

It is suggested that not one, but four different grades of lampbrush chromosome organisation characterise different stages of mitosis and meiosis: (a) where a single chromatid organises symmetrically disposed lateral loops (second meiotic prophase), (b) where the two sister chromatids of a visibly single chromosome organise lateral loops in a laterally asymmetrical fashion, both sets of loops projecting from the same side and away from the face used, in meiosis, for pairing (early first meiotic prophase), (c) where the lateral loops organised by two visibly separate sister chromatids are symmetrically disposed on either side of the chromosome and project away from each other (mitotic prophase and late first meiotic prophase), (d) where the organisation is as in (c) but chromatid axes are intimately fused and form a visibly single strand (female amphibian diffuse diplotene).     

Pds5 is required for homologue pairing and inhibits synapsis of sister chromatids during yeast meiosis

During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.     

The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1

Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.     

Evolution of meiosis of unicellulate and multicellular eucaryotes. Aromorphosis at the cellular level

An attempt was undertaken to apply the concept elaborated for the evolution of multicellular organisms to that of unicellular eucaryotes. The latter's meiosis was formed on the basis of combination on three intracellular processes: 1) homologous DNA recombination, 2) chromosome disjunction with the assistance of mitotic apparatus, and 3) formation of "linear" chromosome elements consisting of specific proteins. Mechanism of homologous chromosome recombination was inherited from the archibacteria, while both the mitotic apparatus and "linear" chromosome elements emerged de novo. These elements appeared (resulting from appearance of the meiosis-specific proteins) as a complication of cohesion filaments, arising at the boundary between the sister chromatids after DNA replication. Homologous chromosome recombination made it possible for the chromosomes of diploid organisms to join pairwise by means of Holliday structures, while temporary blocking of hydrolysis of the linear elements at centromeres made it possible for the kinetochores to acquire unipolarity and for the sister chromatids to move to the same pole. All these provided for reduction of the chromosome number. Such a type of the reduction of chromosome number was retained by the extant imperfect ascomycetes Schizosaccharomyces pombe and Aspergillus nidulans, and by the infusorian Tetrahyrmena thermophila. It was the derivative of specific proteins, i.e. synaptonemal complexes (SCs). that appeared to be aromorphosis; they came to existence due to the pairwise joining of the chromosome "linear" elements by means of protein "zipper". The SCs join homologous chromosomes temporarily at the prophase of meiotic reduction division, thus optimizing condition for the crossing over and chiasma formation. The latter and the kinetochore unipolarity both provide for the chromosome disjunction. Kinetochore unipolarity is caused by the protein shugoshin which appears at meiotic prophase I and blocks cohesin hydrolysis at centromeres when anaphase I begins. This type of reductional division became the basis of the classical meiosis in the overwhelming majority of unicellular and multicellular organisms over all eucaryote kingdoms.     

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.     

Condensin restructures chromosomes in preparation for meiotic divisions

The production of haploid gametes from diploid germ cells requires two rounds of meiotic chromosome segregation after one round of replication. Accurate meiotic chromosome segregation involves the remodeling of each pair of homologous chromosomes around the site of crossover into a highly condensed and ordered structure. We showed that condensin, the protein complex needed for mitotic chromosome compaction, restructures chromosomes during meiosis in Caenorhabditis elegans. In particular, condensin promotes both meiotic chromosome condensation after crossover recombination and the remodeling of sister chromatids. Condensin helps resolve cohesin-independent linkages between sister chromatids and alleviates recombination-independent linkages between homologues. The safeguarding of chromosome resolution by condensin permits chromosome segregation and is crucial for the formation of discrete, individualized bivalent chromosomes.     

The cohesin REC8 prevents illegitimate inter‐sister synaptonemal complex assembly

During meiosis, a specialized chromosome structure is assembled to promote pairing/synapsis of homologous chromosomes and meiotic recombination, a process yielding chiasmata between homologs to ensure accurate segregation. Meiosis‐specific cohesin complexes mediating sister chromatid cohesion play pivotal roles in almost all these events, including synaptonemal complex (SC) formation. In this issue of EMBO Reports, Agostinho and colleagues have examined chromosome axes and SC structures by taking advantage of a hypomorphic Stag3 mutant in which the levels of the cohesin subunit REC8 are partly reduced 6 . Using super‐resolution microscopy, the authors illuminate previously unforeseen chromosome axis structures, showing locally separated axes in regions where REC8 is absent, regardless of RAD21L or RAD21 cohesin localization. Furthermore, they assessed the relationship between sister chromatid cohesion and inter‐sister SC formation, demonstrating that “axial opening” in the REC8‐free region is accompanied by illegitimate SC formation between sister chromatids. This study highlights the physiological importance of REC8 in sister chromatid cohesion and proper SC formation during meiosis, suggesting a new model in which a high density of REC8 deposition along the chromosome prevents illegitimate inter‐sister SC formation.     

OsSGO1 maintains synaptonemal complex stabilization in addition to protecting centromeric cohesion during rice meiosis

Shugoshin is a conserved protein in eukaryotes that protects the centromeric cohesin of sister chromatids from cleavage by separase during meiosis. In this study, we identify the rice (Oryza sativa, 2n=2x=24) homolog of ZmSGO1 in maize (Zea mays), named OsSGO1. During both mitosis and meiosis, OsSGO1 is recruited from nucleoli onto centromeres at the onset of prophase. In the Tos17-insertional Ossgo1-1 mutant, centromeres of sister chromatids separate precociously from each other from metaphase I, which causes unequal chromosome segregation during meiosis II. Moreover, the release of OsSGO1 from nucleoli is completely blocked in Ossgo1-1, which leads to the absence of OsSGO1 in centromeric regions after the onset of mitosis and meiosis. Furthermore, the timely assembly and maintenance of synaptonemal complexes during early prophase I are affected in Ossgo1 mutants. Finally, we found that the centromeric localization of OsSGO1 depends on OsAM1, not other meiotic proteins such as OsREC8, PAIR2, OsMER3, or ZEP1.