RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast

The regulation of higher-order chromosome structure is central to cell division and sexual reproduction. Heterochromatin assembly at the centromeres facilitates both kinetochore formation and sister chromatid cohesion, and the formation of specialized chromatin structures at telomeres serves to maintain the length of telomeric repeats, to suppress recombination, and to aid in formation of a bouquet-like structure that facilitates homologous chromosome pairing during meiosis. In fission yeast, genes encoding the Argonaute, Dicer, and RNA-dependent RNA polymerase factors involved in RNA interference (RNAi) are required for heterochromatin formation at the centromeres and mating type region. In this study, we examine the effects of deletions of the fission yeast RNAi machinery on chromosome dynamics during mitosis and meiosis. We find that the RNAi machinery is required for the accurate segregation of chromosomes. Defects in mitotic chromosome segregation are correlated with loss of cohesin at centromeres. Although the telomeres of RNAi mutants maintain silencing, length, and localization of the heterochromatin protein Swi6, we discovered defects in the proper clustering of telomeres in interphase mitotic cells. Furthermore, a small proportion of RNAi mutant cells display aberrant telomere clustering during meiotic prophase. This study demonstrates that the fission yeast RNAi machinery is required for the proper regulation of chromosome architecture during mitosis and meiosis.     

Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe

In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.     

Meiosis in haploid yeast

Haploid yeast cells normally contain either the MATa or MATα mating-type allele and cannot undergo meiosis and spore formation. If both mating-type alleles are present as a consequence of chromosome III disomy (MATa/MATα), haploids initiate meiosis but do not successfully form spores, probably because the haploid chromosome complement is irregularly partitioned during meiotic nuclear division. We have demonstrated that the ochre-suppressible mutation spo13-1 enables haploid yeast cells disomic for chromosome III and heterozygous at the mating-type locus to complete meiosis and spore formation, yielding two haploid spores. Previous studies have shown that the absence of the wild-type SPO13 gene function permits diploid cells to bypass homologous chromosome segregation at meiosis I and proceed directly to meiosis II. During spo13-1 haploid meiosis, cells enter prophase of meiosis I. Genetic recombination, monitored on the chromosome III disome, occurs at levels similar to those seen in diploids, indicating that the level of exchange between homologs is an autonomous property of individual chromosomes and not dependent on exchange elsewhere in the genome. Exchange is then followed by a single meiosis II equational chromosome division. Recombination in spo13-1 haploids is blocked by the spo11-1 mutation, which also eliminates recombination between homologous chromosomes during conventional diploid meiosis. We conclude that Spo⁺ haploids expressing both a and α mating-type information attempt a SPO13-dependent meiosis I division, and that this division, in the absence of paired homologous chromosomes, is responsible for the failure of such haploids to complete normal gametogenesis. Our observations support the conclusion that initiation and completion of meiosis II and spore formation are not dependent on either completion of meiosis I or the presence of a diploid chromosome complement.     

Initiation of recombination in Saccharomyces cerevisiae haploid meiosis.

In most eukaryotes during prophase I of meiosis, homologous chromosomes pair and recombine by coordinated molecular and cellular processes. To directly test whether or not the early steps of the initiation of recombination depend on the presence of a homologous chromosome, we have examined the formation and processing of DNA double-strand breaks (DSBs, the earliest physical landmark of recombination initiation) in various haploid Saccharomyces cerevisiae strains capable of entering meiosis. We find that DSBs occur in haploid meiosis, showing that the presence of a homolog is not required for DSB formation. DSBs occur at the same positions in haploid and diploid meioses. However, these two types of meiosis exhibit subtle differences with respect to the timing of formation and levels of DSBs. In haploid meiosis, a slower rate of DSB formation and a reduction in the frequency of DSB (at one of the three sites analyzed) were observed. These results might indicate that interactions between homologs play a role in the formation of meiotic DSBs. Furthermore, haploid strains exhibit a pronounced delay in the disappearance of meiotic DSBs compared to diploid strains, which suggests that sister chromatid interactions for DSB repair are inhibited in haploid meiosis.     

Physical association between nonhomologous chromosomes precedes distributive disjunction in yeast.

During meiosis homologous chromosomes normally pair, undergo reciprocal recombination, and then segregate from each other. Distributive disjunction is the meiotic segregation that is observed in the absence of homologous recombination and can occur for both nonrecombinant homologous chromosomes and completely nonhomologous chromosomes. While the mechanism of distributive disjunction is not known, several models have been presented that either involve or are completely independent of interactions between the segregating chromosomes. In this report, we demonstrate that distributive disjunction in Saccharomyces cerevisiae is preceded by an interaction between nonhomologous chromosomes.     

Polo kinase Cdc5 is a central regulator of meiosis I

During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5’s central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern.Polo kinases are central regulators of chromosome segregation and control multiple mitotic events (1). Budding yeast contains a single Polo kinase, CDC5. Unlike in higher eukaryotes, budding yeast CDC5 primarily regulates postmetaphase events, its essential function being to trigger exit from mitosis (2). CDC5 also contributes to the efficient inactivation of cohesins, the protein complexes that hold sister chromatids together until the onset of chromosome segregation. Cdc5 phosphorylates the cohesin subunit Mcd1/Scc1 to facilitate its cleavage by the protease separase (3).CDC5 also regulates the specialized cell division that gives rise to gametes, known as meiosis (4). During meiosis, two consecutive rounds of chromosome segregation follow one round of DNA replication. During meiosis I, homologous chromosomes segregate; during meiosis II, sister chromatids separate (5). The chromosome segregation machinery is modified in three ways to facilitate the unusual meiosis I division. First, the combination of homologous recombination and cohesin complexes distal to the resulting cross-overs mediate the physical linkage of homologous chromosomes, which is essential for their accurate segregation during meiosis I. Second, sister chromatids of each homolog must be segregated to the same pole rather than to opposite poles, as they are during mitosis. The fusion of sister kinetochores by co-orientation factors (the monopolin complex in yeast) facilitates the attachment of microtubules emanating from one spindle pole. Third, cohesin complexes must be lost in a stepwise manner from chromosomes. During meiosis I cohesin complexes are lost from chromosome arms to bring about the segregation of homologous chromosomes (6). The residual cohesins at centromeres facilitate the accurate segregation of sister chromatids during meiosis II. Cdc5 has been implicated in the execution of all three meiosis I-specific events. CDC5 is required for the resolution of double Holliday junctions during homologous recombination (7, 8). Cdc5 also controls the co-orientation of sister chromatids by promoting the association of the monopolin complex with kinetochores (7, 9). Finally, CDC5 has been implicated in regulating the stepwise loss of cohesins (7, 9, 10). Phosphorylation of the cohesin subunit Rec8, a meiosis-specific cohesin subunit that replaces Scc1/Mcd1 in the meiotic cohesin complex, controls the stepwise loss of cohesins from chromosomes. Rec8 phosphorylation is critical for its proteolytic cleavage and removal from chromosome arms during meiosis I (10, 11). Maintaining Rec8 in a dephosphorylated form around centromeric regions protects it from cleavage. This is accomplished by Sgo1, a shugoshin/MEI-S332 family member that recruits protein phosphatase 2A to centromeric regions (12). Our studies have implicated Cdc5 as one, but not the only, protein kinase phosphorylating Rec8 to target it for proteolytic cleavage by separase (10).In addition to controlling meiosis I-specific events, CDC5 also regulates general cell-cycle functions during meiosis I that it does not affect during mitosis. During meiosis I, CDC5 controls separase activity. Degradation of the separase inhibitor securin (Pds1 in yeast) liberates separase to trigger anaphase (5). During meiosis I, but not during mitosis, CDC5 is required for Pds1 degradation (7, 9). How Cdc5 takes on new functions during meiosis I is not understood. Similarly, little is known about whether and how Cdc5 functions during meiosis II because cells depleted for Cdc5 arrest in metaphase I (7, 9).Here we show that CDC5 controls cohesin removal in multiple ways. CDC5-dependent phosphorylation of Rec8 is essential for efficient Rec8 cleavage. Furthermore, Cdc5 triggers the degradation of Spo13, thereby contributing to the dismantling of the cohesin-protective domain around centromeres. Our data further show that despite its central role in meiosis I chromosome segregation, CDC5 is dispensable during meiosis II and does not phosphorylate its meiosis I targets during meiosis II. Our findings indicate that the evolution of additional CDC5 functions is a central aspect of establishing the unique meiotic chromosome segregation pattern.     

From the Cover: PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.     

Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice

Errors in meiotic chromosome segregation are the leading cause of spontaneous abortions and birth defects. Almost all such aneuploidy derives from meiotic errors in females, with increasing maternal age representing a major risk factor. It was recently reported that histones are globally deacetylated in mammalian oocytes during meiosis but not mitosis. In the present study, inhibition of meiotic histone deacetylation was found to induce aneuploidy in fertilized mouse oocytes, which resulted in embryonic death in utero at an early stage of development. In addition, a histone remained acetylated in the oocytes of older (10-month-old) female mice, suggesting that the function for histone deacetylation is decreased in the oocytes of such mice. Thus, histone deacetylation may be involved in the fair distribution of chromosomes during meiotic division. The high incidence of aneuploidy in the embryos of older females may be due to inadequate meiotic histone deacetylation.     

Loss of DNA methylation affects the recombination landscape in Arabidopsis

During sexual reproduction, one-half of the genetic material is deposited in gametes, and a complete set of chromosomes is restored upon fertilization. Reduction of the genetic information before gametogenesis occurs in meiosis, when cross-overs (COs) between homologous chromosomes secure an exchange of their genetic information. COs are not evenly distributed along chromosomes and are suppressed in chromosomal regions encompassing compact, hypermethylated centromeric and pericentromeric DNA. Therefore, it was postulated that DNA hypermethylation is inhibitory to COs. Here, when analyzing meiotic recombination in mutant plants with hypomethylated DNA, we observed unexpected and counterintuitive effects of DNA methylation losses on CO distribution. Recombination was further promoted in the hypomethylated chromosome arms while it was inhibited in heterochromatic regions encompassing pericentromeric DNA. Importantly, the total number of COs was not affected, implying that loss of DNA methylation led to a global redistribution of COs along chromosomes. To determine by which mechanisms altered levels of DNA methylation influence recombination--whether directly in cis or indirectly in trans by changing expression of genes encoding recombination components--we analyzed CO distribution in wild-type lines with randomly scattered and well-mapped hypomethylated chromosomal segments. The results of these experiments, supported by expression profiling data, suggest that DNA methylation affects meiotic recombination in cis. Because DNA methylation exhibits significant variation even within a single species, our results imply that it may influence the evolution of plant genomes through the control of meiotic recombination.     

Long-range organization of tandem arrays of alpha satellite DNA at the centromeres of human chromosomes: high-frequency array-length polymorphism and meiotic stability.

The long-range organization of arrays of alpha satellite DNA at the centromeres of human chromosomes was investigated by pulsed-field gel electrophoresis techniques. Both restriction-site and array-length polymorphisms were detected in multiple individuals and their meiotic segregation was observed in three-generation families. Such variation was detected in all of the alpha satellite arrays examined (chromosomes 1, 3, 7, 10, 11, 16, 17, X, and Y) and thus appears to be a general feature of human centromeric DNA. The length of individual centromeric arrays was found to range from an average of approximately 680 kilobases (kb) for the Y chromosome to approximately 3000 kb for chromosome 11. Furthermore, individual arrays appear to be meiotically stable, since no changes in fragment lengths were observed. In total, we analyzed 84 meiotic events involving approximately 191,000 kb of alpha satellite DNA from six autosomal centromeres without any evidence for recombination within an array. High-frequency array length variation and the potential to detect meiotic recombination within them allow direct comparisons of genetic and physical distances in the region of the centromeres of human chromosomes. The generation of primary consensus physical maps of alpha satellite arrays is a first step in the characterization of the centromeric DNA of human chromosomes.     

Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.     

Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation

Saccharomyces cerevisiae Hop2 and Mnd1 are abundant meiosisspecific chromosomal proteins, and mutations in the corresponding genes lead to defects in meiotic recombination and in homologous chromosome interactions during mid-prophase. Analysis of various double mutants suggests that HOP2, MND1, and DMC1 act in the same genetic pathway for the establishment of close juxtaposition between homologous meiotic chromosomes. Biochemical studies indicate that Hop2 and Mnd1 proteins form a stable heterodimer with a higher affinity for double-stranded than single-stranded DNA, and that this heterodimer stimulates the strand assimilation activity of Dmc1 in vitro. Together, the genetic and biochemical results suggest that Hop2, Mnd1, and Dmc1 are functionally interdependent during meiotic DNA recombination.     

Restriction of ectopic recombination by interhomolog interactions during Saccharomyces cerevisiae meiosis

In Saccharomyces cerevisiae meiosis, recombination occurs frequently between sequences at the same location on homologs (allelic recombination) and can take place between dispersed homologous sequences (ectopic recombination). Ectopic recombination occurs less often than does allelic, especially when homologous sequences are on heterologous chromosomes. To account for this, it has been suggested that homolog pairing (homolog colocalization and alignment) either promotes allelic recombination or restricts ectopic recombination. The latter suggestion was tested by examining ectopic recombination in two cases where normal interhomolog relationships are disrupted. In the first case, one member of a homolog pair was replaced by a homologous (related but not identical) chromosome that has diverged sufficiently to prevent allelic recombination. In the second case, ndj1 mutants were used to delay homolog pairing and synapsis. Both circumstances resulted in a substantial increase in the frequency of ectopic recombination between arg4-containing plasmid inserts located on heterologous chromosomes. These findings suggest that, during normal yeast meiosis, progressive homolog colocalization, alignment, synapsis, and allelic recombination restrict the ability of ectopically located sequences to find each other and recombine. In the absence of such restrictions, the meiotic homology search may encompass the entire genome.     

The synaptonemal complex protein,Zip1, promotes the segregation of nonexchange chromosomes at meiosis I

Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.     

Cohesins are required for meiotic DNA breakage and recombination in Schizosaccharomyces pombe

In preparation for the unique segregation of homologs at the first meiotic division, chromosomes undergo dramatic changes. The meiosis-specific sister chromatid cohesins Rec8 and Rec11 of Schizosaccharomyces pombe are recruited around the time of premeiotic replication, and Rec10, a component of meiosis-specific linear elements, is subsequently added. Here we report that Rec10 is essential for meiosis-specific DNA breakage by Rec12 (Spo11 homolog) and for meiotic recombination. DNA breakage and recombination also depend on the Rec8 and Rec11 cohesins, strictly in some genomic intervals but less so in others. Thus, in addition to their previously recognized role in meiotic chromosome segregation, cohesins have a direct role, as do linear element components, in meiotic recombination by enabling double-strand DNA break formation by Rec12. Our results reveal a pathway, whose regulation is significantly different from that in the distantly related yeast Saccharomyces cerevisiae, for meiosis-specific chromosome differentiation and high-frequency recombination.     

Construction of telocentric chromosomes in Saccharomyces cerevisiae.

We describe a simple method for the construction of large chromosomal deletions in yeast. Diploid yeast cells were transformed with DNA fragments that replace large regions of the chromosomes by homologous recombination. Using this method, we have constructed a telocentric chromosome III in which approximately equal to 100 kilobases (kb) of DNA has been removed from the left arm of the chromosome, so that the centromere is 12 kb from the left telomere. This telocentric chromosome is mitotically stable. Its rate of loss in a diploid strain is 2.5-7.4 X 10(-4) per cell division compared to a rate of loss of 0.36-1.8 X 10(-4) per cell division for a normal chromosome III. It also segregates 2+:2- with fidelity during meiosis. The construction of systematic deletions in a chromosome should be useful in determining the essential features for proper chromosomal segregation and replication.     

Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope

Dynamic repositioning of telomeres is a unique feature of meiotic prophase I that is highly conserved among eukaryotes. At least in fission yeast it was shown to be required for proper alignment and recombination of homologous chromosomes. On entry into meiosis telomeres attach to the nuclear envelope and transiently cluster at a limited area to form a chromosomal bouquet. Telomere clustering is thought to promote chromosome recognition and stable pairing of the homologs. However, the molecular basis of telomere attachment and movement is largely unknown. Here we report that mammalian SUN-domain protein Sun2 specifically localizes to the nuclear envelope attachment sites of meiotic telomeres. Sun2-telomere association is maintained throughout the dynamic movement of telomeres. This association does not require the assembly of chromosomal axial elements or the presence of A-type lamins. Detailed EM analysis revealed that Sun2 is part of a membrane-spanning fibrillar complex that interconnects attached telomeres with cytoplasmic structures. Together with recent findings in fission yeast, our study indicates that the molecular mechanisms required for tethering meiotic telomeres and their dynamic movements during bouquet formation are conserved among eukaryotes.     

A Possible Effect of Heterochromatin on Chromosome Pairing

Rye chromosomes were selectively stained in the meiosis of triticale by means of heterochromatin banding techniques. Compared to wheat chromosomes, rye chromosomes showed reduced pairing at first meiotic metaphase. Within the rye genome this pairing failure was associated with the presence of large, terminal heterochromatic bands. Since these terminal bands of rye chromosomes are late replicating, the effect of heterochromatin could arise from an overlap between the processes of chromosome replication and chromosome pairing.