Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination.

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.

Linear elements are stable structures along the chromosome axis in fission yeast meiosis.

The structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure called a synaptonemal complex (SC) is formed in many eukaryotes. However, in the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), which are structures related to axial elements of the SC, form on the meiotic cohesin-based chromosome axis. The structure of LinEs has been observed using silver-stained electron micrographs or in immunofluorescence-stained spread nuclei. However, the fine structure of LinEs and their dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) of the core components of LinEs (Rec10, Rec25, Rec27, Mug20) and a linE-binding protein Hop1. We found that LinEs form along the chromosome axis and elongate during meiotic prophase. 3D-SIM microscopy revealed that Rec10 localized to meiotic chromosomes in the absence of other LinE proteins, but shaped into LinEs only in the presence of all three other components, the Rec25, Rec27, and Mug20. Elongation of LinEs was impaired in double-strand break-defective rec12- cells. The structure of LinEs persisted after treatment with 1,6-hexanediol and showed slow fluorescence recovery from photobleaching. These results indicate that LinEs are stable structures resembling axial elements of the SC.

Maintenance of meiotic crossover against reduced double-strand break formation in fission yeast lacking histone H2A.Z.

Meiotic crossover (CO) recombination initiates from programmed DNA double-strand breaks (DSBs) around hotspots, and results in reciprocal exchange of chromosome segments between homologous chromosomes (homologs). COs are crucial for most sexually-reproducing organisms because they promote accurate chromosome segregation and create genetic diversity. Therefore, faithful accomplishment of CO formation is ensured in many ways, but the bases of the regulation are not fully understood. Our previous study using fission yeast has revealed that mutants lacking the conserved histone H2A.Z are defective in DSB formation but maintain CO frequency at three loci tested. Here, we tested five additional sites to show that mutants lacking H2A.Z exhibit normal and increased CO frequency at two and three loci, respectively. Examining one of the CO-increased intervals in the mutant revealed that the CO upregulation is mediated at least partly at a recombination intermediate level. In addition, our genetic as well as genome-wide analyses implied a possibility that, even without H2A.Z, COs are maintained by weak and non-hotspot DSBs, which are processed preferentially as CO. These observations provide clues to further our understanding on CO control.

Chromosome-associated RNA-protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe.

Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA-protein complexes is required for robust pairing. Moreover, the lncRNA-protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA-protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.

Histone H2A insufficiency causes chromosomal segregation defects due to anaphase chromosome bridge formation at rDNA repeats in fission yeast.

The nucleosome, composed of DNA and a histone core, is the basic structural unit of chromatin. The fission yeast Schizosaccharomyces pombe has two genes of histone H2A, hta1+ and hta2+; these genes encode two protein species of histone H2A (H2Aα and H2Aß, respectively), which differ in three amino acid residues, and only hta2+ is upregulated during meiosis. However, it is unknown whether S. pombe H2Aα and H2Aß have functional differences. Therefore, in this study, we examined the possible functional differences between H2Aα and H2Aß during meiosis in S. pombe. We found that deletion of hta2+, but not hta1+, causes defects in chromosome segregation and spore formation during meiosis. Meiotic defects in hta2+ deletion cells were rescued by expressing additional copies of hta1+ or by expressing hta1+ from the hta2 promoter. This indicated that the defects were caused by insufficient amounts of histone H2A, and not by the amino acid residue differences between H2Aα and H2Aß. Microscopic observation attributed the chromosome segregation defects to anaphase bridge formation in a chromosomal region at the repeats of ribosomal RNA genes (rDNA repeats). These results suggest that histone H2A insufficiency affects the chromatin structures of rDNA repeats, leading to chromosome missegregation in S. pombe.

The conserved histone variant H2A.Z illuminates meiotic recombination initiation.

Meiotic recombination ensures faithful chromosome segregation and confers genetic diversity to gametes, and thus, is a key DNA-templated reaction not only for sexual reproduction, but also evolution. This recombination is initiated by programmed DNA double strand breaks (DSBs), which are mainly formed at recombination hotspots. As meiotic DSB formation requires multiple proteins, it is regulated by chromatin structure. In particular, DSB occurs in a higher-order chromatin architecture termed "axis-loop", in which many loops protrude from proteinaceous axis. Previous studies have suggested that assembly of this structure is dependent on chromatin binding of cohesin, which in turn recruits proteins implicated in DSB formation. However, roles of chromatin in meiotic DSB formation are not fully characterized. This review article summarizes our recent report showing that the conserved histone H2A variant H2A.Z promotes meiotic DSB formation in fission yeast. Through a series of experiments, we found that, in H2A.Z-lacking mutants, multiple proteins involved in DSB formation, but not cohesin subunits, are less associated with chromatin. Strikingly, nuclei were more compact in the absence of H2A.Z. These observations led us to propose that fission yeast H2A.Z promotes meiotic DSB formation partly through modulating chromosome architecture to enhance interaction between DSB-related proteins and cohesin-loaded chromatin. In addition, biological implications of our findings are discussed, and their relevance to DSB formation in other species as well as to other DNA-related events are also provided.

The histone variant H2A.Z promotes initiation of meiotic recombination in fission yeast.

Meiotic recombination is initiated by programmed formation of DNA double strand breaks (DSBs), which are mainly formed at recombination hotspots. Meiotic DSBs require multiple proteins including the conserved protein Spo11 and its cofactors, and are influenced by chromatin structure. For example, local chromatin around hotspots directly impacts DSB formation. Moreover, DSB is proposed to occur in a higher-order chromatin architecture termed 'axis-loop', in which many loops protrude from cohesin-enriched axis. However, still much remains unknown about how meiotic DSBs are generated in chromatin. Here, we show that the conserved histone H2A variant H2A.Z promotes meiotic DSB formation in fission yeast. Detailed investigation revealed that H2A.Z is neither enriched around hotspots nor axis sites, and that transcript levels of DSB-promoting factors were maintained without H2A.Z. Moreover, H2A.Z appeared to be dispensable for chromatin binding of meiotic cohesin. Instead, in H2A.Z-lacking mutants, multiple proteins involved in DSB formation, such as the fission yeast Spo11 homolog and its regulators, were less associated with chromatin. Remarkably, nuclei were more compact in the absence of H2A.Z. Based on these, we propose that fission yeast H2A.Z promotes meiotic DSB formation partly through modulating chromosome architecture to enhance interaction between DSB-related proteins and cohesin-loaded chromatin.

Microscopic Observation of Living Cells Stained with Fluorescent Probes.

Fluorescence imaging of living cells provides a unique opportunity to follow dynamic behavior of specific molecules under physiological conditions. In the fission yeast Schizosaccharomyces pombe, expression of a target protein genetically fused with a fluorescent protein such as the jellyfish green fluorescent protein (GFP) is widely used. In addition, fluorescent chemical reagents are also used to stain specific molecules (e.g., Hoechst 33324 to stain DNA). Specimens of S. pombe cells for live cell imaging are prepared by either of two methods: sandwiching the cells between glass coverslips and by mounting the cells on a glass-bottom culture dish. For time-lapse observation, it is necessary to immobilize fission yeast cells on the glass surface of the glass-bottom dish because they are nonadherent and tend to move easily as a result of stage movement, convection flow of culture medium, and the contact and pushing of neighboring cells during cell growth. Either concanavalin A or soybean lectin, which bind to S. pombe cell walls, can be used for immobilization. Considerations for sample preparations and observation conditions are described.

Visualization of a Specific Genome Locus by the lacO/LacI-GFP System.

Observing the dynamics of a specific chromosome locus in living cells can provide important information as to the molecular mechanisms underlying events such as chromosome segregation, homologous chromosome pairing, chromosome arrangement, and gene expression. The lacO/LacI-GFP system provides a simple and useful method in which a chromosome locus is visualized by inserting lacO repeat arrays and then expressing an LacI-GFP fusion that specifically binds to the lacO sequence. This system has been adapted for use in Schizosaccharomyces pombe by expressing the LacI-GFP under a promoter of the dis1+ gene. Furthermore, a two-step integration method has been developed that ensures high-efficiency integration of lacO arrays to a desired target position.

A cohesin-based structural platform supporting homologous chromosome pairing in meiosis.

The pairing and recombination of homologous chromosomes during the meiotic prophase is necessary for the accurate segregation of chromosomes in meiosis. However, the mechanism by which homologous chromosomes achieve this pairing has remained an open question. Meiotic cohesins have been shown to affect chromatin compaction; however, the impact of meiotic cohesins on homologous pairing and the fine structures of cohesion-based chromatin remain to be determined. A recent report using live-cell imaging and super-resolution microscopy demonstrated that the lack of meiotic cohesins alters the chromosome axis structures and impairs the pairing of homologous chromosomes. These results suggest that meiotic cohesin-based chromosome axis structures are crucial for the pairing of homologous chromosomes.

Meiotic cohesin-based chromosome structure is essential for homologous chromosome pairing in Schizosaccharomyces pombe.

Chromosome structure is dramatically altered upon entering meiosis to establish chromosomal architectures necessary for the successful progression of meiosis-specific events. An early meiotic event involves the replacement of the non-SMC mitotic cohesins with their meiotic equivalents in most part of the chromosome, forming an axis on meiotic chromosomes. We previously demonstrated that the meiotic cohesin complex is required for chromosome compaction during meiotic prophase in the fission yeast Schizosaccharomyces pombe. These studies revealed that chromosomes are elongated in the absence of the meiotic cohesin subunit Rec8 and shortened in the absence of the cohesin-associated protein Pds5. In this study, using super-resolution structured illumination microscopy, we found that Rec8 forms a linear axis on chromosomes, which is required for the organized axial structure of chromatin during meiotic prophase. In the absence of Pds5, the Rec8 axis is shortened whereas chromosomes are widened. In rec8 or pds5 mutants, the frequency of homologous chromosome pairing is reduced. Thus, Rec8 and Pds5 play an essential role in building a platform to support the chromosome architecture necessary for the spatial alignment of homologous chromosomes.

Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast.

It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or 'knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

The role of chromosomal retention of noncoding RNA in meiosis.

Meiosis is a process of fundamental importance for sexually reproducing eukaryotes. During meiosis, homologous chromosomes pair with each other and undergo homologous recombination, ultimately producing haploid sets of recombined chromosomes that will be inherited by the offspring. Compared with the extensive progress that has been made in understanding the molecular mechanisms underlying recombination, how homologous sequences pair with each other is still poorly understood. The diversity of the underlying mechanisms of pairing present in different organisms further increases the complexity of this problem. Involvement of meiosis-specific noncoding RNA in the pairing of homologous chromosomes has been found in the fission yeast Schizosaccharomyces pombe. Although different organisms may have developed other or additional systems that are involved in chromosome pairing, the findings in S. pombe will provide new insights into understanding the roles of noncoding RNA in meiosis.

Chromosomally-retained RNA mediates homologous pairing.

Pairing and recombination of homologous chromosomes are essential for ensuring correct segregation of chromosomes in meiosis. In S. pombe, chromosomes are first bundled at the telomeres (forming a telomere bouquet) and then aligned by oscillatory movement of the elongated "horsetail" nucleus. Telomere clustering and subsequent chromosome alignment promote pairing of homologous chromosomes. However, this telomere-bundled alignment of chromosomes cannot be responsible for the specificity of chromosome pairing. Thus, there must be some mechanism to facilitate recognition of homologous partners after telomere clustering. Recent studies in S. pombe have shown that RNA transcripts retained on the chromosome, or RNA bodies, may play a role in recognition of homologous chromosomes for pairing. Acting as fiducial markers of homologous loci they would abrogate the need for direct DNA sequence homology searching.

Hexanucleotide motifs mediate recruitment of the RNA elimination machinery to silent meiotic genes.

The selective elimination system blocks the accumulation of meiosis-specific mRNAs during the mitotic cell cycle in fission yeast. These mRNAs harbour a region, the determinant of selective removal (DSR), which is recognized by a YTH-family RNA-binding protein, Mmi1. Mmi1 directs target transcripts to destruction in association with nuclear exosomes. Hence, the interaction between DSR and Mmi1 is crucial to discriminate mitosis from meiosis. Here, we show that Mmi1 interacts with repeats of the hexanucleotide U(U/C)AAAC that are enriched in the DSR. Disruption of this 'DSR core motif' in a target mRNA inhibits its elimination. Tandem repeats of the motif can function as an artificial DSR. Mmi1 binds to it in vitro. Thus, a core motif cluster is responsible for the DSR activity. Furthermore, certain variant hexanucleotide motifs can augment the function of the DSR core motif. Notably, meiRNA, which composes the nuclear Mei2 dot required to suppress Mmi1 activity during meiosis, carries numerous copies of the core/augmenting motifs on its tail and is indeed degraded by the Mmi1/exosome system, indicating its likely role as decoy bait for Mmi1.

Meiosis-specific noncoding RNA mediates robust pairing of homologous chromosomes in meiosis.

Pairing and recombination of homologous chromosomes are essential for ensuring reductional segregation in meiosis. However, the mechanisms by which chromosomes recognize their homologous partners are poorly understood. Here, we report that the sme2 gene encodes a meiosis-specific noncoding RNA that mediates homologous recognition in the fission yeast Schizosaccharomyces pombe. The sme2 locus shows robust pairing from early in meiotic prophase. The sme2 RNA transcripts accumulate at their respective gene loci and greatly enhance pairing of homologous loci: Deletion of the sme2 sequence eliminates this robust pairing, whereas transposition to other chromosomal sites confers robust pairing at those ectopic sites. Thus, we propose that RNA transcripts retained on the chromosome play an active role in recognition of homologous chromosomes for pairing.

Virtual breakdown of the nuclear envelope in fission yeast meiosis.

Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.

From meiosis to postmeiotic events: alignment and recognition of homologous chromosomes in meiosis.

Recombination of homologous chromosomes is essential for correct reductional segregation of homologous chromosomes, which characterizes meiosis. To accomplish homologous recombination, chromosomes must find their homologous partners and pair with them within the spatial constraints of the nucleus. Although various mechanisms have developed in different organisms, two major steps are involved in the process of pairing: first, alignment of homologous chromosomes to bring them close to each other for recognition; and second, recognition of the homologous partner of each chromosome so that they can form an intimate pair. Here, we discuss the various mechanisms used for alignment and recognition of homologous chromosomes in meiosis.

Localization of gene products using a chromosomally tagged GFP-fusion library in the fission yeast Schizosaccharomyces pombe.

We constructed a library of chromosomally-tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3'-end of a particular chromosomal ORF such that the full-length GFP fusion construct is expressed under the control of the original promoter. Integration of the GFP coding sequence at the authentic chromosomal location of each gene was confirmed by PCR. Microscopic screening of these strains detected sufficient levels of GFP signal in 710 strains and allowed assignment of these GFP-fusion gene products with their intracellular localization: 374 proteins were localized in the nucleus, 65 proteins in the nucleolus, 34 proteins at the nuclear periphery, 27 proteins at the plasma membrane and cytoplasmic membranous structures, 24 proteins at the spindle pole body and microtubules, 92 proteins at cytoplasmic structures, and 94 proteins were uniformly distributed throughout the cytoplasm.

Meiotic cohesins modulate chromosome compaction during meiotic prophase in fission yeast.

The meiotic cohesin Rec8 is required for the stepwise segregation of chromosomes during the two rounds of meiotic division. By directly measuring chromosome compaction in living cells of the fission yeast Schizosaccharomyces pombe, we found an additional role for the meiotic cohesin in the compaction of chromosomes during meiotic prophase. In the absence of Rec8, chromosomes were decompacted relative to those of wild-type cells. Conversely, loss of the cohesin-associated protein Pds5 resulted in hypercompaction. Although this hypercompaction requires Rec8, binding of Rec8 to chromatin was reduced in the absence of Pds5, indicating that Pds5 promotes chromosome association of Rec8. To explain these observations, we propose that meiotic prophase chromosomes are organized as chromatin loops emanating from a Rec8-containing axis: the absence of Rec8 disrupts the axis, resulting in disorganized chromosomes, whereas reduced Rec8 loading results in a longitudinally compacted axis with fewer attachment points and longer chromatin loops.