Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa.

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using "meiotic silencing by unpaired DNA" (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)-dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity.

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

Phase separation drives pairing of homologous chromosomes.

Pairing of homologous chromosomes is crucial for ensuring accurate segregation of chromosomes during meiosis. Molecular mechanisms of homologous chromosome pairing in meiosis have been extensively studied in the fission yeast Schizosaccharomyces pombe. In this organism, meiosis-specific noncoding RNA transcribed from specific genes accumulates at the respective gene loci, and chromosome-associated RNA-protein complexes mediate meiotic pairing of homologous loci through phase separation. Pairing of homologous chromosomes also occurs in somatic diploid cells in certain situations. For example, somatic pairing of homologous chromosomes occurs during the early embryogenesis in diptera, and relies on the transcription-associated chromatin architecture. Earlier models also suggest that transcription factories along the chromosome mediate pairing of homologous chromosomes in plants. These studies suggest that RNA bodies formed on chromosomes mediate the pairing of homologous chromosomes. This review summarizes lessons from S. pombe to provide general insights into mechanisms of homologous chromosome pairing mediated by phase separation of chromosome-associated RNA-protein complexes.

Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

Extranuclear Structural Components that Mediate Dynamic Chromosome Movements in Yeast Meiosis.

Telomere-led rapid chromosome movements or rapid prophase movements direct fundamental meiotic processes required for successful haploidization of the genome. Critical components of the machinery that generates rapid prophase movements are unknown, and the mechanism underlying rapid prophase movements remains poorly understood. We identified S. cerevisiae Mps2 as the outer nuclear membrane protein that connects the LINC complex with the cytoskeleton. We also demonstrate that the motor Myo2 works together with Mps2 to couple the telomeres to the actin cytoskeleton. Further, we show that Csm4 interacts with Mps2 and is required for perinuclear localization of Myo2, implicating Csm4 as a regulator of the Mps2-Myo2 interaction. We propose a model in which the newly identified functions of Mps2 and Myo2 cooperate with Csm4 to drive chromosome movements in meiotic prophase by coupling telomeres to the actin cytoskeleton.

Principles of meiotic chromosome assembly revealed in S. cerevisiae.

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

Multicomponent Yeast Two-Hybrid System: Applications to Study Protein-Protein Interactions in SMC Complexes.

Analysis of protein-protein interactions (PPI) is key for the understanding of most protein assemblies including structural maintenance of chromosomes (SMC) complexes. SMC complexes are composed of SMC proteins, kleisin, and kleisin-interacting subunits. These subunits interact in specific ways to constitute and regulate the closed structure of the complexes. Specifically, kleisin molecules bridge the SMC dimers and the kleisin-interacting subunits modulate stability of the bridge. Here we describe a multicomponent version of a yeast two-hybrid (Y2H) method and its application for analysis of the bridging role of the Nse4 kleisin in the SMC5/6 complex. Using this technique, we also show a stabilizing effect of KITE (kleisin-interacting tandem winged-helix element) proteins on SMC5/6.

Cells Escape an Operational Mitotic Checkpoint through a Stochastic Process.

Improperly attached chromosomes activate the mitotic checkpoint that arrests cell division before anaphase. Cells can maintain an arrest for several hours but eventually will resume proliferation, a process we refer to as adaptation. Whether adapting cells bypass an active block or whether the block has to be removed to resume proliferation is not clear. Likewise, it is not known whether all cells of a genetically homogeneous population are equally capable to adapt. Here, we show that the mitotic checkpoint is operational when yeast cells adapt and that each cell has the same propensity to adapt. Our results are consistent with a model of the mitotic checkpoint where adaptation is driven by random fluctuations of APC/CCdc20, the molecular species inhibited by the checkpoint. Our data provide a quantitative framework for understanding how cells overcome a constant stimulus that halts cell cycle progression.

Subtelomeres constitute a safeguard for gene expression and chromosome homeostasis.

The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.

Discovery of genes involved in mitosis, cell division, cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains.

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini-chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non-essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.

Structure of the Pds5-Scc1 Complex and Implications for Cohesin Function.

Sister chromatid cohesion is a fundamental prerequisite to faithful genome segregation. Cohesion is precisely regulated by accessory factors that modulate the stability with which the cohesin complex embraces chromosomes. One of these factors, Pds5, engages cohesin through Scc1 and is both a facilitator of cohesion, and, conversely also mediates the release of cohesin from chromatin. We present here the crystal structure of a complex between budding yeast Pds5 and Scc1, thus elucidating the molecular basis of Pds5 function. Pds5 forms an elongated HEAT repeat that binds to Scc1 via a conserved surface patch. We demonstrate that the integrity of the Pds5-Scc1 interface is indispensable for the recruitment of Pds5 to cohesin, and that its abrogation results in loss of sister chromatid cohesion and cell viability.

Evidence for a dual role of actin in regulating chromosome organization and dynamics in yeast.

Eukaryotic chromosomes undergo movements that are involved in the regulation of functional processes such as DNA repair. To better understand the origin of these movements, we used fluorescence microscopy, image analysis and chromosome conformation capture to quantify the actin contribution to chromosome movements and interactions in budding yeast. We show that both the cytoskeletal and nuclear actin drive local chromosome movements, independently of Csm4, a putative LINC protein. Inhibition of actin polymerization reduces subtelomere dynamics, resulting in more confined territories and enrichment in subtelomeric contacts. Artificial tethering of actin to nuclear pores increased both nuclear pore complex (NPC) and subtelomere motion. Chromosome loci that were positioned away from telomeres exhibited reduced motion in the presence of an actin polymerization inhibitor but were unaffected by the lack of Csm4. We further show that actin was required for locus mobility that was induced by targeting the chromatin-remodeling protein Ino80. Correlated with this, DNA repair by homologous recombination was less efficient. Overall, interphase chromosome dynamics are modulated by the additive effects of cytoskeletal actin through forces mediated by the nuclear envelope and nuclear actin, probably through the function of actin in chromatin-remodeling complexes.

Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152.

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication.

Aurora B prevents chromosome arm separation defects by promoting telomere dispersion and disjunction.

The segregation of centromeres and telomeres at mitosis is coordinated at multiple levels to prevent the formation of aneuploid cells, a phenotype frequently observed in cancer. Mitotic instability arises from chromosome segregation defects, giving rise to chromatin bridges at anaphase. Most of these defects are corrected before anaphase onset by a mechanism involving Aurora B kinase, a key regulator of mitosis in a wide range of organisms. Here, we describe a new role for Aurora B in telomere dispersion and disjunction during fission yeast mitosis. Telomere dispersion initiates in metaphase, whereas disjunction takes place in anaphase. Dispersion is promoted by the dissociation of Swi6/HP1 and cohesin Rad21 from telomeres, whereas disjunction occurs at anaphase after the phosphorylation of condensin subunit Cnd2. Strikingly, we demonstrate that deletion of Ccq1, a telomeric shelterin component, rescued cell death after Aurora inhibition by promoting the loading of condensin on chromosome arms. Our findings reveal an essential role for telomeres in chromosome arm segregation.

Chromosomes rein back the spindle pole body during horsetail movement in fission yeast meiosis.

In meiosis, pairing and recombination of homologous chromosomes are crucial for the correct segregation of chromosomes, and substantial movements of chromosomes are required to achieve homolog pairing. During this process, it is known that telomeres cluster to form a bouquet arrangement of chromosomes. The fission yeast Schizosaccharomyces pombe provides a striking example of bouquet formation, after which the entire nucleus oscillates between the cell poles (these oscillations are generally called horsetail nuclear movements) while the telomeres remain clustered to the spindle pole body (SPB; a centrosome-equivalent structure in fungi) at the leading edge of the moving nucleus. S. pombe mutants defective in telomere clustering frequently form aberrant spindles, such as monopolar or nonpolar spindles, leading to missegregation of the chromosomes at the subsequent meiotic divisions. Here we demonstrate that such defects in meiotic spindle formation caused by loss of meiotic telomere clustering are rescued when nuclear movement is prevented. On the other hand, stopping nuclear movement does not rescue defects in telomere clustering, nor chromosome missgregation even in cells that have formed a bipolar spindle. These results suggest that movement of the SPB without attachment of telomeres leads to the formation of aberrant spindles, but that recovering bipolar spindles is not sufficient for rescue of chromosome missegregation in mutants lacking telomere clustering.

Heterochromatin controls γH2A localization in Neurospora crassa.

In response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called γH2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of γH2A followed by next-generation sequencing (ChIP-seq). γH2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. γH2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.

The fission yeast FANCM ortholog directs non-crossover recombination during meiosis.

The formation of healthy gametes depends on programmed DNA double-strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favor of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to humans suggests that this interplay may be a general feature of meiotic recombination.