Target of Rapamycin Complex 1 (TORC1) signaling promotes growth and aging. Inhibition of TORC1 leads to reduced protein translation, which promotes longevity. TORC1-dependent post-transcriptional regulation of protein translation has been well studied, while analogous transcriptional regulation is less understood. Here we screen fission yeast mutants for resistance to Torin1, which inhibits TORC1 and cell growth. Cells lacking the GATA factor Gaf1 (gaf1Δ) grow normally even in high doses of Torin1. The gaf1Δ mutation shortens the chronological lifespan of non-dividing cells and diminishes Torin1-mediated longevity. Expression profiling and genome-wide binding experiments show that upon TORC1 inhibition, Gaf1 directly upregulates genes for small-molecule metabolic pathways and indirectly represses genes for protein translation. Surprisingly, Gaf1 binds to and downregulates the tRNA genes, so it also functions as a transcription factor for RNA polymerase III. Thus, Gaf1 controls the transcription of both protein-coding and tRNA genes to inhibit translation and growth downstream of TORC1.

Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.

Daniel Jeffares, Jürg Bähler and colleagues report the genome sequences of 161 natural isolates of Schizosaccharomyces pombe, finding moderate genetic diversity and weak global population structure. They also report genome-wide association studies for 223 quantitative traits. Natural variation within species reveals aspects of genome evolution and function. The fission yeast Schizosaccharomyces pombe is an important model for eukaryotic biology, but researchers typically use one standard laboratory strain. To extend the usefulness of this model, we surveyed the genomic and phenotypic variation in 161 natural isolates. We sequenced the genomes of all strains, finding moderate genetic diversity (π = 3 × 10−3 substitutions/site) and weak global population structure. We estimate that dispersal of S. pombe began during human antiquity (∼340 BCE), and ancestors of these strains reached the Americas at ∼1623 CE. We quantified 74 traits, finding substantial heritable phenotypic diversity. We conducted 223 genome-wide association studies, with 89 traits showing at least one association. The most significant variant for each trait explained 22% of the phenotypic variance on average, with indels having larger effects than SNPs. This analysis represents a rich resource to examine genotype-phenotype relationships in a tractable model.

Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5′-3′ exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼0.24% in wild type and ∼1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance.

Exon-skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different slice varaiants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast ( Schizosaccharomyces pombe ), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were ubiquitous but rare compared to canonical splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5’-3’ exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts were down-regulated. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at below 1 copy per cell, even in the absence of nuclear RNA surveillance and late during meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing error-rate was ∼0.24% in wild-type and ∼1.75% in nuclear exonuclease mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing, yet most of these novel splicing events were rare and targeted for nuclear degradation. Analysis of human transcriptomes revealed similar, albeit much weaker trends for pervasive exon-skipping transcripts, some of which being degraded by the nuclear exosome. This study highlights widespread, but low frequency alternative splicing which is targeted by nuclear RNA surveillance.

Both canonical and alternative splicing of RNAs are governed by intronic sequence elements and produce transient lariat structures fastened by branch points within introns. To map precisely the location of branch points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon-skipping branch points. LaSSO thus provides an effective approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated into RNA-seq pipelines.

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects (trans-eQTLs) greatly exceeds the number of local effects (cis-eQTLs) and that non-coding RNAs are as likely to be affected by eQTLs as protein-coding RNAs. We identified a genetic variation of swc5 that modifies the levels of 871 RNAs, with effects on both sense and antisense transcription, and show that this effect most likely goes through a compromised deposition of the histone variant H2A.Z. The strains, methods, and datasets generated here provide a rich resource for future studies.

Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects ( trans- eQTLs) greatly exceeds the number of local effects ( cis- eQTLs) and that non-coding RNAs are as likely to be affected by eQTLs as protein-coding RNAs. We identified a genetic variation of swc 5 that modifies the levels of 871 RNAs, with effects on both sense and antisense transcription, and show that this effect most likely goes through a compromised deposition of the histone variant H 2 A.Z. The strains, methods, and datasets generated here provide a rich resource for future studies. pht both pht 1 and clr 4 . difference was observed between the parents in cdb 4 , which indicates that other genetic events are implicated, as expected since cdb 4 is also associated with another aseQTL (Supplementary Information). For paf 1 and its 3 , effects observed in swc 5 -fs strains (pool and Y 0036 ) were not observed in Δ swc 5 . This may be due to other genetic effects in cis of swc 5 , or to effects of auxotrophic and mating makers which differ between 968 and Δ swc 5 (Supplementary Information).

The acetylation state of histones, controlled by histone acetyltransferases (HATs) and deacetylases (HDACs), profoundly affects DNA transcription and repair by modulating chromatin accessibility to the cellular machinery. The Schizosaccharomyces pombe HDAC Clr6 (human HDAC1) binds to different sets of proteins that define functionally distinct complexes: I, I', and II. Here, we determine the composition, architecture, and functions of a new Clr6 HDAC complex, I'', delineated by the novel proteins Nts1, Mug165, and Png3. Deletion of nts1 causes increased sensitivity to genotoxins and deregulated expression of Tf2 elements, long noncoding RNA, and subtelomeric and stress-related genes. Similar, but more pervasive, phenotypes are observed upon Clr6 inactivation, supporting the designation of complex I'' as a mediator of a key subset of Clr6 functions. We also reveal that with the exception of Tf2 elements, the genome-wide loading sites and loci regulated by Clr6 I″ do not correlate. Instead, Nts1 loads at genes that are expressed in midmeiosis, following oxidative stress, or are periodically expressed. Collective data suggest that Clr6 I'' has (i) indirect effects on gene expression, conceivably by mediating higher-order chromatin organization of subtelomeres and Tf2 elements, and (ii) direct effects on the transcription of specific genes in response to certain cellular or environmental stimuli.

Material Supplemental http://genome.cshlp.org/content/suppl/2014/05/14/gr.166819.113.DC1.html References http://genome.cshlp.org/content/24/7/1169.full.html#ref-list-1 This article cites 46 articles, 22 of which can be accessed free at: Open Access Open Access option. Genome Research Freely available online through the License Commons Creative . http://creativecommons.org/licenses/by/4.0 License (Attribution 4.0 International), as described at , is available under a Creative Commons Genome Research This article, published in

Network analysis provides a powerful framework for the interpretation of genome-wide data. While static network approaches have proved fruitful, there is increasing interest in the insights gained from the analysis of cellular networks under different conditions. In this work, we study the effect of stress on cellular networks in fission yeast. Stress elicits a sophisticated and large scale cellular response, involving a shift of resources from cell growth and metabolism towards protection and maintenance. Previous work has suggested that these changes can be appreciated at the network level. In this paper, we study two types of cellular networks: gene co-regulation networks and weighted protein interaction networks. We show that in response to oxidative stress, the co-regulation networks re-organize towards a more modularised structure: while sets of genes become more tightly co-regulated, co-regulation between these modules is decreased. This shift translates into longer average shortest path length, increased transitivity, and decreased modular overlap in these networks. We also find a similar change in structure in the weighted protein interaction network in response to both oxidative stress and nitrogen starvation, confirming and extending previous findings. These changes in network structure could represent an increase in network robustness and/or the emergence of more specialised functional modules. Additionally, we find stress induces tighter co-regulation of non-coding RNAs, decreased functional importance of splicing factors, as well as changes in the centrality of genes involved in chromatin organization, cytoskeleton organization, cell division, and protein turnover.

Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond.

Data on absolute molecule numbers will empower the modeling, understanding, and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under two key physiological conditions. The integrated data set supports quantitative biology and affords unique insights into cell regulation. Although mRNAs are typically expressed in a narrow range above 1 copy/cell, most long, noncoding RNAs, except for a distinct subset, are tightly repressed below 1 copy/cell. Cell-cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also bring about more switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.

Background Meiosis is the specialized form of the cell cycle by which diploid cells produce the haploid gametes required for sexual reproduction. Initiation and progression through meiosis requires that the expression of the meiotic genes is precisely controlled so as to provide the correct gene products at the correct times. During meiosis, four temporal gene clusters are either induced or repressed by a cascade of transcription factors. Principal Findings In this report a novel copper-fist-type regulator, Cuf2, is shown to be expressed exclusively during meiosis. The expression profile of the cuf2+ mRNA revealed that it was induced during middle-phase meiosis. Both cuf2+ mRNA and protein levels are unregulated by copper addition or starvation. The transcription of cuf2+ required the presence of a functional mei4+ gene encoding a key transcription factor that activates the expression of numerous middle meiotic genes. Microscopic analyses of cells expressing a functional Cuf2-GFP protein revealed that Cuf2 co-localized with both homologous chromosomes and sister chromatids during the meiotic divisions. Cells lacking Cuf2 showed an elevated and sustained expression of several of the middle meiotic genes that persisted even during late meiosis. Moreover, cells carrying disrupted cuf2Δ/cuf2Δ alleles displayed an abnormal morphology of the forespore membranes and a dramatic reduction of spore viability. Significance Collectively, the results revealed that Cuf2 functions in the timely repression of the middle-phase genes during meiotic differentiation.

Unicellular eukaryotes have many advantages as model organisms since they are easy to grow and maintain, have short cell cycles, and there are many tools and approaches available to assist analysis. Two such organisms, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe, have been utilized in the study of NCLs, since several NCL genes and their gene products are conserved in these species. Both have orthologues for the proteins CLN3, designated Btn1p, and cathepsin D, designated Pep4p in S. cerevisiae or Sxa1p in S. pombe. The fission yeast, S. pombe, also has a CLN1 orthologue, designated Ppt1p.

BACKGROUND:The generation of mature mRNAs involves interconnected processes, including transcription by RNA polymerase II (Pol II), modification of histones, and processing of pre-mRNAs through capping, intron splicing, and polyadenylation. These processes are thought to be integrated, both spatially and temporally, but it is unclear how these connections manifest at a global level with respect to chromatin patterns and transcription kinetics. We sought to clarify the relationships between chromatin, transcription and splicing using multiple genome-wide approaches in fission yeast.RESULTS:To investigate these functional interdependencies, we determined Pol II occupancy across all genes using high-density tiling arrays. We also performed ChIP-chip on the same array platform to globally map histone H3 and its H3K36me3 modification, complemented by formaldehyde-assisted isolation of regulatory elements (FAIRE). Surprisingly, Pol II occupancy was higher in introns than in exons, and this difference was inversely correlated with gene expression levels at a global level. Moreover, introns showed distinct distributions of histone H3, H3K36me3 and FAIRE signals, similar to those at promoters and terminators. These distinct transcription and chromatin patterns of intronic regions were most pronounced in poorly expressed genes.CONCLUSIONS:Our findings suggest that Pol II accumulates at the 3' ends of introns, leading to substantial transcriptional delays in weakly transcribed genes. We propose that the global relationship between transcription, chromatin remodeling, and splicing may reflect differences in local nuclear environments, with highly expressed genes being associated with abundant processing factors that promote effective intron splicing and transcriptional elongation.

Batten disease is characterised by lysosomal dysfunction. The most common type of the disease is caused by mutations in the membrane protein CLN3, whose function is unknown. We show that the fission yeast orthologue Btn1p, previously implicated in vacuole function, is required for correct sorting of the vacuole hydrolase carboxypeptidase Y (Cpy1p). This is, in part, due to a defect in trafficking of Vps10p, the sorting receptor for Cpy1p, from the Golgi to the trans-Golgi network in btn1Δ cells. Our data also implicate btn1 in other Vps10-independent Cpy1-sorting pathways. Furthermore, btn1 affects the number, intracellular location and structure of Golgi compartments. We show that the prevacuole location of Btn1p is at the Golgi, because Btn1p colocalises predominantly with the Golgi marker Gms1p in compartments that are sensitive to Brefeldin A. Btn1p function might be linked to that of Vps34p, a phosphatidylinositol 3-kinase, because Btn1p acts as a multicopy suppressor of the severe Cpy1p vacuole protein-sorting defect of vps34Δ cells. Together, these results indicate an important role for Btn1p in the Golgi complex, which affects Golgi homeostasis and vacuole protein sorting. We propose a similar role for CLN3 in mammalian cells.

The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle-regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident.

btn1 , the Schizosaccharomyces pombe orthologue of the human Batten disease gene CLN3 , exerts multiple cellular effects. As well as a role in vacuole pH homoeostasis, we now show that Btn1p is essential for growth at high temperatures. Its absence results in progressive defects at 37°C that culminate in total depolarized growth and cell lysis. These defects are preceded by a progressive failure to correctly polarize sterol‐rich domains after cytokinesis and are accompanied by loss of Myo1p localization. Furthermore, we found that in Sz. pombe , sterol spreading is linked to defective formation/polarization of F‐actin patches and disruption of endocytosis and that these processes are aberrant in btn1 Δ cells. Consistent with a role for Btn1p in polarized growth, Btn1p has an altered location at 37°C and is retained in actin‐dependent endomembrane structures near the cell poles or septum.