With the growing availability of large-scale biological datasets, automated methods of extracting functionally meaningful information from this data are becoming increasingly important. Data relating to functional association between genes or proteins, such as co-expression or functional association, is often represented in terms of gene or protein networks. Several methods of predicting gene function from these networks have been proposed. However, evaluating the relative performance of these algorithms may not be trivial: concerns have been raised over biases in different benchmarking methods and datasets, particularly relating to non-independence of functional association data and test data. In this paper we propose a new network-based gene function prediction algorithm using a commute-time kernel and partial least squares regression (Compass). We compare Compass to GeneMANIA, a leading network-based prediction algorithm, using a number of different benchmarks, and find that Compass outperforms GeneMANIA on these benchmarks. We also explicitly explore problems associated with the non-independence of functional association data and test data. We find that a benchmark based on the Gene Ontology database, which, directly or indirectly, incorporates information from other databases, may considerably overestimate the performance of algorithms exploiting functional association data for prediction.

In the life sciences, many measurement methods yield only the relative abundances of different components in a sample. With such relative-or compositional-data, differential expression needs careful interpretation, and correlation-a statistical workhorse for analyzing pairwise relationships-is an inappropriate measure of association. Using yeast gene expression data we show how correlation can be misleading and present proportionality as a valid alternative for relative data. We show how the strength of proportionality between two variables can be meaningfully and interpretably described by a new statistic ϕ which can be used instead of correlation as the basis of familiar analyses and visualisation methods, including co-expression networks and clustered heatmaps. While the main aim of this study is to present proportionality as a means to analyse relative data, it also raises intriguing questions about the molecular mechanisms underlying the proportional regulation of a range of yeast genes.

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, afford insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress.

We report a high quality and system-wide proteome catalogue covering 71% (3,542 proteins) of the predicted genes of fission yeast, Schizosaccharomyces pombe, presenting the largest protein dataset to date for this important model organism. We obtained this high proteome and peptide (11.4 peptides/protein) coverage by a combination of extensive sample fractionation, high resolution Orbitrap mass spectrometry, and combined database searching using the iProphet software as part of the Trans-Proteomics Pipeline. All raw and processed data are made accessible in the S. pombe PeptideAtlas. The identified proteins showed no biases in functional properties and allowed global estimation of protein abundances. The high coverage of the PeptideAtlas allowed correlation with transcriptomic data in a system-wide manner indicating that post-transcriptional processes control the levels of at least half of all identified proteins. Interestingly, the correlation was not equally tight for all functional categories ranging from r(s) >0.80 for proteins involved in translation to r(s) <0.45 for signal transduction proteins. Moreover, many proteins involved in DNA damage repair could not be detected in the PeptideAtlas despite their high mRNA levels, strengthening the translation-on-demand hypothesis for members of this protein class. In summary, the extensive and publicly available S. pombe PeptideAtlas together with the generated proteotypic peptide spectral library will be a useful resource for future targeted, in-depth, and quantitative proteomic studies on this microorganism.

Gene expression is controlled globally and at multiple levels in response to environmental stress, but the relationships among these dynamic regulatory changes are not clear. Here we analyzed global regulation during different stress conditions in fission yeast, Schizosaccharomyces pombe, combining dynamic genome-wide data on mRNA, translation, and protein profiles.

Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1+) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes. Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged. We report the global gene-expression response of S. pombe to loss of trt1+.

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.

Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.

PomBase (www.pombase.org), the model organism database (MOD) for the fission yeast Schizosaccharomyces pombe, supports research within and beyond the S. pombe community by integrating and presenting genetic, molecular, and cell biological knowledge into intuitive displays and comprehensive data collections. With new content, novel query capabilities, and biologist-friendly data summaries and visualization, PomBase also drives innovation in the MOD community.

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.

Cells balance glycolysis with respiration to support their metabolic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis or fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast. This variant reduced Pyk1 activity and glycolytic flux. Replacing the "low-activity" pyk1 allele in the laboratory strain with the "high-activity" allele was sufficient to increase fermentation and decrease respiration. This metabolic rebalancing triggered systems-level adjustments in the transcriptome and proteome and in cellular traits, including increased growth and chronological lifespan but decreased resistance to oxidative stress. Thus, low Pyk1 activity does not lead to a growth advantage but to stress tolerance. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate kinase isoforms.

Genetically identical cells are known to differ in many physiological parameters such as growth rate and drug tolerance. Metabolic specialization is believed to be a cause of such phenotypic heterogeneity, but detection of metabolically divergent subpopulations remains technically challenging. We developed a proteomics-based technology, termed differential isotope labelling by amino acids (DILAC), that can detect producer and consumer subpopulations of a particular amino acid within an isogenic cell population by monitoring peptides with multiple occurrences of the amino acid. We reveal that young, morphologically undifferentiated yeast colonies contain subpopulations of lysine producers and consumers that emerge due to nutrient gradients. Deconvoluting their proteomes using DILAC, we find evidence for in situ cross-feeding where rapidly growing cells ferment and provide the more slowly growing, respiring cells with ethanol. Finally, by combining DILAC with fluorescence-activated cell sorting, we show that the metabolic subpopulations diverge phenotypically, as exemplified by a different tolerance to the antifungal drug amphotericin B. Overall, DILAC captures previously unnoticed metabolic heterogeneity and provides experimental evidence for the role of metabolic specialization and cross-feeding interactions as a source of phenotypic heterogeneity in isogenic cell populations.

Genetic factors underlying aging are remarkably conserved from yeast to human. The fission yeast Schizosaccharomyces pombe is an emerging genetic model to analyze cellular aging. Chronological lifespan (CLS) has been studied in stationary-phase yeast cells depleted for glucose, which only survive for a few days. Here, we analyzed CLS in quiescent S. pombe cells deprived of nitrogen, which arrest in a differentiated, G0-like state and survive for more than 2 months. We applied parallel mutant phenotyping by barcode sequencing (Bar-seq) to assay pooled haploid deletion mutants as they aged together during long-term quiescence. As expected, mutants with defects in autophagy or quiescence were under-represented or not detected. Lifespan scores could be calculated for 1199 mutants. We focus the discussion on the 48 most long-lived mutants, including both known aging genes in other model systems and genes not previously implicated in aging. Genes encoding membrane proteins were particularly prominent as pro-aging factors. We independently verified the extended CLS in individual assays for 30 selected mutants, showing the efficacy of the screen. We also applied Bar-seq to profile all pooled deletion mutants for proliferation under a standard growth condition. Unlike for stationary-phase cells, no inverse correlation between growth and CLS of quiescent cells was evident. These screens provide a rich resource for further studies, and they suggest that the quiescence model can provide unique, complementary insights into cellular aging.

Genome-wide assays and screens typically result in large lists of genes or proteins. Enrichments of functional or other biological properties within such lists can provide valuable insights and testable hypotheses. To systematically detect these enrichments can be challenging and time-consuming, because relevant data to compare against query gene lists are spread over many different sources. We have developed AnGeLi (Analysis of Gene Lists), an intuitive, integrated web-tool for comprehensive and customized interrogation of gene lists from the fission yeast, Schizosaccharomyces pombe. AnGeLi searches for significant enrichments among multiple qualitative and quantitative information sources, including gene and phenotype ontologies, genetic and protein interactions, numerous features of genes, transcripts, translation, and proteins such as copy numbers, chromosomal positions, genetic diversity, RNA polymerase II and ribosome occupancy, localization, conservation, half-lives, domains, and molecular weight among others, as well as diverse sets of genes that are co-regulated or lead to the same phenotypes when mutated. AnGeLi uses robust statistics which can be tailored to specific needs. It also provides the option to upload user-defined gene sets to compare against the query list. Through an integrated data submission form, AnGeLi encourages the community to contribute additional curated gene lists to further increase the usefulness of this resource and to get the most from the ever increasing large-scale experiments. AnGeLi offers a rigorous yet flexible statistical analysis platform for rich insights into functional enrichments and biological context for query gene lists, thus providing a powerful exploratory tool through which S. pombe researchers can uncover fresh perspectives and unexpected connections from genomic data. AnGeLi is freely available at: www.bahlerlab.info/AnGeLi.

Reactive oxygen species, such as H2O2, can damage cells but also promote fundamental processes, including growth, differentiation and migration. The mechanisms allowing cells to differentially respond to toxic or signaling H2O2 levels are poorly defined. Here we reveal that increasing external H2O2 produces a bi-phasic response in intracellular H2O2. Peroxiredoxins (Prx) are abundant peroxidases which protect against genome instability, ageing and cancer. We have developed a dynamic model simulating in vivo changes in Prx oxidation. Remarkably, we show that the thioredoxin peroxidase activity of Prx does not provide any significant protection against external rises in H2O2. Instead, our model and experimental data are consistent with low levels of extracellular H2O2 being efficiently buffered by other thioredoxin-dependent activities, including H2O2-reactive cysteines in the thiol-proteome. We show that when extracellular H2O2 levels overwhelm this buffering capacity, the consequent rise in intracellular H2O2 triggers hyperoxidation of Prx to thioredoxin-resistant, peroxidase-inactive form/s. Accordingly, Prx hyperoxidation signals that H2O2 defenses are breached, diverting thioredoxin to repair damage.

A systems-level understanding of biological processes and information flow requires the mapping of cellular component interactions, among which protein-protein interactions are particularly important. Fission yeast (Schizosaccharomyces pombe) is a valuable model organism for which no systematic protein-interaction data are available. We exploited gene and protein properties, global genome regulation datasets, and conservation of interactions between budding and fission yeast to predict fission yeast protein interactions in silico. We have extensively tested our method in three ways: first, by predicting with 70-80% accuracy a selected high-confidence test set; second, by recapitulating interactions between members of the well-characterized SAGA co-activator complex; and third, by verifying predicted interactions of the Cbf11 transcription factor using mass spectrometry of TAP-purified protein complexes. Given the importance of the pathway in cell physiology and human disease, we explore the predicted sub-networks centered on the Tor1/2 kinases. Moreover, we predict the histidine kinases Mak1/2/3 to be vital hubs in the fission yeast stress response network, and we suggest interactors of argonaute 1, the principal component of the siRNA-mediated gene silencing pathway, lost in budding yeast but preserved in S. pombe. Of the new high-quality interactions that were discovered after we started this work, 73% were found in our predictions. Even though any predicted interactome is imperfect, the protein network presented here can provide a valuable basis to explore biological processes and to guide wet-lab experiments in fission yeast and beyond. Our predicted protein interactions are freely available through PInt, an online resource on our website (www.bahlerlab.info/PInt).

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.

To provide consistent computable descriptions of phenotype data, PomBase is developing a formal ontology of phenotypes observed in fission yeast.

Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped.