Yeast Deletion Library Research Articles

Role of a novel endoplasmic reticulum-resident glycoprotein Mtc6/Ehg2 in high-pressure growth: stability of tryptophan permease Tat2 in Saccharomyces cerevisiae.

Deep-sea organisms are subjected to extreme conditions; therefore, understanding their adaptive strategies is crucial. We utilize Saccharomyces cerevisiae as a model to investigate pressure-dependent protein regulation and piezo-adaptation. Using yeast deletion library analysis, we identified 6 poorly characterized genes that are crucial for high-pressure growth, forming novel functional modules associated with cell growth. In this study, we aimed to unravel the molecular mechanisms of high-pressure adaptation in S. cerevisiae, focusing on the role of MTC6. MTC6, the gene encoding the novel glycoprotein Mtc6/Ehg2, was found to stabilize tryptophan permease Tat2, ensuring efficient tryptophan uptake and growth under high pressure at 25MPa. The loss of MTC6 led to promoted vacuolar degradation of Tat2, depending on the Rsp5-Bul1 ubiquitin ligase complex. These findings enhance our understanding of deep-sea adaptations and stress biology, with broad implications for biotechnology, environmental microbiology, and evolutionary insights across species.

32994 Pezadeftide is a potent antifungal peptide with rapid fungicidial activity via a unique mechanism of action

Background: Fungal infections of skin, hair, and nails are extremely common and can be caused by a broad range of fungal pathogens including dermatophytes, yeasts, and nondermatophytic molds. Pezadeftide is a plant defensin peptide with potent fungicidal activity against a range of fungal pathogens that cause skin and nail infections, including Trichophyton spp., Candida spp, and a range of nondermatophytic molds. Pezadeftide is currently in development as a new topical treatment for onychomycosis. Methods: The activity of pezadeftide against a range of fungal pathogens was tested using the microbroth dilution method to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). Fluorescent dyes were employed to assess the effect of pezadeftide on various cellular components. To identify potential targets for pezadeftide, a yeast deletion library was screened for fitness in the presence of low concentrations of pezadeftide. Results: Pezadeftide has potent antifungal activity across a broad range of fungal pathogens that cause dermatophytic infections. Pezadeftide inhibits growth at low micromolar concentrations and is fungicidal at similar concentrations, suggesting that its primary mode-of-action is fungicidal. Pezadeftide rapidly enters fungal cells and causes a rapid mitochondrial response that results in hyperpolarization of the mitochondrial membrane. Fusion of the vacuoles and production of reactive oxygen species are observed prior to disruption of the plasma membrane and cell death. Conclusions: Pezadeftide is a potent, broad-spectrum antifungal with a fungicidal mode-of-action. It rapidly kills fungal cells via interaction with the mitochondria, hyperpolarisation of the mitochondrial membrane and production of reactive oxygen species.

A Role for Inositol Pyrophosphates in ER Protein Translocation?

The purpose of this study is to identify proteins involved in endoplasmic reticulum to cytoplasm transport of the Gag protein of the yeast Saccharomyces cerevisiae Ty1 retroelement. Saccharomyces cerevisiae, or bakers/brewer’s yeast, exists as a model organism due to its quick growth time and easily manipulated genome. A multitude of yeast genes are also homologous to ones found in larger eukaryotes. During Ty1 transposition, Gag is translocated by the signal recognition protein (SRP) from the cytoplasm to the ER lumen where it adopts a stable conformation. The stabilized Gag protein is then translocated through an unknown mechanism to the cytoplasm where it aggregates to form virus‐like particles. Screening of a yeast deletion library identified SIW14 as a potential candidate gene involved in Gag translocation. The yeast deletion library was transformed with a Ty1 Gag‐Ura3 fusion protein on a LEU2 selectable plasmid. In wild type cells, the Gag‐Ura3 fusion is transported to the cytoplasm, thus yeast cells are unable to survive on media containing 5‐fluoroorotic acid (5‐FOA), as the URA3 gene converts 5‐FOA to the toxic product 5‐fluorouracil. Cells with an siw14 deletion are viable on 5‐FOA containing media, presumably sequestering the Ty1 Gag‐Ura3 fusion protein in the ER. Siw14p is an inositol phosphatase that hydrolyzes the beta phosphate on 5‐diphospho‐1,2,3,4,6‐pentakisphosphate (5PP‐IP5) to inositol hexaphosphate (IP6). Inositol pyrophosphates are energy‐rich signaling molecules and can act as biological messengers. Vip1p, Ddp1p, and Kcs1p are also proteins involved in the phosphorylation state of inositol pyrophosphates in yeast. inositol heptaphosphate (IP7) or inositol octaphosphate (IP8) are substrates for pyrophosphorylation of phosphorylated serine residues in proximity to acidic amino acids, such as aspartate and glutamate. No energy is expended during pyrophosphorylation; it is dependent on the presence of an Mg2+ divalent ion. We hypothesize that the deletion of a pyrophosphatase kinase results in the inability of an unknown transport protein from being pyrophopshoprylated to facilitate Gag transport across the ER. Strains harboring deletions of siw14, kcs1, vip1, and ddp1 will be assayed for Ty1 transposition. Fluorescence microscopy of deletion and wild type cells containing a fluorescently labeled Gag protein will be observed for Gag localization.

Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) ‘biological process’ terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including ‘cell cycle’ (cdc2, rik1, pas1, and leo1), ‘signaling’ (sck2, oga1, and cki3), and ‘vesicle-mediated transport’ (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the ‘signaling’ GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation

BackgroundDespite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance.ResultsWe find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses.ConclusionsOur results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

Defects in the NuA4 acetyltransferase complex increase stability of the ribosomal RNA gene and extend replicative lifespan.

Genome instability is a cause of cellular senescence. The ribosomal RNA gene repeat (rDNA) is one of the most unstable regions in the genome and its instability is proposed to be a major inducer of cellular senescence and restricted lifespan. We previously conducted a genome-wide screen using a budding yeast deletion library to identify mutants that exhibit a change in the stability of the rDNA region, compared to the wild-type. To investigate the correlation between rDNA stability and lifespan, we examined deletion mutants with very stable rDNA and found that deletion of EAF3, encoding a component of the NuA4 histone acetyltransferase complex, reproducibly resulted in increased stabilization of the rDNA. In the absence of Eaf3, and of other subunits of the NuA4 complex, we observed lower levels of extrachromosomal rDNA circles that are produced by recombination in the rDNA and are thus an indicator of rDNA instability. The replicative lifespan in the eaf3 mutant was extended by ~30%, compared to the wild-type strain. Our findings provide evidence that rDNA stability is correlated with extended replicative lifespan. The eaf3 mutation possibly affects the non-coding transcription in rDNA that regulates rDNA recombination through cohesin dissociation.

Genome-Wide Identification of Host Genes Required for Toxicity of Bacterial Cytolethal Distending Toxin in a Yeast Model.

BackgroundAggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity.ObjectiveTo investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model.MethodsWe transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type.ResultsOut of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity.ConclusionThe genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication.

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen.

Fucoidan, the sulfated fucose-rich polysaccharide derived from brown macroalgae, was reported to display some anti-cancer effects in in vitro and in vivo models that included apoptosis and cell cycle arrest. The proposed mechanisms of action involve enhanced immune surveillance and direct pro-apoptotic effects via the activation of cell signaling pathways that remain largely uncharacterized. This study aimed to identify cellular pathways influenced by fucoidan using an unbiased genetic approach to generate additional insights into the anti-cancer effects of fucoidan. Drug–gene interactions of Undaria pinnatifida fucoidan were assessed by a systematic screen of the entire set of 4,733 halpoid Saccharomyces cerevsiae gene deletion strains. Some of the findings were confirmed using cell cycle analysis and DNA damage detection in non-immortalized human dermal fibroblasts and colon cancer cells. The yeast deletion library screen and subsequent pathway and interactome analysis identified global effects of fucoidan on a wide range of eukaryotic cellular processes, including RNA metabolism, protein synthesis, sorting, targeting and transport, carbohydrate metabolism, mitochondrial maintenance, cell cycle regulation, and DNA damage repair-related pathways. Fucoidan also reduced clonogenic survival, induced DNA damage and G1-arrest in colon cancer cells, while these effects were not observed in non-immortalized human fibroblasts. Our results demonstrate global effects of fucoidan in diverse cellular processes in eukaryotic cells and further our understanding about the inhibitory effect of Undaria pinnatifida fucoidan on the growth of human cancer cells.

Anti-silencing factor Epe1 associates with SAGA to regulate transcription within heterochromatin.

Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.

A website to identify shared genes in Saccharomyces cerevisiae homozygous deletion library screens

BackgroundThe homozygous yeast deletion library includes approximately 4800 diploid strains each containing one deleted non-essential gene. Hundreds of publications have arisen through experimentation using this genome-wide biological resource. As part of this work over 677 genesets have been collated from these experiments representing the phenotypic responses of the library to a diverse set of chemical and physical challenges.DescriptionA website called the Saccharomyces cerevisiae Homozygous Deletion Library Tools (ScHo DeLiTo-96) has been developed with the primary goal of browsing and identifying genes shared between these responsive phenotypes (available at yeastdb.org). Geneset comparisons have been performed for each phenotype against all others to identify common genes. Genesets and other curated information are stored in a relational database and a website interface allows users to query and browse the data in an intuitive way to reveal commonality between selected phenotypic responses. The most commonly occurring genes in all of the stored phenotypes are highly over-represented in the GO slim term “cellular ion homeostasis” indicating that genes shared between phenotypes may highlight a common cellular response. Additionally, user derived genesets can be uploaded and intersected against the stored data to reveal common responses which may otherwise have been obscure.ConclusionThese tools provide a simple method to perform niche enquiries between datasets derived from the yeast deletion library.

Chronological Lifespan in Yeast Is Dependent on the Accumulation of Storage Carbohydrates Mediated by Yak1, Mck1 and Rim15 Kinases.

Upon starvation for glucose or any other macronutrient, yeast cells exit from the mitotic cell cycle and acquire a set of characteristics that are specific to quiescent cells to ensure longevity. Little is known about the molecular determinants that orchestrate quiescence entry and lifespan extension. Using starvation-specific gene reporters, we screened a subset of the yeast deletion library representing the genes encoding ‘signaling’ proteins. Apart from the previously characterised Rim15, Mck1 and Yak1 kinases, the SNF1/AMPK complex, the cell wall integrity pathway and a number of cell cycle regulators were shown to be necessary for proper quiescence establishment and for extension of chronological lifespan (CLS), suggesting that entry into quiescence requires the integration of starvation signals transmitted via multiple signaling pathways. The CLS of these signaling mutants, and those of the single, double and triple mutants of RIM15, YAK1 and MCK1 correlates well with the amount of storage carbohydrates but poorly with transition-phase cell cycle status. Combined removal of the glycogen and trehalose biosynthetic genes, especially GSY2 and TPS1, nearly abolishes the accumulation of storage carbohydrates and severely reduces CLS. Concurrent overexpression of GSY2 and TSL1 or supplementation of trehalose to the growth medium ameliorates the severe CLS defects displayed by the signaling mutants (rim15Δyak1Δ or rim15Δmck1Δ). Furthermore, we reveal that the levels of intracellular reactive oxygen species are cooperatively controlled by Yak1, Rim15 and Mck1, and the three kinases mediate the TOR1-regulated accumulation of storage carbohydrates and CLS extension. Our data support the hypothesis that metabolic reprogramming to accumulate energy stores and the activation of anti-oxidant defence systems are coordinated by Yak1, Rim15 and Mck1 kinases to ensure quiescence entry and lifespan extension in yeast.

Characterization of aggregate load and pattern in living yeast cells by flow cytometry.

Protein aggregation is both a hallmark of and a driving force for a number of diseases. It is therefore important to identify the nature of these aggregates and the mechanism(s) by which the cell counteracts their detrimental properties. Currently, the study of aggregation in vivo is performed primarily using fluorescently tagged versions of proteins and analyzing the aggregates by fluorescence microscopy. While this strategy is considered the gold standard, it has several limitations, particularly with respect to its suitability for high-throughput screening (HTS). Here, using a GFP fusion of the well-characterized yeast prion amyloid protein [PSI+], we demonstrate that flow cytometry, which utilizes the same physical principles as fluorescence microscopy, can be used to determine the aggregate load and pattern in live and fixed yeast cells. Furthermore, our approach can easily be applied to high-throughput analyses such as screenings with a yeast deletion library.

Genome-wide screen of fission yeast mutants for sensitivity to 6-azauracil, an inhibitor of transcriptional elongation.

6-Azauracil (6 AU) inhibits enzymes in nucleoside synthesis and depletes the intracellular GTP/UTP pool. Mutations in transcriptional elongation machinery, as well as mutations in a variety of other pathways, exaggerate the growth defect of cells in the presence of 6 AU. Thus, identification of mutations that render cells sensitive to 6 AU will benefit study on the basis of 6 AU-sensitive phenotype. Here we performed a genome-wide screen of a fission yeast deletion library. Of 3235 single-gene deletions, 66 mutants displayed at least 50% drop of fitness in the presence of 6 AU and 60 mutants were reported for the first time; five deletions showed synthetic decrease of fitness when combined with deletion of set3(+) , which encodes a transcriptional regulator. Genes conferring tolerance to 6 AU were enriched in various processes, especially in chromosome segregation. Accordingly, genes encoding subunits of CLRC complex and spindle pole body were over-represented. Mutants were subjected to an in vivo transcript length-dependent reporter assay to assess the potential roles of deleted genes in transcriptional elongation. As with the deletions known to affect elongation, nab2Δ, nxt1Δ, rhp18Δ, SPAC24C9.08Δ, clr3Δ and ncs1Δset3Δ mutants exhibited defects in expressing long transcripts. New 6 AU-sensitive mutants identified here will help to elucidate the mechanism of action of 6 AU in the cells. Meanwhile, our study revealed novel genes potentially involved in transcriptional elongation and provided valuable targets for transcription study.

Gcn5 Modulates the Cellular Response to Oxidative Stress and Histone Deacetylase Inhibition.

To identify chemical genetic interactions underlying the mechanism of action of histone deacetylase inhibitors (HDACi) a yeast deletion library was screened for hypersensitive deletion mutants that confer increased sensitivity to the HDACi, CG-1521. The screen demonstrated that loss of GCN5 or deletion of components of the Gcn5 histone acetyltransferase (HAT) complex, SAGA, sensitizes yeast to CG-1521-induced cell death. Expression profiling after CG-1521 treatment reveals increased expression of genes involved in metabolism and oxidative stress response, and oxidative stress response mutants are hypersensitive to CG-1521 treatment. Accumulation of reactive oxygen species and increased cell death are enhanced in the gcn5Δ deletion mutant, and are abrogated by anti-oxidants, indicating a central role of oxidative stress in CG-1521-induced cell death. In human cell lines, siRNA mediated knockdown of GCN5 or PCAF, or chemical inhibition of GCN5 enzymatic activity, increases the sensitivity to CG-1521 and SAHA. These data suggest that the combination of HDAC and GCN5/PCAF inhibitors can be used for cancer treatment.

Tiled ChrI RHS collection: a pilot high-throughput screening tool for identification of allelic variants.

Reciprocal hemizygosity analysis is a genetic technique that allows phenotypic determination of the allelic effects of a gene in a genetically uniform background. Expanding this single gene technique to generate a genome-wide collection is termed as reciprocal hemizygosity scanning (RHS). The RHS collection should circumvent the need for linkage mapping and provide the power to identify all possible allelic variants for a given phenotype. However, the published RHS collections based on the existing genome-wide haploid deletion library reported a high rate of false positives. In this study, we report de novo construction of a RHS collection that is not based on the yeast deletion library. This collection has been constructed for the shortest yeast chromosome, ChrI. Using this ChrI RHS collection, we identified 13 allelic variants for the previously mapped loci and novel allelic variants for the growth differences in different environments. A few of these novel variants, which were fine mapped to a gene level, identified novel genetic variation for the previously studied environmental conditions. The availability of a genome-wide RHS collection would thus help us uncover a comprehensive list of allelic variants and better our understanding of the molecular pathways modulating a quantitative trait.

Abstract 5535: Gcn5 modulates the cellular response to oxidative stress induced by histone deacetylase inhibitors

Abstract Aberrant histone post-translational modifications including acetylation are common in human cancers and result in dysregulation of gene expression. Our laboratory has focused on studying the effects of hydroxamic acid based HDAC inhibitors (HDACis) including TSA, SAHA and CG-1521. We have employed a whole genome yeast deletion library screen to gain insight into the mechanism of the HDAC inhibitor CG-1521 and identify potential combinatorial drug targets. Gene ontology enrichment analysis shows that sensitive strains are highly enriched in processes regulating chromatin remodeling and transcription. Deletions of components of the SAGA HAT complex are highly overrepresented in the sensitive strains, including the catalytic subunit Gcn5. The gcn5 deletion strain displays increased sensitivity to CG-1521 induced cell death compared to the wild type strain, suggesting that the SAGA complex is required to reduce the effect of CG-1521 on cell growth. Gene expression analysis indicates that CG-1521 elicits an oxidative stress response in both the wild type and the gcn5 deletion strain. Gcn5 deletion substantially increases the level of oxidative stress induced by CG-1521 as measured by flow cytometry. In the colorectal cancer cell line HT29, treatment with CG-1521 is accompanied by an increase in oxidative stress and co-treatment with the Gcn5 specific inhibitor MB-3 or RNAi-mediated knockdown of the human Gcn5 homologs, Gcn5 and PCAF, enhances the growth-inhibitory effect of CG-1521. These studies have shown that disruption of the SAGA HAT complex with MB-3 or RNAi-mediated knockdown sensitizes tumor cells to CG-1521. Taken together, our work suggests that the combination of selective HAT and HDAC inhibitors may be an effective treatment of cancer. Citation Format: Ann-Christin Gaupel, Thomas J. Begley, Martin P. Tenniswood. Gcn5 modulates the cellular response to oxidative stress induced by histone deacetylase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5535. doi:10.1158/1538-7445.AM2014-5535

Tls1 regulates splicing of shelterin components to control telomeric heterochromatin assembly and telomere length.

Heterochromatin preferentially forms at repetitive DNA elements through RNAi-mediated targeting of histone-modifying enzymes. It was proposed that splicing factors interact with the RNAi machinery or regulate the splicing of repeat transcripts to directly participate in heterochromatin assembly. Here, by screening the fission yeast deletion library, we comprehensively identified factors required for telomeric heterochromatin assembly, including a novel gene tls1+. Purification of Tls1 and mass spectrometry analysis of its interacting proteins show that Tls1 associates with the spliceosome subunit Brr2. RNA sequencing analysis shows that the splicing of a subset of mRNAs are affected in tls1Δ cells, including mRNAs of shelterin components rap1+ and poz1+. Importantly, replacing rap1+ and poz1+ with their cDNAs significantly alleviated heterochromatin defects of tls1Δ cells, suggesting that the missplicing of shelterin components is the cause of such defects, and that splicing factors regulate telomeric heterochromatin through the proper splicing of heterochromatin factors. In addition to its role in telomeric heterochromatin assembly, Tls1-mediated splicing of shelterin mRNAs also regulates telomere length. Given that its human homologue C9ORF78 also associates with the spliceosome and is overexpressed in multiple cancer cell lines, our results suggest that C9ORF78 overexpression might alter the proper splicing of genes during cancer progression.

Elimination of damaged mitochondria through mitophagy reduces mitochondrial oxidative stress and increases tolerance to trichothecenes.

Trichothecene mycotoxins are natural contaminants of small grain cereals and are encountered in the environment, posing a worldwide threat to human and animal health. Their mechanism of toxicity is poorly understood, and little is known about cellular protection mechanisms against trichothecenes. We previously identified inhibition of mitochondrial protein synthesis as a novel mechanism for trichothecene-induced cell death. To identify cellular functions involved in trichothecene resistance, we screened the Saccharomyces cerevisiae deletion library for increased sensitivity to nonlethal concentrations of trichothecin (Tcin) and identified 121 strains exhibiting higher sensitivity than the parental strain. The largest group of sensitive strains had significantly higher reactive oxygen species (ROS) levels relative to the parental strain. A dose-dependent increase in ROS levels was observed in the parental strain treated with different trichothecenes, but not in a petite version of the parental strain or in the presence of a mitochondrial membrane uncoupler, indicating that mitochondria are the main site of ROS production due to toxin exposure. Cytotoxicity of trichothecenes was alleviated after treatment of the parental strain and highly sensitive mutants with antioxidants, suggesting that oxidative stress contributes to trichothecene sensitivity. Cotreatment with rapamycin and trichothecenes reduced ROS levels and cytotoxicity in the parental strain relative to the trichothecene treatment alone, but not in mitophagy deficient mutants, suggesting that elimination of trichothecene-damaged mitochondria by mitophagy improves cell survival. These results reveal that increased mitophagy is a cellular protection mechanism against trichothecene-induced mitochondrial oxidative stress and a potential target for trichothecene resistance.

High throughput screening identifies modulators of histone deacetylase inhibitors.

BackgroundPrevious studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521.ResultsScreening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5Δ strains show a G1 delay after CG-1521 treatment, however the gcn5Δ strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5Δ strain.ConclusionGenome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-528) contains supplementary material, which is available to authorized users.

The proper splicing of RNAi factors is critical for pericentric heterochromatin assembly in fission yeast.

Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres.