Genetic-interaction screens uncover novel biological roles and regulators of transcription factors in fission yeast.

In Schizosaccharomyces pombe, systematic analyses of single transcription factor deletion or overexpression strains have made substantial advances in determining the biological roles and target genes of transcription factors, yet these characteristics are still relatively unknown for over a quarter of them. Moreover, the comprehensive list of proteins that regulate transcription factors remains incomplete. To further characterize Schizosaccharomyces pombe transcription factors, we performed synthetic sick/lethality and synthetic dosage lethality screens by synthetic genetic array. Examination of 2,672 transcription factor double deletion strains revealed a sick/lethality interaction frequency of 1.72%. Phenotypic analysis of these sick/lethality strains revealed potential cell cycle roles for several poorly characterized transcription factors, including SPBC56F2.05, SPCC320.03, and SPAC3C7.04. In addition, we examined synthetic dosage lethality interactions between 14 transcription factors and a miniarray of 279 deletion strains, observing a synthetic dosage lethality frequency of 4.99%, which consisted of known and novel transcription factor regulators. The miniarray contained deletions of genes that encode primarily posttranslational-modifying enzymes to identify putative upstream regulators of the transcription factor query strains. We discovered that ubiquitin ligase Ubr1 and its E2/E3-interacting protein, Mub1, degrade the glucose-responsive transcriptional repressor Scr1. Loss of ubr1+ or mub1+ increased Scr1 protein expression, which resulted in enhanced repression of flocculation through Scr1. The synthetic dosage lethality screen also captured interactions between Scr1 and 2 of its known repressors, Sds23 and Amk2, each affecting flocculation through Scr1 by influencing its nuclear localization. Our study demonstrates that sick/lethality and synthetic dosage lethality screens can be effective in uncovering novel functions and regulators of Schizosaccharomyces pombe transcription factors.

Using a chemical genetic screen to enhance our understanding of the antimicrobial properties of copper.

The competitive toxic and stress-inducing nature of copper necessitates systems that sequester and export this metal from the cytoplasm of bacterial cells. Several predicted mechanisms of toxicity include the production of reactive oxygen species, thiol depletion, DNA, and iron-sulfur cluster disruption. Accompanying these mechanisms include pathways of homeostasis such as chelation, oxidation, and transport. Still, the mechanisms of copper resistance and sensitivity are not fully understood. Furthermore, studies fail to recognize that the response to copper is likely a result of numerous mechanisms, as in the case for homeostasis, in which proteins and enzymes work as a collective to maintain appropriate copper concentrations. In this study, we used the Keio collection, an array of 3985 Escherichia coli mutants, each with a deleted non-essential gene, to gain a better understanding of the effects of prolonged exposure to copper. In short, we recovered two copper homeostatic genes involved in transporting and assembling that are required in mediating prolonged copper stress under the conditions assessed. The gene coding for the protein TolC was uncovered as a sensitive hit, and we demonstrated that tolC, an outer membrane efflux channel, is key in mitigating copper sensitivity. Additionally, the activity of tRNA processing was enriched along with the deletion of several proteins involved in importing generated copper tolerance. Lastly, key genes belonging to central carbon metabolism and nicotinamide adenine dinucleotide biosynthesis were uncovered as tolerant hits. Overall, this study shows that copper sensitivity and tolerance are a result of numerous mechanisms acting in combination within the cell.

Fission yeast RNA-binding proteins Puf2 and Puf4 are involved in repression of ferrireductase Frp1 expression in response to iron.

This study identifies a post-transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA-binding proteins in Schizosaccharomyces pombe. Cells expressing Puf2 and Puf4 stimulate decay of the frp1+ mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1+ mRNA is stabilized in puf2Δ puf4Δ mutant cells under iron-replete conditions. As a result, puf2Δ puf4Δ cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP-frp1+ 3'UTR RNAs was generated using a reporter gene containing the 3' untranslated region (UTR) of frp1+ that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1+ 3'UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM-homology RNA-binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1+ 3'UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1+ mRNA under basal and iron-replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.

Plant PUF RNA-binding proteins: A wealth of diversity for post-transcriptional gene regulation.

PUF proteins are a conserved group of sequence-specific RNA-binding proteins that typically function to negatively regulate mRNA stability and translation. PUFs are well characterized at the molecular, structural and functional levels in Drosophila, Caenorhabditis elegans, budding yeast and human systems. Although usually encoded by small gene families, PUFs are over-represented in the plant genome, with up to 36 genes identified in a single species. PUF gene expansion in plants has resulted in extensive variability in gene expression patterns, diversity in predicted RNA-binding domain structure, and novel combinations of key amino acids involved in modular nucleotide binding. Reports on the characterization of plant PUF structure and function continue to expand, and include RNA target identification, subcellular distribution, crystal structure, and molecular mechanisms. Arabidopsis PUF mutant analysis has provided insight into biological function, and has identified roles related to development and environmental stress tolerance. The diversity of plant PUFs implies an extensive role for this family of proteins in post-transcriptional gene regulation. This diversity also holds the potential for providing novel RNA-binding domains that could be engineered to produce designer PUFs to alter the metabolism of target RNAs in the cell.

Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli.

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron⁻sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

Exposure to naphthenic acids and the acid extractable organic fraction from oil sands process-affected water alters the subcellular structure and dynamics of plant cells.

Oil sands surface mining generates vast quantities of oil sands process-affected water (OSPW) as a by-product of bitumen extraction. The acid extractable organic (AEO) fraction of OSPW contains several contaminants, including naphthenic acids (NAs). While responses of living organisms to NA and AEO exposure have been described at the developmental, physiological, metabolic and gene expression levels, the effects of these compounds at the cellular and subcellular level are limited. Using live cell fluorescence microscopy and a suite of fluorescent marker proteins, we studied the intracellular responses of the plant cell cytoskeleton and several membrane-bound organelles to NA and AEO treatments. A rapid disassembly of cortical microtubules and a decrease in dynamics associated with actin filaments was observed in response to these treatments. Concomitantly, the integrity and dynamics of mitochondria, peroxisomes, Golgi stacks, and endoplasmic reticulum were also altered. AEO treatments were the most toxic to cells and resulted in the accumulation reactive oxygen species. This study provides foundational evidence for intracellular responses to NA and AEO exposure using two evolutionarily diverse model plant cell types. This cellular assay could be used to identify the most toxic components of AEO sub-fractions, and assist in determining the effectiveness of OSPW remediation efforts.

Using a Chemical Genetic Screen to Enhance Our Understanding of the Antibacterial Properties of Silver.

It is essential to understand the mechanisms by which a toxicant is capable of poisoning the bacterial cell. The mechanism of action of many biocides and toxins, including numerous ubiquitous compounds, is not fully understood. For example, despite the widespread clinical and commercial use of silver (Ag), the mechanisms describing how this metal poisons bacterial cells remains incomplete. To advance our understanding surrounding the antimicrobial action of Ag, we performed a chemical genetic screen of a mutant library of Escherichia coli—the Keio collection, in order to identify Ag sensitive or resistant deletion strains. Indeed, our findings corroborate many previously established mechanisms that describe the antibacterial effects of Ag, such as the disruption of iron-sulfur clusters containing proteins and certain cellular redox enzymes. However, the data presented here demonstrates that the activity of Ag within the bacterial cell is more extensive, encompassing genes involved in cell wall maintenance, quinone metabolism and sulfur assimilation. Altogether, this study provides further insight into the antimicrobial mechanism of Ag and the physiological adaption of E. coli to this metal.

Differential Hepatic Gene Expression Profile of Male Fathead Minnows Exposed to Daily Varying Dose of Environmental Contaminants Individually and in Mixture.

Environmental contaminants are known to impair reproduction, metabolism and development in wild life and humans. To investigate the mechanisms underlying adverse effects of contaminants, fathead minnows were exposed to a number of endocrine disruptive chemicals (EDCs) including Nonylphenol (NP), bisphenol-A (BPA), Di(2-ethylhexyl) phthalate (DEHP), and a mixture of the three chemicals for 21 days, followed by determination of the liver transcriptome by expression microarrays. Pathway analysis revealed a distinct mode of action for the individual chemicals and their mixture. The results showed expression changes in over 980 genes in response to exposure to these EDC contaminants individually and in mixture. Ingenuity Pathway core and toxicity analysis were used to identify the biological processes, pathways and the top regulators affected by these compounds. A number of canonical pathways were significantly altered, including cell cycle & proliferation, lipid metabolism, inflammatory, innate immune response, stress response, and drug metabolism. We identified 18 genes that were expressed in all individual and mixed treatments. Relevant candidate genes identified from expression microarray data were verified using quantitative PCR. We were also able to identify specific genes affected by NP, BPA, and DEHP individually, but were also affected by exposure to the mixture of the contaminants. Overall the results of this study provide novel information on the adverse health impact of contaminants tested based on pathway analysis of transcriptome data. Furthermore, the results identify a number of new biomarkers that can potentially be used for screening environmental contaminants.

Embryonic exposure to model naphthenic acids delays growth and hatching in the pond snail Lymnaea stagnalis.

Naphthenic acids (NAs), a class of structurally diverse carboxylic acids with often complex ring structures and large aliphatic tail groups, are important by-products of many petrochemical processes including the oil sands mining activity of Northern Alberta. While it is evident that NAs have both acute and chronic harmful effects on many organisms, many aspects of their toxicity remain to be clarified. Particularly, while substantive data sets have been collected on NA toxicity in aquatic prokaryote and vertebrate model systems, to date, nothing is known about the toxic effects of these compounds on the embryonic development of aquatic invertebrate taxa, including freshwater mollusks. This study examines under laboratory conditions the toxicity of NAs extracted from oil sands process water (OSPW) and the low-molecular weight model NAs cyclohexylsuccinic acid (CHSA), cyclohexanebutyric acid (CHBA), and 4-tert-butylcyclohexane carboxylic acid (4-TBCA) on embryonic development of the snail Lymnaea stagnalis, a common freshwater gastropod with a broad Palearctic distribution. Evidence is provided for concentration-dependent teratogenic effects of both OSPW-derived and model NAs with remarkably similar nominal threshold concentrations between 15 and 20 mg/L and 28d EC50 of 31 mg/L. In addition, the data provide evidence for substantial toxicokinetic differences between CHSA, CHBA and 4-TBCA. Together, our study introduces Lymnaea stagnalis embryonic development as an effective model to assay NA-toxicity and identifies molecular architecture as a potentially important toxicokinetic parameter in the toxicity of low-molecular weight NA in embryonic development of aquatic gastropods.

Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.

Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast.

Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1 These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1(+)increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of theO-mannosyltransferase encoding gene omh1(+) Loss of omh1(+)abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1(+)resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2(+) or cbf12(+) This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network.

Biostimulation of Oil Sands Process-Affected Water with Phosphate Yields Removal of Sulfur-Containing Organics and Detoxification.

The ability to mitigate toxicity of oil sands process-affected water (OSPW) for return into the environment is an important issue for effective tailings management in Alberta, Canada. OSPW toxicity has been linked to classical naphthenic acids (NAs), but the toxic contribution of other acid-extractable organics (AEOs) remains unknown. Here, we examine the potential for in situ bioremediation of OSPW AEOs by indigenous algae. Phosphate biostimulation was performed in OSPW to promote the growth of indigenous photosynthetic microorganisms and subsequent toxicity and chemical changes were determined. After 12 weeks, the AEO fraction of phosphate-biostimulated OSPW was significantly less toxic to the fission yeast Schizosaccharomyces pombe than unstimulated OSPW. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) analysis of the AEO fraction in phosphate-biostimulated OSPW showed decreased levels of SO3 class compounds, including a subset that may represent linear arylsulfonates. A screen with S. pombe transcription factor mutant strains for growth sensitivity to the AEO fraction or sodium dodecylbenzenesulfonate revealed a mode of toxic action consistent with oxidative stress and detrimental effects on cellular membranes. These findings demonstrate a potential algal-based in situ bioremediation strategy for OSPW AEOs and uncover a link between toxicity and AEOs other than classical NAs.

Identification of detoxification pathways in plants that are regulated in response to treatment with organic compounds isolated from oil sands process-affected water.

Bitumen mining in the Athabasca oil sands region of northern Alberta results in the accumulation of large volumes of oil sands process-affected water (OSPW). The acid-extractable organic (AEO) fraction of OSPW contains a variety of compounds, including naphthenic acids, aromatics, and sulfur- and nitrogen-containing compounds that are toxic to aquatic and terrestrial organisms. We have studied the effect of AEO treatment on the transcriptome of root and shoot tissues in seedlings of the model plant, Arabidopsis thaliana. Several genes encoding enzymes involved in the xenobiotic detoxification pathway were upregulated, including cytochrome P450s (CYPs), UDP-dependent glycosyltransferases (UGTs), glutathione-S-transferases (GSTs), and membrane transporters. In addition, gene products involved in oxidative stress, ß-oxidation, and glucosinolate degradation were also upregulated, indicating other potential mechanisms of the adaptive response to AEO exposure. These results provide insight into the pathways that plants use to detoxify the organic acid component of OSPW. Moreover, this study advances our understanding of genes that could be exploited to potentially develop phytoremediation and biosensing strategies for AEO contaminants resulting from oil sands mining.

The NuA4 complex promotes translesion synthesis (TLS)-mediated DNA damage tolerance.

Lesions in DNA can block replication fork progression, leading to its collapse and gross chromosomal rearrangements. To circumvent such outcomes, the DNA damage tolerance (DDT) pathway becomes engaged, allowing the replisome to bypass a lesion and complete S phase. Chromatin remodeling complexes have been implicated in the DDT pathways, and here we identify the NuA4 remodeler, which is a histone acetyltransferase, to function on the translesion synthesis (TLS) branch of DDT. Genetic analyses in Saccharomyces cerevisiae showed synergistic sensitivity to MMS when NuA4 alleles, esa1-L254P and yng2Δ, were combined with the error-free bypass mutant ubc13Δ. The loss of viability was less pronounced when NuA4 complex mutants were disrupted in combination with error-prone/TLS factors, such as rev3Δ, suggesting an epistatic relationship between NuA4 and error-prone bypass. Consistent with cellular viability measurements, replication profiles after exposure to MMS indicated that small regions of unreplicated DNA or damage were present to a greater extent in esa1-L254P/ubc13Δ mutants, which persist beyond the completion of bulk replication compared to esa1-L254P/rev3Δ. The critical role of NuA4 in error-prone bypass is functional even after the bulk of replication is complete. Underscoring this observation, when Yng2 expression is restricted specifically to G2/M of the cell cycle, viability and TLS-dependent mutagenesis rates were restored. Lastly, disruption of HTZ1, which is a target of NuA4, also resulted in mutagenic rates of reversion on level with esa1-L254P and yng2Δ mutants, indicating that the histone variant H2A.Z functions in vivo on the TLS branch of DDT.

Systematic genetic analysis of transcription factors to map the fission yeast transcription-regulatory network.

Mapping transcriptional-regulatory networks requires the identification of target genes, binding specificities and signalling pathways of transcription factors. However, the characterization of each transcription factor sufficiently for deciphering such networks remains laborious. The recent availability of overexpression and deletion strains for almost all of the transcription factor genes in the fission yeast Schizosaccharomyces pombe provides a valuable resource to better investigate transcription factors using systematic genetics. In the present paper, I review and discuss the utility of these strain collections combined with transcriptome profiling and genome-wide chromatin immunoprecipitation to identify the target genes of transcription factors.

The effect of oil sands process-affected water and naphthenic acids on the germination and development of Arabidopsis.

Oil sands mining in the Athabasca region of northern Alberta results in the production of large volumes of oil sands process-affected water (OSPW). We have evaluated the effects of OSPW, the acid extractable organic (AEO) fraction of OSPW, and individual naphthenic acids (NAs) on the germination and development of the model plant, Arabidopsis thaliana (Arabidopsis). The surrogate NAs that were selected for this study were petroleum NAs that have been used in previous toxicology studies and may not represent OSPW NAs. A tricyclic diamondoid NA that was recently identified as a component of OSPW served as a model NA in this study. Germination of Arabidopsis seeds was not inhibited when grown on medium containing up to 75% OSPW or by 50mgL(-1) AEO. However, simultaneous exposure to three simple, single-ringed surrogate NAs or a double-ringed surrogate NA had an inhibitory effect on germination at a concentration of 10mgL(-1), whereas inhibition of germination by the diamondoid model NA was observed only at 50mgL(-1). Seedling root growth was impaired by treatment with low concentrations of OSPW, and exposure to higher concentrations of OSPW resulted in increased growth inhibition of roots and primary leaves, and caused bleaching of cotyledons. Treatment with single- or double-ringed surrogate NAs at 10mgL(-1) severely impaired seedling growth. AEO or diamondoid NA treatment was less toxic, but resulted in severely impaired growth at 50mgL(-1). At low NA concentrations there was occasionally a stimulatory effect on root and shoot growth, possibly owing to the broad structural similarity of some NAs to known plant growth regulators such as auxins. This report provides a foundation for future studies aimed at using Arabidopsis as a biosensor for toxicity and to identify genes with possible roles in NA phytoremediation.

Functional characterization of fission yeast transcription factors by overexpression analysis.

In Schizosaccharomyces pombe, over 90% of transcription factor genes are nonessential. Moreover, the majority do not exhibit significant growth defects under optimal conditions when deleted, complicating their functional characterization and target gene identification. Here, we systematically overexpressed 99 transcription factor genes with the nmt1 promoter and found that 64 transcription factor genes exhibited reduced fitness when ectopically expressed. Cell cycle defects were also often observed. We further investigated three uncharacterized transcription factor genes (toe1(+)-toe3(+)) that displayed cell elongation when overexpressed. Ectopic expression of toe1(+) resulted in a G1 delay while toe2(+) and toe3(+) overexpression produced an accumulation of septated cells with abnormalities in septum formation and nuclear segregation, respectively. Transcriptome profiling and ChIP-chip analysis of the transcription factor overexpression strains indicated that Toe1 activates target genes of the pyrimidine-salvage pathway, while Toe3 regulates target genes involved in polyamine synthesis. We also found that ectopic expression of the putative target genes SPBC3H7.05c, and dad5(+) and SPAC11D3.06 could recapitulate the cell cycle phenotypes of toe2(+) and toe3(+) overexpression, respectively. Furthermore, single deletions of the putative target genes urg2(+) and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15, and rds1(+), could suppress the phenotypes of toe1(+) and toe2(+) overexpression, respectively. This study implicates new transcription factors and metabolism genes in cell cycle regulation and demonstrates the potential of systematic overexpression analysis to elucidate the function and target genes of transcription factors in S. pombe.

Deciphering the transcriptional-regulatory network of flocculation in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP-chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2(+) and deletion of rfl1(+) resulted in strong flocculation and transcriptional upregulation of gsf2(+)/pfl1(+) and several other putative flocculin genes (pfl2(+)-pfl9(+)). Overexpression of the pfl(+) genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl(+) overexpression. Among the pfl1(+) genes, only loss of gsf2(+) abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2(+) or adn3(+) overexpression was likely mediated by the transcriptional activation of cell wall-remodeling genes including gas2(+), psu1(+), and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2(+) expression in an inhibitory feed-forward loop involving mbx2(+). These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall-remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.

Conserved rules govern genetic interaction degree across species.

BACKGROUND: Synthetic genetic interactions have recently been mapped on a genome scale in the budding yeast Saccharomyces cerevisiae, providing a functional view of the central processes of eukaryotic life. Currently, comprehensive genetic interaction networks have not been determined for other species, and we therefore sought to model conserved aspects of genetic interaction networks in order to enable the transfer of knowledge between species. RESULTS: Using a combination of physiological and evolutionary properties of genes, we built models that successfully predicted the genetic interaction degree of S. cerevisiae genes. Importantly, a model trained on S. cerevisiae gene features and degree also accurately predicted interaction degree in the fission yeast Schizosaccharomyces pombe, suggesting that many of the predictive relationships discovered in S. cerevisiae also hold in this evolutionarily distant yeast. In both species, high single mutant fitness defect, protein disorder, pleiotropy, protein-protein interaction network degree, and low expression variation were significantly predictive of genetic interaction degree. A comparison of the predicted genetic interaction degrees of S. pombe genes to the degrees of S. cerevisiae orthologs revealed functional rewiring of specific biological processes that distinguish these two species. Finally, predicted differences in genetic interaction degree were independently supported by differences in co-expression relationships of the two species. CONCLUSIONS: Our findings show that there are common relationships between gene properties and genetic interaction network topology in two evolutionarily distant species. This conservation allows use of the extensively mapped S. cerevisiae genetic interaction network as an orthology-independent reference to guide the study of more complex species.