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.

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.

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.

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.

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.

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.

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.

Chromatin- and transcription-related factors repress transcription from within coding regions throughout the Saccharomyces cerevisiae genome.

Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.

Genetic interactions of MAF1 identify a role for Med20 in transcriptional repression of ribosomal protein genes.

Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription under multiple conditions. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis.

Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryotes.

Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of approximately 220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that approximately 29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.

Using expression profiling data to identify human microRNA targets.

We demonstrate that paired expression profiles of microRNAs (miRNAs) and mRNAs can be used to identify functional miRNA-target relationships with high precision. We used a Bayesian data analysis algorithm, GenMiR++, to identify a network of 1,597 high-confidence target predictions for 104 human miRNAs, which was supported by RNA expression data across 88 tissues and cell types, sequence complementarity and comparative genomics data. We experimentally verified our predictions by investigating the result of let-7b downregulation in retinoblastoma using quantitative reverse transcriptase (RT)-PCR and microarray profiling: some of our verified let-7b targets include CDC25A and BCL7A. Compared to sequence-based predictions, our high-scoring GenMiR++ predictions had much more consistent Gene Ontology annotations and were more accurate predictors of which mRNA levels respond to changes in let-7b levels.

Comprehensive genetic analysis of transcription factor pathways using a dual reporter gene system in budding yeast.

The development and application of genomic reagents and techniques has fuelled progress in our understanding of regulatory networks that control gene expression in eukaryotic cells. However, a full description of the network of regulator-gene interactions that determine global gene expression programs remains elusive and will require systematic genetic as well as biochemical assays. Here, we describe a functional genomics approach that combines reporter technology, genome-wide array-based reagents and high-throughput imaging to discover new regulators controlling gene expression patterns in Saccharomyces cerevisiae. Our strategy utilizes the synthetic genetic array (SGA) method to systematically introduce promoter-GFP (green fluorescent protein) reporter constructs along with a control promoter-RFP (red fluorescent protein) gene into the array of approximately 4500 viable yeast deletion mutants. Fluorescence intensities from each reporter are assayed from individual colonies arrayed on solid agar plates using a scanning fluorimager and the ratio of GFP to RFP intensity reveals deletion mutants that cause differential GFP expression. We are exploiting this screening approach to construct a detailed map describing the interplay of regulators controlling the eukaryotic cell cycle. The method is extensible to any transcription factor or signalling pathway for which an appropriate reporter gene can be devised.

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.

Identification of transcription factor targets by phenotypic activation and microarray expression profiling in yeast.

A major obstacle to identify physiological transcriptional targets is that the conditions that induce the majority of yeast transcription factors (TFs) are unknown. Microarray analyses of deletion mutants indicate that most TFs are inactive under standard growth conditions. To overcome this, we screened an ordered array of yeast open reading frames (ORFs) to identify TFs that confer reduced fitness upon overexpression, suggesting that overexpression results in an activated state (phenotypic activation). Approximately one-third of all yeast TFs exhibited this phenotype. Here, we describe in detail our methodology to characterize these TF overexpression strains including microarray expression profiling, data analysis, and motif searching. Our analyses show that in many cases, the differentially regulated genes correspond to physiological functions and known targets of well-characterized TFs. The expected binding sites of several TFs were also identified in the promoters of these genes. Moreover, novel DNA-binding sequences and putative targets were identified for less-characterized TFs. These results demonstrate that phenotypic activation is an effective approach to rapidly characterize TFs on a large scale, which should also be feasible in other organisms.

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 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.

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.

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.

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.

Transcriptional networks: reverse-engineering gene regulation on a global scale.

A major objective in post-genome research is to fully understand the transcriptional control of each gene and the targets of each transcription factor. In yeast, large-scale experimental and computational approaches have been applied to identify co-regulated genes, cis regulatory elements, and transcription factor DNA binding sites in vivo. Methods for modeling and predicting system behavior, and for reconciling discrepancies among data types, are being explored. The results indicate that a complete and comprehensive yeast transcriptional network will ultimately be achieved.