"Chemogenomics: Tools for Protein Families" and "Chemical Genomics: Chemical and Biological Integration"

Pharmacogenomics. Jan, 2003  |  Pubmed ID: 12517281

Chemogenomic Profiling: Identifying the Functional Interactions of Small Molecules in Yeast

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2004  |  Pubmed ID: 14718668

We demonstrate the efficacy of a genome-wide protocol in yeast that allows the identification of those gene products that functionally interact with small molecules and result in the inhibition of cellular proliferation. Here we present results from screening 10 diverse compounds in 80 genome-wide experiments against the complete collection of heterozygous yeast deletion strains. These compounds include anticancer and antifungal agents, statins, alverine citrate, and dyclonine. In several cases, we identified previously known interactions; furthermore, in each case, our analysis revealed novel cellular interactions, even when the relationship between a compound and its cellular target had been well established. In addition, we identified a chemical core structure shared among three therapeutically distinct compounds that inhibit the ERG24 heterozygous deletion strain, demonstrating that cells may respond similarly to compounds of related structure. The ability to identify on-and-off target effects in vivo is fundamental to understanding the cellular response to small-molecule perturbants.

Mechanisms of Haploinsufficiency Revealed by Genome-wide Profiling in Yeast

Genetics. Apr, 2005  |  Pubmed ID: 15716499

Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that approximately 3% of all approximately 5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders.

Genome-wide Requirements for Resistance to Functionally Distinct DNA-damaging Agents

PLoS Genetics. Aug, 2005  |  Pubmed ID: 16121259

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of approximately 4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

Introns Regulate RNA and Protein Abundance in Yeast

Genetics. Sep, 2006  |  Pubmed ID: 16816425

The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast.

A Unique and Universal Molecular Barcode Array

Nature Methods. Aug, 2006  |  Pubmed ID: 16862133

Molecular barcode arrays allow the analysis of thousands of biological samples in parallel through the use of unique 20-base-pair (bp) DNA tags. Here we present a new barcode array, which is unique among microarrays in that it includes at least five replicates of every tag feature. The use of smaller dispersed replicate features dramatically improves performance versus a single larger feature and allows the correction of previously undetectable hybridization defects.

Experimental Approaches to Identify Genetic Networks

Current Opinion in Biotechnology. Oct, 2006  |  Pubmed ID: 16962766

Systems biology offers the promise of a fully integrated view of cellular physiology. To realize this potential requires the analysis of diverse genome-wide datasets and the incorporation of these analyses into integrated networks. In the past decade, the budding yeast Saccharomyces cerevisiae has provided the benchmark for the design of such large-scale experiments. Many of these experimental approaches have been adopted and adapted to study other systems, including worm, fly, fish and mammalian cultured cells, using an ingenious set of molecular tools.

Chemical Genomic Profiling for Identifying Intracellular Targets of Toxicants Producing Parkinson's Disease

Toxicological Sciences : an Official Journal of the Society of Toxicology. Jan, 2007  |  Pubmed ID: 17043098

The yeast deletion collection includes approximately 4700 strains deleted for both copies of every nonessential gene. This collection is a powerful resource for identifying the cellular pathways that functionally interact with drugs. In the present study, the complete pool of approximately 4700 barcoded homozygous deletion strains of Saccharomyces cerevisiae were surveyed to identify genes/pathways interacting with 1-methyl-4-phenylpyridinium (MPP(+)) and N,N-dimethyl-4-4-bipiridinium (paraquat), neurotoxicants that can produce Parkinson's disease. Each yeast mutant is molecularly "barcoded" the collections can be grown competitively and ranked for sensitivity by microarray hybridization. Analysis data from these screens allowed us to determine that the multivesicular body pathway is an important element of toxicity induced by both MPP(+) and paraquat. When yeast genes that when deleted showed sensitivity to MPP(+) and paraquat toxicity were analyzed for their homology to human genes, 80% were found to have highly conserved human homologs (with e < 10(-8)). Future work will address if these human genes may also functionally interact with MPP(+) and paraquat toxicity.

Systematic Pathway Analysis Using High-resolution Fitness Profiling of Combinatorial Gene Deletions

Nature Genetics. Feb, 2007  |  Pubmed ID: 17206143

Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single- and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination-derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.

Accelerating the Discovery of Biologically Active Small Molecules Using a High-throughput Yeast Halo Assay

Journal of Natural Products. Mar, 2007  |  Pubmed ID: 17291044

The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.

A High-resolution Atlas of Nucleosome Occupancy in Yeast

Nature Genetics. Oct, 2007  |  Pubmed ID: 17873876

We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.

Examining Protein Protein Interactions Using Endogenously Tagged Yeast Arrays: the Cross-and-capture System

Genome Research. Dec, 2007  |  Pubmed ID: 17989249

Comprehensive approaches to detect protein-protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p-Mms1p and Sae2p-Mre11p. The Rtt101p-Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies.

Genome-wide Analysis of Barcoded Saccharomyces Cerevisiae Gene-deletion Mutants in Pooled Cultures

Nature Protocols. 2007  |  Pubmed ID: 18007632

The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.

Chemical-genetic Approaches for Exploring the Mode of Action of Natural Products

Progress in Drug Research. Fortschritte Der Arzneimittelforschung. Progrès Des Recherches Pharmaceutiques. 2008  |  Pubmed ID: 18416308

Determining the mode of action of bioactive compounds, including natural products, is a central problem in chemical biology. Because many genes are conserved from the yeast Saccharomyces cerevisiae to humans and a number of powerful genomics tools and methodologies have been developed for this model system, yeast is making a major contribution to the field of chemical genetics. The set of barcoded yeast deletion mutants, including the set of approximately 5000 viable haploid and homozygous diploid deletion mutants and the complete set of approximately 6000 heterozygous deletion mutants, containing the set of approximately 1000 essential genes, are proving highly informative for identifying chemical-genetic interactions and deciphering compound mode of action. Gene deletions that render cells hypersensitive to a specific drug identify pathways that buffer the cell against the toxic effects of the drug and thereby provide clues about both gene and compound function. Moreover, compounds that show similar chemical-genetic profiles often perturb similar target pathways. Gene dosage can be exploited to discover connections between compounds and their targets. For example, haploinsufficiency profiling of an antifungal compound, in which the set of approximately 6000 heterozygous diploid deletion mutants are scored for hypersensitivity to a compound, may identify the target directly. Creating deletion mutant collections in other fungal species, including the major human fungal pathogen Candida albicans, will expand our chemical genomics tool set, allowing us to screen for antifungal lead drugs directly. The yeast deletion mutant collection is also being exploited to map large-scale genetic interaction data obtained from genome-wide synthetic lethal screens and the integration of this data with chemical genetic data should provide a powerful system for linking compounds to their target pathway. Extensive application of chemical genetics in yeast has the potential to develop a small molecule inhibitor for the majority of all approximately 6000 yeast genes.

The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes

Science (New York, N.Y.). Apr, 2008  |  Pubmed ID: 18420932

Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes ( approximately 80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.

Identification of Small Molecule Inhibitors of Pseudomonas Aeruginosa Exoenzyme S Using a Yeast Phenotypic Screen

PLoS Genetics. Feb, 2008  |  Pubmed ID: 18454192

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

The Extensive and Condition-dependent Nature of Epistasis Among Whole-genome Duplicates in Yeast

Genome Research. Jul, 2008  |  Pubmed ID: 18463300

Since complete redundancy between extant duplicates (paralogs) is evolutionarily unfavorable, some degree of functional congruency is eventually lost. However, in budding yeast, experimental evidence collected for duplicated metabolic enzymes and in global physical interaction surveys had suggested widespread functional overlap between paralogs. While maintained functional overlap is thought to confer robustness against genetic mutation and facilitate environmental adaptability, it has yet to be determined what properties define paralogs that can compensate for the phenotypic consequence of deleting a sister gene, how extensive this epistasis is, and how adaptable it is toward alternate environmental states. To this end, we have performed a comprehensive experimental analysis of epistasis as indicated by aggravating genetic interactions between paralogs resulting from an ancient whole-genome duplication (WGD) event occurring in the budding yeast Saccharomyces cerevisiae, and thus were able to compare properties of large numbers of epistatic and non-epistatic paralogs with identical evolutionary times since divergence. We found that more than one-third (140) of the 399 examinable WGD paralog pairs were epistatic under standard laboratory conditions and that additional cases of epistasis became obvious only under media conditions designed to induce cellular stress. Despite a significant increase in within-species sequence co-conservation, analysis of protein interactions revealed that paralogs epistatic under standard laboratory conditions were not more functionally overlapping than those non-epistatic. As experimental conditions had an impact on the functional categorization of paralogs deemed epistatic and only a fraction of potential stress conditions have been interrogated here, we hypothesize that many epistatic relationships remain unresolved.

An Integrated Platform of Genomic Assays Reveals Small-molecule Bioactivities

Nature Chemical Biology. Aug, 2008  |  Pubmed ID: 18622389

Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.

Yeast Barcoders: a Chemogenomic Application of a Universal Donor-strain Collection Carrying Bar-code Identifiers

Nature Methods. Aug, 2008  |  Pubmed ID: 18622398

The ability to perform complex bioassays in parallel enables experiments that are otherwise impossible because of throughput and cost constraints. For example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is bar-coded with unique DNA sequences. It is, however, time-consuming and expensive to individually bar-code individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique bar codes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of bar-coded 'decreased abundance by mRNA perturbation' (DAmP) loss-of-function strains comprising 87.1% of all essential yeast genes. These experiments validate both the Barcoders and the DAmP strain collection as useful tools for genome-wide chemical-genetic assays.

Off-target Effects of Psychoactive Drugs Revealed by Genome-wide Assays in Yeast

PLoS Genetics. 2008  |  Pubmed ID: 18688276

To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes.

Yeast Chemical Genomics and Drug Discovery: an Update

Trends in Pharmacological Sciences. Oct, 2008  |  Pubmed ID: 18755517

The Saccharomyces cerevisiae sequencing project (the first eukaryotic genome decoded) was completed in 1995 and, subsequently, the first version of the yeast knockout collection was made available in 2002. Since then, many diverse studies have applied these resources to understand drug mechanism of action and to identify novel drug targets and target pathways. In this update of an earlier review, we present a snapshot of the current state of chemical genomic approaches in yeast, propose a set of integrated chemical genomic assays to move the field forward and consider its near-term future.

Combination Chemical Genetics

Nature Chemical Biology. Nov, 2008  |  Pubmed ID: 18936752

Predicting the behavior of living organisms is an enormous challenge given their vast complexity. Efforts to model biological systems require large datasets generated by physical binding experiments and perturbation studies. Genetic perturbations have proven important and are greatly facilitated by the advent of comprehensive mutant libraries in model organisms. Small-molecule chemical perturbagens provide a complementary approach, especially for systems that lack mutant libraries, and can easily probe the function of essential genes. Though single chemical or genetic perturbations provide crucial information associating individual components (for example, genes, proteins or small molecules) with pathways or phenotypes, functional relationships between pathways and modules of components are most effectively obtained from combined perturbation experiments. Here we review the current state of and discuss some future directions for 'combination chemical genetics', the systematic application of multiple chemical or mixed chemical and genetic perturbations, both to gain insight into biological systems and to facilitate medical discoveries.

Chemical-genetic Profiling of Imidazo[1,2-a]pyridines and -pyrimidines Reveals Target Pathways Conserved Between Yeast and Human Cells

PLoS Genetics. Nov, 2008  |  Pubmed ID: 19043571

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical-genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure-activity relationships.

A Library of Yeast Transcription Factor Motifs Reveals a Widespread Function for Rsc3 in Targeting Nucleosome Exclusion at Promoters

Molecular Cell. Dec, 2008  |  Pubmed ID: 19111667

The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes. One-third of the binding specificities have not been previously reported. Several binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially approximately 100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters.

Identification of Genes Involved in the Toxic Response of Saccharomyces Cerevisiae Against Iron and Copper Overload by Parallel Analysis of Deletion Mutants

Toxicological Sciences : an Official Journal of the Society of Toxicology. Jan, 2008  |  Pubmed ID: 17785683

Iron and copper are essential nutrients for life as they are required for the function of many proteins but can be toxic if present in excess. Accumulation of these metals in the human body as a consequence of overload disorders and/or high environmental exposures has detrimental effects on health. The budding yeast Saccharomyces cerevisiae is an accepted cellular model for iron and copper metabolism in humans primarily because of the high degree of conservation between pathways and proteins involved. Here we report a systematic screen using yeast deletion mutants to identify genes involved in the toxic response to growth-inhibitory concentrations of iron and copper sulfate. We aimed to understand the cellular responses to toxic concentrations of these two metals by analyzing the different subnetworks and biological processes significantly enriched with these genes. Our results indicate the presence of two different detoxification pathways for iron and copper that converge toward the vacuole. The product of several of the identified genes in these pathways form molecular complexes that are conserved in mammals and include the retromer, endosomal sorting complex required for transport (ESCRT) and AP-3 complexes, suggesting that the mechanisms involved can be extrapolated to humans. Our data also suggest a disruption in ion homeostasis and, in particular, of iron after copper exposure. Moreover, the identification of treatment-specific genes associated with biological processes such as DNA double-strand break repair for iron and tryptophan biosynthesis for copper suggests differences in the mechanisms by which these two metals are toxic at high concentrations.

Combining Chemical Genomics Screens in Yeast to Reveal Spectrum of Effects of Chemical Inhibition of Sphingolipid Biosynthesis

BMC Microbiology. 2009  |  Pubmed ID: 19144191

Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug.

Knocking Sense into Regulatory Pathways

Nature Biotechnology. Feb, 2009  |  Pubmed ID: 19204694

Computationally Driven, Quantitative Experiments Discover Genes Required for Mitochondrial Biogenesis

PLoS Genetics. Mar, 2009  |  Pubmed ID: 19300474

Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.

Novel Insights into Iron Metabolism by Integrating Deletome and Transcriptome Analysis in an Iron Deficiency Model of the Yeast Saccharomyces Cerevisiae

BMC Genomics. 2009  |  Pubmed ID: 19321002

Iron-deficiency anemia is the most prevalent form of anemia world-wide. The yeast Saccharomyces cerevisiae has been used as a model of cellular iron deficiency, in part because many of its cellular pathways are conserved. To better understand how cells respond to changes in iron availability, we profiled the yeast genome with a parallel analysis of homozygous deletion mutants to identify essential components and cellular processes required for optimal growth under iron-limited conditions. To complement this analysis, we compared those genes identified as important for fitness to those that were differentially-expressed in the same conditions. The resulting analysis provides a global perspective on the cellular processes involved in iron metabolism.

Precise Gene-dose Alleles for Chemical Genetics

Genetics. Jun, 2009  |  Pubmed ID: 19332878

Modulating gene dose is an effective way to alter protein levels and modify phenotypes to understand gene function. In addition, combining gene-dose alleles with chemical perturbation can provide insight into drug-gene interactions. Here, we present a strategy that combines diverse loss-of-function alleles to systematically modulate gene dose in Saccharomyces cerevisiae. The generated gene dosage allele set expands the genetic toolkit for uncovering novel phenotypes.

A Molecular Barcoded Yeast ORF Library Enables Mode-of-action Analysis of Bioactive Compounds

Nature Biotechnology. Apr, 2009  |  Pubmed ID: 19349972

We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.

Self-reporting Cells

BioTechniques. Apr, 2009  |  Pubmed ID: 19480631

Advances in biomarker discovery, synthetic biology and next-generation genomics promise to make designer cells a reality in the near future. Such cells can be engineered to report on diverse aspects of their physiology, potentially opening up new avenues of biological inquiry and enhancing our understanding of cell function.

Chemogenomic Approaches to Elucidation of Gene Function and Genetic Pathways

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19521822

The approximately 6,000 strains in the yeast deletion collection can be studied in a single culture by using a microarray to detect the 20 bp DNA "barcodes" or "tags" contained in each strain. Barcode intensities measured by microarray are compared across time-points or across conditions to analyze the relative fitness of each strain. The development of this pooled fitness assay has greatly facilitated the functional annotation of the yeast genome by making genome-wide gene-deletion studies faster and easier, and has led to the development of high throughput methods for studying drug action in yeast. Pooled screens can be used for identifying gene functions, measuring the functional relatedness of gene pairs to group genes into pathways, identifying drug targets, and determining a drug's mechanism of action. This process involves five main steps: preparing aliquots of pooled cells, pooled growth, isolation of genomic DNA and PCR amplification of the barcodes, array hybridization, and data analysis. In addition to yeast fitness applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, siRNA vectors, and molecular inversion probes.

Alternative Splicing of PTC7 in Saccharomyces Cerevisiae Determines Protein Localization

Genetics. Sep, 2009  |  Pubmed ID: 19564484

It is well established that higher eukaryotes use alternative splicing to increase proteome complexity. In contrast, Saccharomyces cerevisiae, a single-cell eukaryote, conducts predominantly regulated splicing through retention of nonfunctional introns. In this article we describe our discovery of a functional intron in the PTC7 (YHR076W) gene that can be alternatively spliced to create two mRNAs that code for distinct proteins. These two proteins localize to different cellular compartments and have distinct cellular roles. The protein translated from the spliced mRNA localizes to the mitochondria and its expression is carbon-source dependent. In comparison, the protein translated from the unspliced mRNA contains a transmembrane domain, localizes to the nuclear envelope, and mediates the toxic effects of Latrunculin A exposure. In conclusion, we identified a definitive example of functional alternative splicing in S. cerevisiae that confers a measurable fitness benefit.

Quantitative Phenotyping Via Deep Barcode Sequencing

Genome Research. Oct, 2009  |  Pubmed ID: 19622793

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

Two-color Cell Array Screen Reveals Interdependent Roles for Histone Chaperones and a Chromatin Boundary Regulator in Histone Gene Repression

Molecular Cell. Aug, 2009  |  Pubmed ID: 19683497

We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess consequences of genetic perturbations on gene expression. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at core histone promoters. We found that activation of histone (HTA1) gene expression involves both relief of Rtt106-mediated repression by the activity of the histone acetyltransferase Rtt109 and restriction of Rtt106 to the promoter region by the bromodomain-containing protein Yta7. We propose that the maintenance of Asf1/HIR/Rtt106-mediated repressive chromatin domains is the primary mechanism of cell-cycle regulation of histone promoters. Our data suggest that this pathway may represent a chromatin regulatory mechanism that is broadly used across the genome.

A Comparative Analysis of DNA Barcode Microarray Feature Size

BMC Genomics. 2009  |  Pubmed ID: 19825181

Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity.

You Too Can Play with an Edge

Nature Methods. Nov, 2009  |  Pubmed ID: 19876015

Recent Advances and Method Development for Drug Target Identification

Trends in Pharmacological Sciences. Feb, 2010  |  Pubmed ID: 20004028

Although it is commonly recognized that most drugs cause inhibition or activation of function by physically binding to one or more gene products, the direct interactions of bioactive small molecules with specific gene products, or targets, is often not well characterized. From a therapeutic perspective, it is nevertheless essential to know a drug's binding partner(s) to understand the mechanism of action and anticipate possible side effects to avoid costly clinical failures. This knowledge is increasingly important as the prevalence of polypharmacy expands to include drugs that engage multiple targets. This review provides a succinct overview of several recent approaches that employ genetics, proteomics, expression profiling or bioinformatics procedures for the systematic characterization of the targets of bioactive compounds. The continuous improvement and advancement of existing technologies is critically discussed and we offer a perspective on the future of innovative emerging new generation technologies.

Integrating High-throughput Genetic Interaction Mapping and High-content Screening to Explore Yeast Spindle Morphogenesis

The Journal of Cell Biology. Jan, 2010  |  Pubmed ID: 20065090

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.

The Genetic Landscape of a Cell

Science (New York, N.Y.). Jan, 2010  |  Pubmed ID: 20093466

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

Systematic Analysis of Genome-wide Fitness Data in Yeast Reveals Novel Gene Function and Drug Action

Genome Biology. 2010  |  Pubmed ID: 20226027

We systematically analyzed the relationships between gene fitness profiles (co-fitness) and drug inhibition profiles (co-inhibition) from several hundred chemogenomic screens in yeast. Co-fitness predicted gene functions distinct from those derived from other assays and identified conditionally dependent protein complexes. Co-inhibitory compounds were weakly correlated by structure and therapeutic class. We developed an algorithm predicting protein targets of chemical compounds and verified its accuracy with experimental testing. Fitness data provide a novel, systems-level perspective on the cell.

Genotype to Phenotype: a Complex Problem

Science (New York, N.Y.). Apr, 2010  |  Pubmed ID: 20413493

We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.

Highly-multiplexed Barcode Sequencing: an Efficient Method for Parallel Analysis of Pooled Samples

Nucleic Acids Research. Jul, 2010  |  Pubmed ID: 20460461

Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such 'Bar-seq' assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.

A Universal TagModule Collection for Parallel Genetic Analysis of Microorganisms

Nucleic Acids Research. Aug, 2010  |  Pubmed ID: 20494978

Systems-level analyses of non-model microorganisms are limited by the existence of numerous uncharacterized genes and a corresponding over-reliance on automated computational annotations. One solution to this challenge is to disrupt gene function using DNA tag technology, which has been highly successful in parallelizing reverse genetics in Saccharomyces cerevisiae and has led to discoveries in gene function, genetic interactions and drug mechanism of action. To extend the yeast DNA tag methodology to a wide variety of microorganisms and applications, we have created a universal, sequence-verified TagModule collection. A hallmark of the 4280 TagModules is that they are cloned into a Gateway entry vector, thus facilitating rapid transfer to any compatible genetic system. Here, we describe the application of the TagModules to rapidly generate tagged mutants by transposon mutagenesis in the metal-reducing bacterium Shewanella oneidensis MR-1 and the pathogenic yeast Candida albicans. Our results demonstrate the optimal hybridization properties of the TagModule collection, the flexibility in applying the strategy to diverse microorganisms and the biological insights that can be gained from fitness profiling tagged mutant collections. The publicly available TagModule collection is a platform-independent resource for the functional genomics of a wide range of microbial systems in the post-genome era.

Most "dark Matter" Transcripts Are Associated with Known Genes

PLoS Biology. May, 2010  |  Pubmed ID: 20502517

A series of reports over the last few years have indicated that a much larger portion of the mammalian genome is transcribed than can be accounted for by currently annotated genes, but the quantity and nature of these additional transcripts remains unclear. Here, we have used data from single- and paired-end RNA-Seq and tiling arrays to assess the quantity and composition of transcripts in PolyA+ RNA from human and mouse tissues. Relative to tiling arrays, RNA-Seq identifies many fewer transcribed regions ("seqfrags") outside known exons and ncRNAs. Most nonexonic seqfrags are in introns, raising the possibility that they are fragments of pre-mRNAs. The chromosomal locations of the majority of intergenic seqfrags in RNA-Seq data are near known genes, consistent with alternative cleavage and polyadenylation site usage, promoter- and terminator-associated transcripts, or new alternative exons; indeed, reads that bridge splice sites identified 4,544 new exons, affecting 3,554 genes. Most of the remaining seqfrags correspond to either single reads that display characteristics of random sampling from a low-level background or several thousand small transcripts (median length = 111 bp) present at higher levels, which also tend to display sequence conservation and originate from regions with open chromatin. We conclude that, while there are bona fide new intergenic transcripts, their number and abundance is generally low in comparison to known exons, and the genome is not as pervasively transcribed as previously reported.

A Predictive Model for Drug Bioaccumulation and Bioactivity in Caenorhabditis Elegans

Nature Chemical Biology. Jul, 2010  |  Pubmed ID: 20512140

The resistance of Caenorhabditis elegans to pharmacological perturbation limits its use as a screening tool for novel small bioactive molecules. One strategy to improve the hit rate of small-molecule screens is to preselect molecules that have an increased likelihood of reaching their target in the worm. To learn which structures evade the worm's defenses, we performed the first survey of the accumulation and metabolism of over 1,000 commercially available drug-like small molecules in the worm. We discovered that fewer than 10% of these molecules accumulate to concentrations greater than 50% of that present in the worm's environment. Using our dataset, we developed a structure-based accumulation model that identifies compounds with an increased likelihood of bioavailability and bioactivity, and we describe structural features that facilitate small-molecule accumulation in the worm. Preselecting molecules that are more likely to reach a target by first applying our model to the tens of millions of commercially available compounds will undoubtedly increase the success of future small-molecule screens with C. elegans.

A Survey of Yeast Genomic Assays for Drug and Target Discovery

Pharmacology & Therapeutics. Aug, 2010  |  Pubmed ID: 20546776

Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach.

Genome-wide Screen in Saccharomyces Cerevisiae Identifies Vacuolar Protein Sorting, Autophagy, Biosynthetic, and TRNA Methylation Genes Involved in Life Span Regulation

PLoS Genetics. Jul, 2010  |  Pubmed ID: 20657825

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-wide Analysis of Chromatin Structure

PLoS Genetics. Sep, 2010  |  Pubmed ID: 20824081

Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function.

Exploring Gene Function and Drug Action Using Chemogenomic Dosage Assays

Methods in Enzymology. 2010  |  Pubmed ID: 20946813

In this chapter, we describe a series of genome-wide, cell-based assays that provide a solid basis for understanding drug-gene interactions, gene function, and for defining the mechanism of action of small molecules. Each of these assays takes advantage of the ability to grow complex pools competitively and to use high-density microarrays that report the results of such screens. The assays described here take advantage of alterations in gene dosage of Saccharomyces cerevisiae, and include HIP (haploinsufficiency profiling), HOP (homozygous profiling), and MSP (multicopy suppression profiling) as genetic tools to understand gene function and drug mechanism. The common experimental theme is that, in each assay, strains are pooled and screened in parallel to investigate the relative contribution of each gene product to sensitivity or resistance to a drug or environmental perturbation across the genome in a single assay. Further, the compendium of results from these screens can inform large-scale network analysis of genetic function, gene-gene interactions, and mechanism of drug action.

Gene Annotation and Drug Target Discovery in Candida Albicans with a Tagged Transposon Mutant Collection

PLoS Pathogens. 2010  |  Pubmed ID: 20949076

Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.

Endosomal Protein Sorting and Autophagy Genes Contribute to the Regulation of Yeast Life Span

Autophagy. Nov, 2010  |  Pubmed ID: 20953148

Accumulating evidence from various organisms points to a role for autophagy in the regulation of life span. By performing a genome-wide screen to identify novel life span determinants in Saccharomyces cerevisiae, we have obtained further insights into the autophagy-related and -unrelated degradation processes that may be important for preventing cellular senescence. The generation of multivesicular bodies and their fusion with the vacuole in the endosomal pathway emerged as novel cell functions involved in yeast chronological survival and longevity extension.

Extensive Role of the General Regulatory Factors, Abf1 and Rap1, in Determining Genome-wide Chromatin Structure in Budding Yeast

Nucleic Acids Research. Mar, 2011  |  Pubmed ID: 21081559

The packaging of eukaryotic DNA into chromatin has profound consequences for gene regulation, as well as for other DNA transactions such as recombination, replication and repair. Understanding how this packaging is determined is consequently a pressing problem in molecular genetics. DNA sequence, chromatin remodelers and transcription factors affect chromatin structure, but the scope of these influences on genome-wide nucleosome occupancy patterns remains uncertain. Here, we use high resolution tiling arrays to examine the contributions of two general regulatory factors, Abf1 and Rap1, to nucleosome occupancy in Saccharomyces cerevisiae. These factors have each been shown to bind to a few hundred promoters, but we find here that thousands of loci show localized regions of altered nucleosome occupancy within 1 h of loss of Abf1 or Rap1 binding, and that altered chromatin structure can occur via binding sites having a wide range of affinities. These results indicate that DNA-binding transcription factors affect chromatin structure, and probably dynamics, throughout the genome to a much greater extent than previously appreciated.

Knocking out Multigene Redundancies Via Cycles of Sexual Assortment and Fluorescence Selection

Nature Methods. Feb, 2011  |  Pubmed ID: 21217751

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

Systematic Exploration of Essential Yeast Gene Function with Temperature-sensitive Mutants

Nature Biotechnology. Apr, 2011  |  Pubmed ID: 21441928

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.

The Synthetic Genetic Interaction Network Reveals Small Molecules That Target Specific Pathways in Sacchromyces Cerevisiae

Molecular BioSystems. Jun, 2011  |  Pubmed ID: 21487606

High-throughput elucidation of synthetic genetic interactions (SGIs) has contributed to a systems-level understanding of genetic robustness and fault-tolerance encoded in the genome. Pathway targets of various compounds have been predicted by comparing chemical-genetic synthetic interactions to a network of SGIs. We demonstrate that the SGI network can also be used in a powerful reverse pathway-to-drug approach for identifying compounds that target specific pathways of interest. Using the SGI network, the method identifies an indicator gene that may serve as a good candidate for screening a library of compounds. The indicator gene is selected so that compounds found to produce sensitivity in mutants deleted for the indicator gene are likely to abrogate the target pathway. We tested the utility of the SGI network for pathway-to-drug discovery using the DNA damage checkpoint as the target pathway. An analysis of the compendium of synthetic lethal interactions in yeast showed that superoxide dismutase 1 (SOD1) has significant SGI connectivity with a large subset of DNA damage checkpoint and repair (DDCR) genes in Saccharomyces cerevisiae, and minimal SGIs with non-DDCR genes. We screened a sod1Δ strain against three National Cancer Institute (NCI) compound libraries using a soft agar high-throughput halo assay. Fifteen compounds out of ∼3100 screened showed selective toxicity toward sod1Δ relative to the isogenic wild type (wt) strain. One of these, 1A08, caused a transient increase in growth in the presence of sublethal doses of DNA damaging agents, suggesting that 1A08 inhibits DDCR signaling in yeast. Genome-wide screening of 1A08 against the library of viable homozygous deletion mutants further supported DDCR as the relevant targeted pathway of 1A08. When assayed in human HCT-116 colorectal cancer cells, 1A08 caused DNA-damage resistant DNA synthesis and blocked the DNA-damage checkpoint selectively in S-phase.

The SWI/SNF Complex Acts to Constrain Distribution of the Centromeric Histone Variant Cse4

The EMBO Journal. May, 2011  |  Pubmed ID: 21505420

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.

A Comprehensive Platform for Highly Multiplexed Mammalian Functional Genetic Screens

BMC Genomics. 2011  |  Pubmed ID: 21548937

Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.

Dosage Suppression Genetic Interaction Networks Enhance Functional Wiring Diagrams of the Cell

Nature Biotechnology. Jun, 2011  |  Pubmed ID: 21572441

Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.

Design, Synthesis, and Characterization of a Highly Effective Hog1 Inhibitor: a Powerful Tool for Analyzing MAP Kinase Signaling in Yeast

PloS One. 2011  |  Pubmed ID: 21655328

The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

Design, Synthesis and Characterization of a Highly Effective Inhibitor for Analog-sensitive (as) Kinases

PloS One. 2011  |  Pubmed ID: 21698101

Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1(T100G)). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

New Azole Antifungal Agents with Novel Modes of Action: Synthesis and Biological Studies of New Tridentate Ligands Based on Pyrazole and Triazole

European Journal of Medicinal Chemistry. Sep, 2011  |  Pubmed ID: 21723647

The synthesis and extensive biological study of two new tridentates ligands based on pyrazole and triazole are described. The antifungal activity against the budding yeast cells of the newly synthesized compounds was determined. These compounds were toxic to yeast cells. Cell cycle analysis suggested that treatment with these compounds impairs cell division in G1 of the cell cycle. Using yeast-based functional genomics technologies, we found that these compounds tolerance requires DNA repair pathway and SKI complex function. We have also found that the PKC1 heterozygous deletion strain was the most sensitive to these compounds using HaploInsufficiency Profiling, suggesting that the Pkc1 protein may be the target for these compounds. These results strongly suggest that these compounds induce DNA damage and thus exert a different mechanism of action compared to other azole derivatives. These two compounds might therefore represent promising lead compounds for further development of antifungal drugs for human therapy.

Signature-tagged Mutagenesis to Characterize Genes Through Competitive Selection of Bar-coded Genome Libraries

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21815096

The availability of collections of genome-wide deletion mutants greatly accelerates systematic analyses of gene function. However, each of the thousands of genes that comprise a genome must be phenotyped individually unless they can be assayed in parallel and subsequently deconvolved. To this end, unique molecular identifiers have been developed for a variety of microbes. Specifically, the addition of DNA "tags," or "bar codes," to each mutant allows all mutants in a collection to be pooled and phenotyped in parallel, greatly increasing experimental throughput. In this chapter, we provide an overview of current methodologies used to create such tagged mutant collections and outline how they can be applied to understand gene function, gene-gene interactions, and drug-gene interactions. Finally, we present a methodology that uses universal TagModules, capable of bar coding a wide range of microorganisms, and demonstrate its reduction to practice by creating tagged mutant collections in the pathogenic yeast Candida albicans.

The Automated Cell: Compound and Environment Screening System (ACCESS) for Chemogenomic Screening

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21863492

The automated cell, compound and environment screening system (ACCESS) was developed as an automated platform for chemogenomic research. In the yeast Saccharomyces cerevisiae, a number of genomic screens rely on the modulation of gene dose to determine the mode of action of bioactive compounds or the effects of environmental/compound perturbations. These and other phenotypic experiments have been shown to benefit from high-resolution growth curves and a highly automated controlled environment system that enables a wide range of multi-well assays that can be run over many days without any manual intervention. Furthermore, precise control of drug dosing, timing of drug exposure, and precise timing of cell harvesting at specific generation times are important for optimal results. Some of these benefits include the ability to derive fine distinctions between growth rates of mutant strains (1) and the discovery of novel compounds and drug targets (2). The automation has also enabled large-scale screening projects with over 100,000 unique compounds screened to date including a thousand genome-wide screens (3). The ACCESS system also has a diverse set of software tools to enable users to set up, run, annotate, and evaluate complex screens with minimal training.

Displaying Chemical Information on a Biological Network Using Cytoscape

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21877291

Cytoscape is an open-source software package that is widely used to integrate and visualize diverse data sets in biology. This chapter explains how to use Cytoscape to integrate open-source chemical information with a biological network. By visualizing information about known compound-target interactions in the context of a biological network of interest, one can rapidly identify novel avenues to perturb the system with compounds and, for example, potentially identify therapeutically relevant targets. Herein, two different protocols are explained in detail, with no prior knowledge of Cytoscape assumed, which demonstrate how to incorporate data from the ChEMBL database with either a gene-gene or a protein-protein interaction network. ChEMBL is a very large, open-source repository of compound-target information available from the European Molecular Biology Laboratory.

Evolution of Nucleosome Occupancy: Conservation of Global Properties and Divergence of Gene-specific Patterns

Molecular and Cellular Biology. Nov, 2011  |  Pubmed ID: 21896781

To examine the role of nucleosome occupancy in the evolution of gene expression, we measured the genome-wide nucleosome profiles of four yeast species, three belonging to the Saccharomyces sensu stricto lineage and the more distantly related Candida glabrata. Nucleosomes and associated promoter elements at C. glabrata genes are typically shifted upstream by ∼20 bp, compared to their orthologs from sensu stricto species. Nonetheless, all species display the same global organization features first described for Saccharomyces cerevisiae: a stereotypical nucleosome organization along genes and a division of promoters into those that contain or lack a pronounced nucleosome-depleted region (NDR), with the latter displaying a more dynamic pattern of gene expression. Despite this global similarity, however, nucleosome occupancy at specific genes diverged extensively between sensu stricto and C. glabrata orthologs (∼50 million years). Orthologs with dynamic expression patterns tend to maintain their lack of NDR, but apart from that, sensu stricto and C. glabrata orthologs are nearly as similar in nucleosome occupancy patterns as nonorthologous genes. This extensive divergence in nucleosome occupancy contrasts with a conserved pattern of gene expression. Thus, while some evolutionary changes in nucleosome occupancy contribute to gene expression divergence, nucleosome occupancy often diverges extensively with apparently little impact on gene expression.

Curcumin Inhibits Growth of Saccharomyces Cerevisiae Through Iron Chelation

Eukaryotic Cell. Nov, 2011  |  Pubmed ID: 21908599

Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G(1) phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.

Nucleosome-coupled Expression Differences in Closely-related Species

BMC Genomics. 2011  |  Pubmed ID: 21942931

Genome-wide nucleosome occupancy is negatively related to the average level of transcription factor motif binding based on studies in yeast and several other model organisms. The degree to which nucleosome-motif interactions relate to phenotypic changes across species is, however, unknown.

A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

PLoS Genetics. Oct, 2011  |  Pubmed ID: 22028670

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.

Compound Prioritization Methods Increase Rates of Chemical Probe Discovery in Model Organisms

Chemistry & Biology. Oct, 2011  |  Pubmed ID: 22035796

Preselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in Saccharomyces cerevisiae and identified ~7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided.

Dafadine Inhibits DAF-9 to Promote Dauer Formation and Longevity of Caenorhabditis Elegans

Nature Chemical Biology. Dec, 2011  |  Pubmed ID: 22057127

The DAF-9 cytochrome P450 is a key regulator of dauer formation, developmental timing and longevity in the nematode Caenorhabditis elegans. Here we describe the first identified chemical inhibitor of DAF-9 and the first reported small-molecule tool that robustly induces dauer formation in typical culture conditions. This molecule (called dafadine) also inhibits the mammalian ortholog of DAF-9(CYP27A1), suggesting that dafadine can be used to interrogate developmental control and longevity in other animals.

Systematic Exploration of Synergistic Drug Pairs

Molecular Systems Biology. 2011  |  Pubmed ID: 22068327

Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs. Drug synergy can result when drugs target the products of genes that act in parallel pathways ('specific synergy'). Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes. Alternatively, 'promiscuous synergy' can arise when one drug non-specifically increases the effects of many other drugs, for example, by increased bioavailability. To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae. We found 38 antifungal synergies, 37 of which were novel. While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs. Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected. While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non-specific promiscuous synergy.

Inhibition of Mitochondrial Translation As a Therapeutic Strategy for Human Acute Myeloid Leukemia

Cancer Cell. Nov, 2011  |  Pubmed ID: 22094260

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

Multiple Means to the Same End: the Genetic Basis of Acquired Stress Resistance in Yeast

PLoS Genetics. Nov, 2011  |  Pubmed ID: 22102822

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H(2)O(2)). Surprisingly, there was little overlap in the genes required for acquisition of H(2)O(2) tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H(2)O(2) in each case. Integrative network analysis of these results with respect to protein-protein interactions, synthetic-genetic interactions, and functional annotations identified many processes not previously linked to H(2)O(2) tolerance. We tested and present several models that explain the lack of overlap in genes required for H(2)O(2) tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance.

Restriction of Histone Gene Transcription to S Phase by Phosphorylation of a Chromatin Boundary Protein

Genes & Development. Dec, 2011  |  Pubmed ID: 22156209

The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.

Bugs, Drugs and Chemical Genomics

Nature Chemical Biology. Jan, 2012  |  Pubmed ID: 22173359

The serendipitous discovery of penicillin inspired intensive research into how small molecules affect basic cellular processes and their potential to treat disease. Biochemical and genetic approaches have been fundamental for clarifying small-molecule modes of action. Genomic technologies have permitted the use of chemical-genetic strategies that comprehensively study compound-target relationships in the context of a living cell, providing a systems biology view of both the cellular targets and the interdependent networks that respond to chemical stress. These studies highlight the fact that in vitro determinations of mechanism rarely translate into a complete understanding of drug behavior in the cell. Here, we review key discoveries that gave rise to the field of chemical genetics, with particular attention to chemical-genetic strategies developed for bakers' yeast, their extension to clinically relevant microbial pathogens, and the potential of these approaches to affect antimicrobial drug discovery.

Genomic Approaches for Determining Nucleosome Occupancy in Yeast

Methods in Molecular Biology (Clifton, N.J.). 2012  |  Pubmed ID: 22183606

The basic unit of chromatin is double-stranded DNA wrapped around nucleosome core particles, the -classic "beads-on-a-string" described by Kornberg and colleagues. The history of chromatin studies has experienced many peaks, from the earliest studies by Miescher to the biochemical studies of the 1960s and 1970s, the appreciation for the influence of histone modifications in controlling gene expression in the 1990s to the genome-wide studies that began in 2006 and show no signs of abating with the introduction of next generation sequencing technologies. Genome-wide studies not only have provided a base line to understand relationships between chromatin structure and gene function but also have begun to provide new insights into chromatin remodelling. Here, we describe the use of genome-wide approaches to determining nucleosome occupancy in yeast.

Mitochondrial Electron Transport is the Cellular Target of the Oncology Drug Elesclomol

PloS One. 2012  |  Pubmed ID: 22253786

Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS) and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s) and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting.

Dafadine Inhibits DAF-9 to Promote Dauer Formation and Longevity of Caenorhabditis Elegans

Nature Chemical Biology. 2012  |  Pubmed ID: 22337098