A gene network's capacity to process information, so as to bind past events to future actions, depends on its structure and logic. From previous and new microarray measurements in Saccharomyces cerevisiae following gene deletions and overexpressions, we identify a core gene regulatory network (GRN) of functional interactions between 328 genes and the transfer functions of each gene. Inferred connections are verified by gene enrichment.

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

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

Many bacterial pathogens use the type III secretion system to inject "effector" proteins into host cells. Here, we report the identification of a 24 member effector protein family found in pathogens including Salmonella, Shigella, and enteropathogenic E. coli. Members of this family subvert host cell function by mimicking the signaling properties of Ras-like GTPases. The effector IpgB2 stimulates cellular responses analogous to GTP-active RhoA, whereas IpgB1 and Map function as the active forms of Rac1 and Cdc42, respectively. These effectors do not bind guanine nucleotides or have sequences corresponding the conserved GTPase domain, suggesting that they are functional but not structural mimics. However, several of these effectors harbor intracellular targeting sequences that contribute to their signaling specificities. The activities of IpgB2, IpgB1, and Map are dependent on an invariant WxxxE motif found in numerous effectors leading to the speculation that they all function by a similar molecular mechanism.

Many disease states result from gene overexpression, often in a specific genetic context. To explore gene overexpression phenotypes systematically, we assembled an array of 5280 yeast strains, each containing an inducible copy of an S. cerevisiae gene, covering >80% of the genome. Approximately 15% of the overexpressed genes (769) reduced growth rate. This gene set was enriched for cell cycle-regulated genes, signaling molecules, and transcription factors. Overexpression of most toxic genes resulted in phenotypes different from known deletion mutant phenotypes, suggesting that overexpression phenotypes usually reflect a specific regulatory imbalance rather than disruption of protein complex stoichiometry. Global overexpression effects were also assayed in the context of a cyclin-dependent kinase mutant (pho85Delta). The resultant gene set was enriched for Pho85p targets and identified the yeast calcineurin-responsive transcription factor Crz1p as a substrate. Large-scale application of this approach should provide a strategy for identifying target molecules regulated by specific signaling pathways.

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

Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts. We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling. We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes. We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1). In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed. We furthermore show that these strains are compatible with automated genetic analysis. This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.

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

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

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

Synthetic genetic interactions have recently been mapped on a genome scale in the budding yeast Saccharomyces cerevisiae, providing a functional view of the central processes of eukaryotic life. Currently, comprehensive genetic interaction networks have not been determined for other species, and we therefore sought to model conserved aspects of genetic interaction networks in order to enable the transfer of knowledge between species.

Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates.

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

We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.