Project description:The goal of the study was to determine the direct targets of four oleate-responsive transcription factors (Oaf1p, Pip2p, Oaf3p, Adr1p) in the presence and absence of the fatty acid oleate. This data is part of a larger study outlined below: A novel network topology-based clustering approach was used to characterize a dynamic transcriptional regulatory network responsive to the fatty acid oleate. Condition-specific large-scale chromatin localization data were generated for four fatty-acid related regulators, and used to construct physical interaction networks. Data integration and simple statistical analysis of dynamic network architecture led to the identification of a regulatory role for a previously uncharacterized protein, and unique mechanism for coordination and regulation of multiple biological responses through the formation of related parallel combinatorial control motifs. Temporal coordination and specificity are achieved through the ability of a regulator (Oaf1p) to have multiple context-specific activities under the same condition. The analysis suggests that active paths for a transcriptional response can be highly interconnected in parallel and involve multiple functions for individual regulators through combinatorial control. Keywords: ChIP-chip, growth condition
Project description:Deciphering gene regulatory mechanisms through the analysis of high-throughput expression data is a challenging computational problem. Previous computational studies have used large expression datasets in order to resolve fine patterns of coexpression, producing clusters or modules of potentially coregulated genes. These methods typically examine promoter sequence information, such as DNA motifs or transcription factor occupancy data, in a separate step after clustering. We needed an alternative and more integrative approach to study the oxygen regulatory network in Saccharomyces cerevisiae using a small dataset of perturbation experiments. Mechanisms of oxygen sensing and regulation underlie many physiological and pathological processes, and only a handful of oxygen regulators have been identified in previous studies. We used a new machine learning algorithm called MEDUSA to uncover detailed information about the oxygen regulatory network using genome-wide expression changes in response to perturbations in the levels of oxygen, heme, Hap1, and Co2+. MEDUSA integrates mRNA expression, promoter sequence, and ChIP-chip occupancy data to learn a model that accurately predicts the differential expression of target genes in held-out data. We used a novel margin-based score to extract significant condition-specific regulators and assemble a global map of the oxygen sensing and regulatory network. This network includes both known oxygen and heme regulators, such as Hap1, Mga2, Hap4, and Upc2, as well as many new candidate regulators. MEDUSA also identified many DNA motifs that are consistent with previous experimentally identified transcription factor binding sites. Because MEDUSA's regulatory program associates regulators to target genes through their promoter sequences, we directly tested the predicted regulators for OLE1, a gene specifically induced under hypoxia, by experimental analysis of the activity of its promoter. In each case, deletion of the candidate regulator resulted in the predicted effect on promoter activity, confirming that several novel regulators identified by MEDUSA are indeed involved in oxygen regulation. MEDUSA can reveal important information from a small dataset and generate testable hypotheses for further experimental analysis.
Project description:These three replicates were analyzed in "Genomewide identification of Sko1 target promoters reveals a regulatory network that operates in response to osmotic stress in Saccharomyces cerevisiae. ", by Proft M, Gibbons FD, Copeland M, Roth FP, Struhl K; published in Eukaryot Cell. 2005 Aug;4(8):1343-52. A new analysis algorithm for Chip-chip data ('Chipper') is described in Genome Biology. Manuscript entitled "Chipper: discovering transcription-factor targets from chromatin immunoprecipitation microarrays using variance stabilization." by FD Gibbons, M Proft, K Struhl, and FP Roth. Accepted, no publication date as yet. Keywords: ChIP-chip
Project description:The goal of the study was to determine the expression profiles of transcription factor deletion strains in the presence of oleate. The data were used for a larger study described below: A novel network topology-based clustering approach was used to characterize a dynamic transcriptional regulatory network responsive to the fatty acid oleate. Condition-specific large-scale chromatin localization data were generated for four fatty-acid related regulators, and used to construct physical interaction networks. Data integration and simple statistical analysis of dynamic network architecture led to the identification of a regulatory role for a previously uncharacterized protein, and unique mechanism for coordination and regulation of multiple biological responses through the formation of related parallel combinatorial control motifs. Temporal coordination and specificity are achieved through the ability of a regulator (Oaf1p) to have multiple context-specific activities under the same condition. The analysis suggests that active paths for a transcriptional response can be highly interconnected in parallel and involve multiple functions for individual regulators through combinatorial control. Keywords: expression analysis of gene deletion strains
Project description:The intracellular metabolome of S. cerevisiae mutants in the gene AYT1 were measured under glucose growth conditions, as well as growth on oleate.
Project description:To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of established glucose signalling and metabolic pathway members in Saccharomyces cerevisiae by DNA microarrays. These deletion mutants do not induce pathway-specific transcriptional responses reflecting the tight interconnection between pathways of the glucose regulatory system. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. The study reveals both known and unknown relationships within and between individual pathways and their members. Metabolic components of the glucose regulatory system are most frequently affected at the transcriptional level. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. Tps2 and Tsl1, two enzymes involved in trehalose biosynthesis, are predicted to be the most downstream transcriptional components. This prediction is further validated by epistasis analysis of Tps2 double mutants. Taken together, this suggests that changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.
Project description:6-Nonadecynoic acid (6-NDA), a plant-derived acetylenic acid, exhibits strong inhibitory activity against the human fungal pathogens Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae to investigate the mechanism of action of this compound. 6-NDA elicited a transcriptome response indicative of fatty acid stress, altering the expression of genes known to be affected when yeast cells are grown in the presence of oleate. Mutants of S. cerevisiae lacking transcription factors that regulate fatty acid beta-oxidation showed increased sensitivity to 6-NDA. Fatty acid profile analysis indicated that 6-NDA inhibited the formation of fatty acids longer than 14 carbons in length. In addition, the growth inhibitory effect of 6-NDA was rescued in the presence of exogenously supplied oleate. To investigate the response of a pathogenic fungal species to 6-NDA, transcriptional profiling and biochemical analyses were also conducted in C. albicans. The transcriptional response and fatty acid profile of C. albicans were comparable to those obtained in S. cerevisiae, and the rescue of growth inhibition with exogenous oleate was also observed in C. albicans. In addition, 6-NDA enhanced the potency of the antifungal drug fluconazole in a fluconazole-resistant clinical isolate of C. albicans. Collectively, our results indicate that the antifungal activity of 6-NDA is mediated by a disruption in fatty acid homeostasis, and that this compound has potential utility in combination therapy in the treatment of drug-resistant fungal infections.