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Growth competition experiment on the Yeast Homozygous Diploid Deletion Pool to investigate which ORFs conferred an advantage/disadvantage when growing on both standard and high concentration of oleic acid

ABSTRACT: The combination of genetic analysis and genome-wide expression profiles make Saccharomyces cerevisiae the cradle of Systems Biology. The way S. cerevisiae uses carbon sources has long been a landmark for studies concerning cellular responses to environmental conditions. S. cerevisiae conserves the entire machinery for utilization of fatty acids, yet with a different compartmentalization compared to higher eukaryotes. We propose S. cerevisiae as a model to understand how perixosomal compartmentalization of oleate metabolism co-evolved with the transcriptional regulatory networks of FA metabolism. With this aim, we performed growth competition experiments in oleate on the pooled collection of all viable yeast null mutants. We integrated results of competitive fitness with transcriptional profiles in oleate, identifying two genes previously not directly associated to FA metabolism: OAR1 and FET3. For the first time we show the importance of iron metabolism not only in maintaining functional mitochondria, but also in influencing peroxisome wellness.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE21620 | GEO | 2012/04/26

SECONDARY ACCESSION(S): PRJNA126999

REPOSITORIES: GEO

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Project description:The combination of genetic analysis and genome-wide expression profiles make Saccharomyces cerevisiae the cradle of Systems Biology. The way S. cerevisiae uses carbon sources has long been a landmark for studies concerning cellular responses to environmental conditions. S. cerevisiae conserves the entire machinery for utilization of fatty acids, yet with a different compartmentalization compared to higher eukaryotes. We propose S. cerevisiae as a model to understand how perixosomal compartmentalization of oleate metabolism co-evolved with the transcriptional regulatory networks of FA metabolism. With this aim, we performed growth competition experiments in oleate on the pooled collection of all viable yeast null mutants. We integrated results of competitive fitness with transcriptional profiles in oleate, identifying two genes previously not directly associated to FA metabolism: OAR1 and FET3. For the first time we show the importance of iron metabolism not only in maintaining functional mitochondria, but also in influencing peroxisome wellness. An aliquot of the Yeast Homozygous Diploid Deletion Pool (95401.H1Pool, Invitrogen), containing equal amounts of each barcoded viable knock-out mutant from the Saccharomyces Genome Deletion project, was grown in the presence of G418 in YPD medium for 24h, then aliquots of 5x10^7 cells were harvested and stored at -80M-BM-0C. From the same pooled culture, equal amounts of cells were washed and transferred to YP medium added with oleic acid 0.1% and 5%. We collected samples of cells 24h and 72h after carbon source metabolic shift, and genomic DNA was extracted. Each experiment was performed in biological duplicate. The upTAG and downTAG regions were amplified separately in PCR reactions, one for each experimental condition, using specific primers. Amplified upTAG and downTAG sequences were combined in equal amounts and used to probe high-density oligonucleotide arrays (Affymetrix GeneChip_DNA_Tag3). Data analysis was performed as previously described (Pierce et al., 2007). In order to adapt this data analysis protocol to our experimental design, and to the use of GeneChip_DNA_Tag3 arrays, some changes to the procedure have been improved.

Project description:Subtelomeric chromatin is subject to evolutionarily conserved complex epigenetic regulation and is implicated in numerous aspects of cellular function including formation of heterochromatin, regulation of different stress response pathways, and control of lifespan. Subtelomeric DNA is characterized by the presence of specific repeated segments that serve to propagate silencing activities or to protect chromosomal regions from spreading epigenetic control. Using condition-specific genome wide chromatin immunoprecipitation and expression data, we show that several yeast transcription factors regulate subtelomeric silencing in response to various environmental stimuli through conditional association with proto-silencing regions called X elements. In this context, some factors control the propagation of silencing toward centromeres in response to stimuli affecting stress responses and metabolism, whereas others appear to influence boundaries of silencing, regulating telomere-proximal genes in Y’ elements. The factors implicated here have previously been shown to control adjacent genes at intrachromosomal positions, suggesting dual functionality of the factors and a possible mechanism of coordinating intrachromosomal gene expression with subtelomeric silencing. These data suggest a fundamental mechanism to coordinate telomere biology related to aging and adaptation with cellular environment and the activities of other cellular processes. These are Chip-CHIP data for myc tagged Oaf1p transcription factor from S. cerevisiae grown in the presence or absence of the fatty acid oleate. ChIP-CHIP analysis was performed to determine the genomic distribution of Oaf1p transcription factor in the BY4742 yeast strain after growth in 0.1% glucose, or in the presence of the fatty acid oleate. Three biological replicates for each growth condition (in the presence of low glucose or 5 h after a shift to medium containing oleate as a carbon source). ChIP samples were amplified by PCR, labelled and hybridized to 50-mer tiling arrays covering both strands of the entire yeast genome at a 64 bp resolution.

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Growth competition experiment on the Yeast Homozygous Diploid Deletion Pool to investigate which ORFs conferred an advantage/disadvantage when growing on both standard and high concentration of oleic acid

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