Project description:Cbf1 is a basic helix-loop-helix transcription factor which regulates the expression of many genes of the sulphur assimilation pathway in yeast. Gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis, is constitutively elevated in cbf1 mutants indicating that Cbf1 normally represses GSH1 expression. We used transcriptional profiling to show that a number of antioxidant and stress genes are similarly repressed by Cbf1, consistent with the high oxidant tolerance of cbf1 mutants. Our data indicate that Cbf1 plays a key role in the regulation of gene expression during oxidative stress conditions induced by exposure to hydrogen peroxide. The Yap1 transcription factor induces GSH1 expression in response to hydrogen peroxide in a mechanism that does not require the Met4 transcription factor to overcome the inhibitory action of Cbf1. We show that hydrogen peroxide does not affect Cbf1 occupancy on the GSH1 promoter, but results in specific modification of Cbf1 by phosphorylation. Our data indicate that casein kinase (CK2) can directly phosphorylate Cbf1 in vitro. Furthermore, CK2 activity is required to phosphorylate Cbf1 and induce GSH1 expression in yeast cells in response to hydrogen peroxide. CK2 mutants are sensitive to hydrogen peroxide consistent with a role for CK2 in regulating the cellular response to oxidative stress. CK2 is a ubiquitous serine/threonine protein kinase and the finding that it is regulated by oxidative stress is particularly interesting, since there is increasing evidence that it is involved in a number of pathological conditions including cancer, neurodegenerative and cardiovascular diseases.
Project description:Adaptation to hydrogen peroxide in Saccharomyces cerevisiae is profiled with expression arrays. Adaptation describes the process in which a mild dose of toxin (in this case, hydrogen peroxide) is able to protect against a later acute dose. Here, we study two adaptive protocols (0.1 mM H2O2 and 0.1 + 0.4 mM H2O2) and one acute protocol (0.4 mM H2O2) to identify processes uniquely involved in adaptation. Predictions from these studies are validated in expression profiling of deletion mutants of the transcription factors Yap1, Mga2, and Rox1.
Project description:ppGpp accumulation caused by ectopic expression of RelA in Saccharomyces cerevisiae gave rise to marked changes in gene expression with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the function-unknown hypothetical gene YBR072C-A, followed by many other known stress-responsive genes. ppGpp acuumulation resulted in enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, and high temperature.
Project description:The yeast Snt2 protein helps coordinate the transcriptional response to hydrogen-peroxide mediated oxidative stress (rapamycin or DMSO)
Project description:This project aims to identify novel RNA binding proteins in the baker's yeast , Saccharomyces cerevisiae, involved in the oxidative stress,. Since interactions between RNAs and proteins may be transient, yeast cells, either untreated and growth in rich media and exposed to 0.5 mM hydrogen peroxide for 15 minutes were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringent conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples where we performed competition assays with polyadenylic acid
Project description:Caloric restriction extends lifespan, an effect once thought to involve attenuation of reactive oxygen species (ROS) generated by aerobic metabolism. However, recent evidence suggests that caloric restriction may in fact raise ROS levels, which in turn provides protection from acute doses of oxidant through a process called adaptation. To shed light on the molecular mechanisms of adaptation, we designed a series of genome-wide deletion fitness screens to identify genes involved in adaptation to hydrogen peroxide.