Project description:Contains gene expression profiles of yeast single and double deletion mutants of gene-specific transcription factors. Genetic interactions were studied by comparing gene expression changes of double mutants with gene expression changes in the respective single mutants. Pairs of gene-specific transcription factors were chosen based on previous evidence for epistasis, including synthetic genetic interactions as well as common DNA binding.
Project description:Contains gene expression profiles of yeast single and double deletion mutants of gene-specific transcription factors. Genetic interactions were studied by comparing gene expression changes of double mutants with gene expression changes in the respective single mutants. Pairs of gene-specific transcription factors were chosen based on previous evidence for epistasis, including synthetic genetic interactions as well as common DNA binding.
Project description:Sit4 is a PP2A-like Ser/Thr protein phosphatase implicated in the regulation of cellular processes important for cell survival during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0 complex. In addition, SIT4 exhibited a negative genetic interaction with VSP27, as sit4∆vps27∆ double mutants had a shortened lifespan compared to sit4∆ and vps27∆ single mutants. Our results also show that Vps27 did not increase sit4∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4∆ cells, as sit4∆vps27∆ double mutants exhibited high iron levels, impaired mitochondrial respiration and mitochondrial DNA (mtDNA) depletion. These findings show for the first-time a cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan.
Project description:In Saccharomyces cerevisiae, specific genes physically relocate from the nucleoplasm to the nuclear periphery concomitant with transcriptional activation, where they associate with the nuclear pore complex (NPC). We took a genomics approach in order to gain insight into the universality and physiological relevance of the interaction between active genes and the NPC. Using synthetic genetic array (SGA) approach, we identified interactions between components of the SAGA histone acetyltransferase complex and the Mlp and Nup60 subunits of the NPC. Cells lacking these SAGA and NPC components display growth defects under optimal growth conditions, in which cells are grown on rich medium containing the preferred sugar glucose as a carbon source and incubated at their optimal temperature. That growth defects were observed under these non-stress conditions suggests that these interactions are indicative of defects in normal cell physiology. These results are consistent with a model where physical interactions between the NPC and SAGA are important for transcription of constitutively expressed genes. To test this hypothesis, we used microarray analysis to assess changes global transcript levels in the absence of Nup60, Ada2, or Nup60 and Ada2. Microarray analysis reveals that the growth defect in these double mutants is correlated with a synthetic reduction in steady-state transcript levels for numerous genes that are strongly expressed in wildtype cells. These results suggest that SAGA and Nup60 cooperate in transcriptional regulation, and are consistent with a model for global regulation of SAGA-dependent transcription at the NPC. Wildype, single deletion, and double deletion cells were inoculated to equal starting ODs in 50 mL YEPD media and grown at 30C with shaking. Cells were collected at mid-log phase, washed with chilled water, and stored at -80C for later processing. RNA was isolated using TRIzol reagent according to the manufacturer's protocol, with modification for yeast cells.