Dissecting the yeast heat shock response by CRISPRi/a
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ABSTRACT:
ORGANISM(S): synthetic construct
PROVIDER: GSE155455 | GEO | 2021/07/01
REPOSITORIES: GEO
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Project description:Protein homeostasis and cellular fitness in the presence of proteotoxic stress is promoted by heat shock factor 1 (HSF1), which controls basal and stress-induced expression of molecular chaperones and other targets. The major heat shock proteins Hsp70 and Hsp90 in turn participate in a negative feedback loop that ensures appropriate coordination of the heat shock response with environmental conditions. Features of this regulatory circuit in the budding yeast Saccharomyces cerevisiae have been recently defined, most notably regarding direct interaction between Hsf1 and the constitutively expressed Hsp70 Ssa1. We sought to further explore the complex nature of Ssa1/Hsf1 regulation. Ssa1 is found to interact independently with both the previously defined CE2 site in the Hsf1 carboxyl-terminal transcriptional activation domain as well as a novel site that we identify within the amino-terminal activation domain. Consistent with both sites bearing a recognition signature for Hsp70, we demonstrate that Ssa1 contacts Hsf1 using its substrate binding domain and abolishing either regulatory site results in loss of Ssa1 association. Removing Hsp70 regulation of Hsf1 results in global dysregulation of Hsf1 transcriptional activity, with synergistic effects when both sites are disrupted together on both gene expression and cellular fitness. Finally, we find that Hsp70 interacts with both transcriptional activation domains of Hsf1 in the related yeast Lachancea kluyveri, implying a conserved mechanism of regulation to promote cellular proteostasis.
2019-04-16 | GSE129832 | GEO
Project description:Yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. Keywords: Stress response
2005-09-21 | GSE3315 | GEO
Project description:Yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. Series containes 3 independent experiments.
2010-06-30 | E-GEOD-3315 | biostudies-arrayexpress
Project description:From preliminary experiments, HSP70 deficient MEF cells display moderate thermotolerance to a severe heatshock of 45.5 degrees after a mild preshock at 43 degrees, even in the absence of hsp70 protein. We would like to determine which genes in these cells are being activated to account for this thermotolerance. Keywords: thermal stress, heat shock response, knockout, cell culture, hsp70
2009-04-09 | GSE11120 | GEO
Project description:Thermotolerance is a crucial virulence attribute for Cryptococcus neoformans, which causes fatal fungal meningitis in humans. A protein kinase, Sch9, suppresses the thermotolerance of C. neoformans but its regulatory mechanism remains unknown. Here we elucidated the Sch9-dependent and -independent signaling networks for modulating the thermotolerance of C. neoformans through a genome-wide transcriptome analysis and reverse genetics approaches. We found that more than 1,800 genes were under transcriptional control during temperature upshift. Genes encoding for molecular chaperones and heat shock proteins were mainly upregulated, while those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process Sch9 was found to regulate basal or induced expression levels of some temperature-responsive genes. Interestingly we found that Sch9 was involved in transcriptional regulation of the Ire1 kinase, which is a key sensor for the unfolded protein response pathway, and was found to be involved in ER stress response. Most notably, our data demonstrated that expression of HSF1, encoding a heat shock transcription factor 1, was downregulated during temperature upshift and Sch9 suppresses its downregulation. In spite of such expression patterns, Hsf1 was essential for growth and its overexpression indeed promoted the thermotolerance of C. neoformans, suggesting dual roles of Hsf1 in thermotolerance. This idea was supported by additional transcriptome analysis with HSF1 overexpression strain, which revealed that Hsf1 served as both activator and repressor. Hsf1 promoted genes such as Hsp104 and Hsp70 (Ssa1 and Ssa2), both of which were found to be highly upregulated during temperature upshift and required for thermotolerance, while Hsf1 repressed genes involved in oxidative stress and thermotolerance.
2015-06-01 | GSE66509 | GEO
Project description:Thermotolerance is a crucial virulence attribute for Cryptococcus neoformans, which causes fatal fungal meningitis in humans. A protein kinase, Sch9, suppresses the thermotolerance of C. neoformans but its regulatory mechanism remains unknown. Here we elucidated the Sch9-dependent and -independent signaling networks for modulating the thermotolerance of C. neoformans through a genome-wide transcriptome analysis and reverse genetics approaches. We found that more than 1,800 genes were under transcriptional control during temperature upshift. Genes encoding for molecular chaperones and heat shock proteins were mainly upregulated, while those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process Sch9 was found to regulate basal or induced expression levels of some temperature-responsive genes. Interestingly we found that Sch9 was involved in transcriptional regulation of the Ire1 kinase, which is a key sensor for the unfolded protein response pathway, and was found to be involved in ER stress response. Most notably, our data demonstrated that expression of HSF1, encoding a heat shock transcription factor 1, was downregulated during temperature upshift and Sch9 suppresses its downregulation. In spite of such expression patterns, Hsf1 was essential for growth and its overexpression indeed promoted the thermotolerance of C. neoformans, suggesting dual roles of Hsf1 in thermotolerance. This idea was supported by additional transcriptome analysis with HSF1 overexpression strain, which revealed that Hsf1 served as both activator and repressor. Hsf1 promoted genes such as Hsp104 and Hsp70 (Ssa1 and Ssa2), both of which were found to be highly upregulated during temperature upshift and required for thermotolerance, while Hsf1 repressed genes involved in oxidative stress and thermotolerance.
2015-06-01 | GSE66508 | GEO
Project description:Dissecting the yeast heat shock response by CRISPRi/a
| PRJNA649790 | ENA
Project description:From preliminary experiments, HSP70 deficient MEF cells display moderate thermotolerance to a severe heatshock of 45.5 degrees after a mild preshock at 43 degrees, even in the absence of hsp70 protein. We would like to determine which genes in these cells are being activated to account for this thermotolerance. Experiment Overall Design: Two cell lines are analyzed - hsp70 knockout and hsp70 rescue cells. 6 microarrays from the (-/-)knockout cells are analyzed (3 Pretreated vs 3 unheated controls). For the (+/+) rescue cells, 4 microarrays are used (2 pretreated and 2 unheated controls). Cells were plated at 3k/well in a 96 well plate, covered with a gas permeable sealer and heat shocked at 43degrees for 30 minutes at the 20 hr time point. The RNA was harvested at 3hrs after heat treatment.
2009-04-17 | E-GEOD-11120 | biostudies-arrayexpress
Project description:Yeast DNA microarray was used to assess and compare the global expression profile of strains harboring different family members of the major cytosolic Hsp70 family. Viability of a yeast strain deleted for all genes encoding members of the Hsp70-Ssa family (Ssa1/2/3/4) was maintained by the presence of a single Ssa family member expressed ectopically from a plasmid vector. The Hsp70-Ssa family constitutes the main source of Hsp70 molecular chaperone activity in the yeast cell. A yeast cell must actively express a member of this family to remain viable. Hsp70-Ssa are highly conserved both within yeast and amongst other species. Ssa1 and 2 are 97% identical at amino acid level and 80% identical to Ssa3 and 4. The aim of this study was to attribute specific functions to single Ssa family members by identifying specific genes or gene families whose expression was altered in the presence (or absence) of Ssa family members. Eight independent RNA samples were pooled to represent a single biological sample for expression analysis. For example, the single sample analyzed for cells harboring only Ssa1 is a pooled sample of eight independent RNA extractions. Hybridization was performed for cells harboring either Ssa1, Ssa2, Ssa3 or Ssa4 as the sole cytosolic Hsp70-Ssa family member. Gene expression profiles of Ssa2/3/4 were all compared to Ssa1.
2012-12-31 | E-GEOD-32433 | biostudies-arrayexpress
Project description:In Drosophila larvae, acquired synaptic thermotolerance following heat shock has previously been shown to correlate with the induction of heat shock proteins (Hsps) including HSP70. We tested the hypothesis that synaptic thermotolerance would be significantly diminished in a temperature-sensitive strain (hsf4) which has been reported not to be able to produce inducible Hsps in response to heat shock. Contrary to our hypothesis, considerable thermoprotection was still observed at hsf4 larval synapses following heat shock. To investigate the cause of this thermoprotection, we conducted DNA microarray experiments to identify heat-induced transcript changes in these organisms. Transcripts of the hsp83, dnaJ-1(hsp40) and gstE1 genes were significantly up-regulated in hsf4 larvae after heat shock. In addition, increases in the levels of Hsp83 and DnaJ-1 proteins but not in the inducible form of Hsp70 were detected by Western blotting. The mode of heat shock administration differentially affected the relative transcript and translational changes for these chaperones. These results indicate that the compensatory up-regulation of constitutively expressed Hsps, in the absence of the synthesis of inducible Hsps including HSP70, could still provide substantial thermoprotection to both synapses and the whole organism. Keywords: heat shock response
2005-12-01 | GSE2998 | GEO