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Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response.

Gutin J, Sadeh A, Rahat A, Aharoni A, Friedman N - Mol. Syst. Biol. (2015)

Bottom Line: The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program.We established a high-throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single-knockout and 1,566 double-knockout strains that carry an HSP12-GFP allele as a reporter for Msn2/4 activity.Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three-way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Computer Science & Engineering Institute of Life Sciences Hebrew University, Jerusalem, Israel.

No MeSH data available.


Related in: MedlinePlus

Using double-knockout library to uncover pathway structureUsing the SGA methodology, we constructed a double-knockout library of 30 * 56 strains. Each one of the strains contains deletions of two genes and the HSP12-GFP reporter.The library was screened under five conditions (Fig1B). The raw data of HSP12-GFP expression (log) after the exposure to heat stress is shown. Overall, the average effect of the double perturbation resembles the measured effect of the single perturbation.Epistatic interactions in the data that match the well-established structure of the cAMP/PKA pathway. HSP12-GFP expression after heat stress is shown. Δras2 is epistatic over Δira2, implicating that Ira2 is upstream of Ras2. Δpde2 is epistatic over Δras2, implicating that Ras2 is upstream of Pde2.Consensus matrix of the epistasis interactions in our library. We identified all the epistatic interactions in three stress conditions (Materials and Methods). We color double perturbations in which the epistasis observed in at least two of the stress conditions. Row epistasis defined as the epistasis of the gene denoting the row over the gene denoting the column. Column epistasis is vice versa. Double perturbation pairs in which the direction of the epistasis interaction has changed between the conditions are colored in black. The upper row and the rightmost column present the single perturbations effects (red/green is increased/decreased HSP12-GFP levels relative to WT).
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fig03: Using double-knockout library to uncover pathway structureUsing the SGA methodology, we constructed a double-knockout library of 30 * 56 strains. Each one of the strains contains deletions of two genes and the HSP12-GFP reporter.The library was screened under five conditions (Fig1B). The raw data of HSP12-GFP expression (log) after the exposure to heat stress is shown. Overall, the average effect of the double perturbation resembles the measured effect of the single perturbation.Epistatic interactions in the data that match the well-established structure of the cAMP/PKA pathway. HSP12-GFP expression after heat stress is shown. Δras2 is epistatic over Δira2, implicating that Ira2 is upstream of Ras2. Δpde2 is epistatic over Δras2, implicating that Ras2 is upstream of Pde2.Consensus matrix of the epistasis interactions in our library. We identified all the epistatic interactions in three stress conditions (Materials and Methods). We color double perturbations in which the epistasis observed in at least two of the stress conditions. Row epistasis defined as the epistasis of the gene denoting the row over the gene denoting the column. Column epistasis is vice versa. Double perturbation pairs in which the direction of the epistasis interaction has changed between the conditions are colored in black. The upper row and the rightmost column present the single perturbations effects (red/green is increased/decreased HSP12-GFP levels relative to WT).

Mentions: To systematically map genetic interactions, we generated a “mini epistatic map” (Schuldiner et al, 2005; Collins et al, 2007) of 30 query strains against 56 target strains (Fig3A, Materials and Methods). The resulting strains contained two mutant alleles and the HSP12-GFP reporter gene. In total, the library contained 1,566 strains. We next analyzed Hsp12-GFP levels, as described above (Fig1BB), in these strains under three stress conditions and in mid-log and post-diauxic shift growth conditions (Fig3B, Materials and Methods, Table EV3). In general, the double-deletion response is consistent with the single-deletion measurements (Fig1). Comparing the average effect of a mutant across all its corresponding double mutants is in agreement with the effect of the single mutant (Fig EV5A).


Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response.

Gutin J, Sadeh A, Rahat A, Aharoni A, Friedman N - Mol. Syst. Biol. (2015)

Using double-knockout library to uncover pathway structureUsing the SGA methodology, we constructed a double-knockout library of 30 * 56 strains. Each one of the strains contains deletions of two genes and the HSP12-GFP reporter.The library was screened under five conditions (Fig1B). The raw data of HSP12-GFP expression (log) after the exposure to heat stress is shown. Overall, the average effect of the double perturbation resembles the measured effect of the single perturbation.Epistatic interactions in the data that match the well-established structure of the cAMP/PKA pathway. HSP12-GFP expression after heat stress is shown. Δras2 is epistatic over Δira2, implicating that Ira2 is upstream of Ras2. Δpde2 is epistatic over Δras2, implicating that Ras2 is upstream of Pde2.Consensus matrix of the epistasis interactions in our library. We identified all the epistatic interactions in three stress conditions (Materials and Methods). We color double perturbations in which the epistasis observed in at least two of the stress conditions. Row epistasis defined as the epistasis of the gene denoting the row over the gene denoting the column. Column epistasis is vice versa. Double perturbation pairs in which the direction of the epistasis interaction has changed between the conditions are colored in black. The upper row and the rightmost column present the single perturbations effects (red/green is increased/decreased HSP12-GFP levels relative to WT).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4631200&req=5

fig03: Using double-knockout library to uncover pathway structureUsing the SGA methodology, we constructed a double-knockout library of 30 * 56 strains. Each one of the strains contains deletions of two genes and the HSP12-GFP reporter.The library was screened under five conditions (Fig1B). The raw data of HSP12-GFP expression (log) after the exposure to heat stress is shown. Overall, the average effect of the double perturbation resembles the measured effect of the single perturbation.Epistatic interactions in the data that match the well-established structure of the cAMP/PKA pathway. HSP12-GFP expression after heat stress is shown. Δras2 is epistatic over Δira2, implicating that Ira2 is upstream of Ras2. Δpde2 is epistatic over Δras2, implicating that Ras2 is upstream of Pde2.Consensus matrix of the epistasis interactions in our library. We identified all the epistatic interactions in three stress conditions (Materials and Methods). We color double perturbations in which the epistasis observed in at least two of the stress conditions. Row epistasis defined as the epistasis of the gene denoting the row over the gene denoting the column. Column epistasis is vice versa. Double perturbation pairs in which the direction of the epistasis interaction has changed between the conditions are colored in black. The upper row and the rightmost column present the single perturbations effects (red/green is increased/decreased HSP12-GFP levels relative to WT).
Mentions: To systematically map genetic interactions, we generated a “mini epistatic map” (Schuldiner et al, 2005; Collins et al, 2007) of 30 query strains against 56 target strains (Fig3A, Materials and Methods). The resulting strains contained two mutant alleles and the HSP12-GFP reporter gene. In total, the library contained 1,566 strains. We next analyzed Hsp12-GFP levels, as described above (Fig1BB), in these strains under three stress conditions and in mid-log and post-diauxic shift growth conditions (Fig3B, Materials and Methods, Table EV3). In general, the double-deletion response is consistent with the single-deletion measurements (Fig1). Comparing the average effect of a mutant across all its corresponding double mutants is in agreement with the effect of the single mutant (Fig EV5A).

Bottom Line: The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program.We established a high-throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single-knockout and 1,566 double-knockout strains that carry an HSP12-GFP allele as a reporter for Msn2/4 activity.Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three-way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Computer Science & Engineering Institute of Life Sciences Hebrew University, Jerusalem, Israel.

No MeSH data available.


Related in: MedlinePlus