Role of heat shock transcription factor in Saccharomyces cerevisiae oxidative stress response

Abstract

The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates the transcription of a set of genes that contain heat shock elements (HSEs) in their promoters and function in diverse cellular processes, including protein folding. Here, we show that Hsf1 activates the transcription of various target genes when cells are treated with oxidizing reagents, including the superoxide anion generators menadione and KO(2) and the thiol oxidants diamide and 1-chloro-2,4-dinitrobenzene (CDNB). Similar to heat shock, the oxidizing reagents are potent inducers of both efficient HSE binding and extensive phosphorylation of Hsf1. The inducible phosphorylation of Hsf1 is regulated by the intramolecular domain-domain interactions and affects HSE structure-specific transcription. Unlike the heat shock, diamide, or CDNB response, menadione or KO(2) activation of Hsf1 is inhibited by cyclic-AMP-dependent protein kinase (PKA) activity, which negatively regulates the activator functions of other transcriptional regulators implicated in the oxidative stress response. These results demonstrate that Hsf1 is a member of the oxidative stress-responsive activators and that PKA is a general negative regulator in the superoxide anion response.

FIG. 1.

Oxidative-stress-induced transcription of various genes. (A) Menadione response. Wild-type cells (strain HS170T) harboring HSE4Ptt-CYC1-lacZ were grown in ESD medium lacking uracil at 28°C and treated with the indicated concentrations of menadione for 30 min (left side) or with 0.3 mM menadione for the indicated times (right side). Total RNA was prepared from the cells and subjected to RT-PCR analysis with primers specific for lacZ, BTN2, CUP1, TRX2, CTT1, and the ACT1 control. The graphs show the mRNA levels normalized to that of ACT1 mRNA, which was 100%. (B) Diamide response. Cells were treated with the indicated concentrations of diamide for 30 min (left side) or with 3.0 mM diamide for the indicated times (right side). RT-PCR analysis was conducted as described above. (C) t-bH₂O₂ response. Cells were treated with the indicated concentrations of t-bH₂O₂ for 15 min. RT-PCR analysis was conducted as described above.

FIG. 2.

Effects of transcription factor mutations on menadione- or diamide-induced transcription. (A) mRNA levels of various genes in tetO-HSF1 cells. tetO-HSF1 cells were grown at 28°C in SD medium supplemented with 20 μg/ml histidine, 30 μg/ml leucine, and 20 μg/ml methionine. For the upper part of the panel, doxycycline (Dox) was added to a final concentration of 10 μg/ml and cells were grown for the indicated times. Extracts were prepared from the cells and subjected to immunoblot analysis with an anti-Hsf1 serum. The leftmost lane shows the amount of Hsf1 protein in HSF1 wild-type cells. For the lower part of the panel, cells cultured in the absence (− Dox) or presence (+ Dox) of 10 μg/ml doxycycline for 14 h were left untreated (C) or were treated with 0.3 mM menadione for 30 min (MD) or 3.0 mM diamide for 30 min (DA) or were grown at 39°C for 15 min (HS). Total RNA was prepared from the cells and subjected to RT-PCR analysis. (B) mRNA levels of various genes in skn7Δ yap1Δ and msn2Δ msn4Δ mutant cells. Wild-type (WT) and skn7Δ yap1Δ and msn2Δ msn4Δ mutant cells harboring HSE4Ptt-CYC1-lacZ were grown in ESD medium lacking uracil at 28°C in the absence (C) or presence of 0.3 mM menadione for 30 min (MD) or that of 3.0 mM diamide for 30 min (DA). Total RNA was prepared from the cells and subjected to RT-PCR analysis.

FIG. 3.

Effects of mutations in cAMP-PKA pathway genes on stress-induced transcription. (Top) wild-type (+) or bcy1Δ mutant (−) cells were grown in ESD medium at 28°C in the absence (C) or presence of 0.3 mM menadione (MD), 3.0 mM diamide (DA), 5.0 mM KO₂, or 0.25 mM CDNB for 30 min or were grown at 39°C for 15 min (HS). (Middle) tpk1 tpk2 tpk3 msn2 msn4 mutant cells harboring the empty vector (−) or YCp-TPK1 (+) were grown as described above, except that KO₂ was added to a final concentration of 3.0 mM. Total RNA was prepared from the cells and subjected to RT-PCR analysis. (Bottom) The mRNA levels in bcy1Δ mutant and PKA-deficient cells relative to those in cognate wild-type cells are expressed as the mean ± the standard deviation of at least four independent experiments. Asterisks indicate significant differences (P < 0.01) when mutant cells are compared with wild-type cells as determined by the Student t test. Note that the relative TRX2 mRNA levels in heat-shocked cells are not shown because the levels were not substantially induced under the conditions tested.

FIG. 4.

Stress-induced changes in Hsf1 activity. (A) Binding of Hsf1 to target genes under stress conditions. Wild-type cells were grown in ESD medium at 28°C in the absence (C) or presence of 0.3 mM menadione (MD), 3.0 mM diamide (DA), 5.0 mM KO₂, or 0.25 mM CDNB (CD) for 30 min or that of 0.6 mM t-bH₂O₂ for 15 min (HP) or were grown at 39°C for 15 min (HS). Chromatin immunoprecipitation analysis was carried out with an anti-Hsf1 serum. The Input and anti-Hsf1 panels show the PCR products amplified from the extracts before immunoprecipitation (1.0% of each sample used for immunoprecipitation) and from the immunoprecipitates, respectively. The lower part of the panel shows the immunoprecipitation (IP)/input ratios, which are expressed as the mean ± the standard deviation of three independent experiments. (B) Phosphorylation of Hsf1 under stress conditions. Wild-type cells were treated with various stressors as described above. Extracts were prepared from the cells and subjected to immunoblot analysis with an anti-Hsf1 serum.

FIG. 5.

Effects of hsf1 mutations on the menadione or diamide response. (A) Schematic diagram of structural motifs of Hsf1 and Hsf1 mutant constructs. The regions indicated are as follows: AR1 and AR2, activation domains; DBD, DNA-binding domain; HR-A/B, hydrophobic repeat regions A and B; CE2, conserved element 2; CTM, C-terminal modulator. Numbers represent amino acid positions. Hsf1-ba1 contains changes of arginine to glutamate at amino acids 826 and 830 (R826E and R830E), and Hsf1-CE2Δ-ba1 contains the ba1 mutation and lacks the CE2 domain. (B) Phosphorylation of Hsf1 derivatives. Wild-type and HSF1, hsf1-ba1, and hsf1-CE2Δ-ba1 mutant cells were grown in ESD medium at 28°C in the absence (C) or presence of 0.3 mM menadione for 30 min (MD) or 3.0 mM diamide for 30 min (DA) or were grown at 39°C for 15 min (HS). Extracts were prepared from the cells and subjected to immunoblot analysis with an anti-Hsf1 serum. (C) mRNA levels of Hsf1 target genes in hsf1 mutant cells. Cells were grown in ESD medium at 28°C in the absence (C) or presence of 0.3 mM menadione for 45 min (MD) or 3.0 mM diamide for 45 min (DA) or were grown at 39°C for 20 min (HS). Total RNA was prepared from the cells and subjected to RT-PCR analysis. (D) Growth of hsf1 mutant cells on medium containing menadione or diamide. Cells at an A₆₀₀ of 0.5 were serially diluted fivefold, and 2 μl was spotted onto YPD medium (control) or YPD medium containing 40 μM menadione or 2.0 mM diamide and incubated at 28°C for 2 days.