Changes in mRNA levels under various heat shock conditions. (A) Schematic representation of the thermal treatments. (B) RT-PCR analysis of various mRNAs. Wild-type cells (strain HS170T) bearing the HSE4Ptt-CYC1-lacZ reporter gene were grown in ESD medium lacking uracil and were heat-shocked as shown in panel A. Control cells (C) were grown at 28°C, and heat-shocked cells (HS) were incubated at 37 or 39°C for 15 min. Total RNA prepared from the cells was subjected to RT-PCR analysis. Transcription factors that activated the mRNA synthesis from each gene are shown to the left of each panel. The ADH1 gene encoding alcohol dehydrogenase was used as a control.

Effects of transcription factor mutations on induced thermotolerance. (A) Induced thermotolerance of wild-type and mutant cells. Wild-type, hsf1-R206S-H220R, and msn2Δ msn4Δ cells (open circles) were grown in ESD medium at 28°C. For analysis of cells harboring the ADH1-HSP104 expression plasmid (filled circles), ESD medium lacking uracil was used. Cell cultures were shifted to 37°C for 60 min and then to 48°C. At the indicated times, aliquots of cells were diluted into ESD medium and plated onto YPD medium to determine cell survival rates. Values represent the means and standard deviations of the results of at least three experiments. (B) RT-PCR analysis of various mRNAs. Wild-type HSF1 cells (lanes 1 to 7) and hsf1-R206S-H220R cells (lanes 8 to 14) expressing HSP104 were grown in ESD medium lacking uracil under condition 3. Total RNA prepared from the cells was subjected to RT-PCR analysis. C, control cells; HS, heat-shocked cells.

Effects of transcription factor mutations on basal thermotolerance. (A) Basal thermotolerance of wild-type and mutant cells. Cell survival was analyzed as described for Fig. 2A except that cells were directly exposed to 48°C without preconditioning. Values represent the means and standard deviations of the results of at least three experiments. (B) Schematic representation of the thermal treatments. (C) RT-PCR analysis of various mRNAs. Wild-type HSF1 cells (lanes 1 to 7) and hsf1-R206S-H220R cells (lanes 8 to 14) expressing HSP104 were grown in ESD medium lacking uracil and were heat-shocked as shown in panel B. Total RNA prepared from the cells was subjected to RT-PCR analysis. C, control cells. (D) Growth of wild-type and mutant cells after severe heat shock. Wild-type (open circles), hsf1-R206S-H220R (filled circles), and msn2Δ msn4Δ (open squares) cells expressing HSP104 were grown in ESD medium lacking uracil at 28°C. Cell cultures were shifted to 48°C for 6 min and then returned to 28°C. At the indicated times, aliquots of cells were diluted into ESD medium and plated onto YPD medium to determine cell growth. Cell numbers were plotted relative to those of the 28°C incubation at 0 min. Values represent the means and standard deviations of the results of at least three experiments.

Regulation of activator function of Hsf1. (A) Immunoblot analysis of Hsf1. Wild-type cells were grown in YPD medium under conditions 1 (lanes 1 to 5), 2 (lanes 6 to 10), and 3 (lanes 11 to 17). Wild-type cells expressing HSP104 were grown in ESD medium lacking uracil under condition 5 (lanes 18 to 21). Extracts were prepared from the cells and subjected to immunoblot analysis with an anti-Hsf1 serum. The positions of hyper- and hypophosphorylated Hsf1 are shown to the right. C, control cells; HS, heat-shocked cells. (B) Chromatin immunoprecipitation analysis of Hsf1 target promoters. Wild-type cells were grown in YPD medium under conditions 3 (lanes 1 to 6 and 18 to 23) and 4 (lanes 7 to 12). Wild-type cells expressing HSP104 were grown in ESD medium lacking uracil under condition 5 (lanes 13 to 17 and 24 to 28). Chromatin immunoprecipitation analysis was carried out with an anti-Hsf1 serum (lanes 2 to 6, 8 to 12, and 14 to 17) or an anti-Rpb3 antibody (lanes 19 to 23 and 25 to 28). The input (IN) lanes (lanes 1, 7, 13, 18, and 24) show the PCR products amplified from the extracts before immunoprecipitation (1.0% of each sample used for immunoprecipitation). For analysis of the anti-Rpb3 immunoprecipitates, the telomere on the right arm of chromosome VI (TEL) was used as a control. (C) RT-PCR analysis of various genes in cells treated with AZC or MG132. Wild-type cells grown in YPD medium (lanes 1 and 3) were heat shocked at 39°C for 20 min (lane 2) or were treated with 20 mM AZC for 1 h (lane 4). erg6 null mutant cells (strain W303erg6Δ) were grown in ESD medium in the presence of either 50 μM MG132 (lane 6) or 0.1% dimethyl sulfoxide (lane 5) for 30 min. Total RNA prepared from the cells was subjected to RT-PCR analysis.

Schematic representation of regulation of thermotolerance by Hsf1 and Msn2/4. Cells acquire thermotolerance upon mild heat shock, during which Hsf1 and Msn2/4 induce expression of proteins involved in protection of cellular components against heat inactivation. In severely heat-shocked cells, promoter-bound, hyperphosphorylated Hsf1 is unable to activate transcription because RNA polymerase II (RNAPII) is inactive. It is unknown whether the transcriptional activity of Msn2/4 is activated by severe heat shock. In recovering cells, misfolded proteins appear to activate Hsf1, which induces expression of proteins involved in reactivation and/or degradation of heat-inactivated components. The transcriptional activity of Msn2/4 is not subject to delayed upregulation. More details are described in the text.