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. 2002 Feb 5;99(3):1200-5.
doi: 10.1073/pnas.032681299. Epub 2002 Jan 29.

Dynamic association of transcriptional activation domains and regulatory regions in Saccharomyces cerevisiae heat shock factor

Tianxin Chen  1 , Carl S Parker
Affiliations

Dynamic association of transcriptional activation domains and regulatory regions in Saccharomyces cerevisiae heat shock factor

Tianxin Chen et al. Proc Natl Acad Sci U S A. .

Abstract

In Saccharomyces cerevisiae, the heat shock transcription factor (HSF) is thought to be a homotypic trimer that is bound to the promoters of heat shock protein (HSP) genes at both normal and heat shock temperatures. Exposure to heat shock greatly and rapidly induces HSF transcriptional activity without further increasing DNA-binding affinity. It is believed that HSF is under negative regulation at normal growth temperatures, but the detailed mechanism by which HSF is activated is still not clear. We report the analysis of mutations in a conserved arginine (residue 274) at the C-terminal end of the DNA-binding domain (DBD). Two mutations significantly increase both basal activity of HSF at normal temperatures and induced activity on heat shock. We demonstrate by coimmunoprecipitation experiments that the mutations reduce the association between the DNA-binding domain/oligomerization domain and the transcription activation domains. Our studies suggest that the DNA-binding domain of HSF can interact with activation domains directly, and this interaction is important for the repression of HSF activity under normal growth conditions. Destabilizing this interaction by heat or by mutations results in HSF transcriptional activation. We propose that Arg-274 is critical for intramolecular repression of HSF activity in normally growing cells.

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Figures

Figure 1
Figure 1
Transcriptional activities of mutant HSFs. (A) Transcriptional activities of wild-type and mutant ScHSFs are measured by levels of HSP70 mRNA by using primer extension. Primer extension products are indicated (HSP70, 309–313 nucleotides; actin, 198–200 nucleotides). Lanes 1–3, wild-type HSF; lanes 4–6, dbd-mut HSF; lanes 7–9, R274K HSF; lanes 10–12, R274G HSF. NS, non-shock, cells grown at 25°C. HS, heat shock, cells subjected to heat shock at 40°C for 30 min. R, recovery, cells put back to 25°C for 1 h after heat shock to allow recovery. (B) Western blotting analysis of wild-type and mutant HSF proteins. Yeast cell extracts were subjected to SDS/PAGE and transferred to nitrocellulose filter. The protein blot was probed with anti-FLAG antibody (Sigma) fused to the C terminus of HSFs.
Figure 2
Figure 2
DNA-binding assay. Gel mobility shift assay of wild-type and mutant DBDs to labeled HSE is shown. A mutant HSE with nGAAnnTTCn repeats changed to nGAAnnGGCn was also used to ensure binding specificity (lanes 5, 10, 15, and 20). Increasing amounts of unlabeled HSE were added for competition assay. The numbers shown at the bottom of each lane are the ratios of unlabeled HSE to [32P]HSE. Major DBD–HSE complexes are shown by the lower arrow. Mutant DBDs also form higher-order complexes (upper arrow). Note that microgram quantities of HSFs are used in these experiments because the recombinant proteins bind to the HSE less well than the native HSF.
Figure 3
Figure 3
Oligomerization state of wild-type and mutant HSFs. One microgram of recombinant wild-type and mutant DBDs was cross-linked by DSS and analyzed by Western blotting analysis. Wild-type DBD exited as an equilibrium of monomer, dimer, and trimer (lane 1). Mutant DBDs also formed hexamer (lanes 2, 3, and 4).
Figure 4
Figure 4
Binding affinities between DNA-binding domain and activation domains correlate with HSF transcriptional activities. (A) Schematic diagram of domain organization of S. cerevisiae HSF. Amino acid endpoints for each region, as well as their proposed functions, are indicated. (AI, AII, and AIII represent three constitutive activation domains. DBD, DNA-binding domain; IRL, isoleusine repeat (oligomerization domain). This diagram is adapted from the work of Nieto-Sotelo et al. (10). (B) Coimmunoprecipitation of activation domains with wild-type and mutant DBDs. Recombinant wild-type and mutant DBDs were immobilized by anti-GST antibody-coupled protein G Sepharose. Recombinant activation domain I (AI), activation domain II (AII), and activation domain III (AIII) were labeled with [γ-32P]ATP and PKA catalytic subunit. 32P-labeled AI (lanes 1–5), AII (lanes 6–10), and AIII (lanes 11–15) were precipitated by immobilized DBDs. Equal amounts of anti-GST antibody-coupled protein G Sepharose without DBD were used as control to assess nonspecific interactions (lanes 1, 6, and 11). (C) Western blotting analysis of immobilized wild-type and mutant DBDs used in coimmunoprecipitation.
Figure 5
Figure 5
Kinetics of wild-type and dbd-mut HSF activation. (A) Primer extension analysis of the activation kinetics of wild-type and dbd-mut HSFs. Cells were harvested after 0, 5, 15, 30, or 180 min of heat shock, and HSP70 mRNA levels were measured by primer extension. Actin mRNA was measured at the same time as internal control. dbd-mut HSF (lanes 6–10) is activated slower than wild type (lanes 1–5). (B) Kinetics plot of wild-type and dbd-mut HSFs activation. The units are arbitrary. The ratio of HSP70 mRNA transcription to actin mRNA transcription at 5 min of heat shock in wild-type cells was set to 1, and all of the other quantitative data (obtained from PhosphorImager plate) were normalized accordingly. The x axis is drawn logarithmically. The transcriptional activities of dbd-mut HSF after 30 min of heat shock were not shown, because progressive cell death caused inaccurate measurement.
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