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. 1998 Mar 1;12(5):654-66.
doi: 10.1101/gad.12.5.654.

Molecular chaperones as HSF1-specific transcriptional repressors

Y Shi  1 , D D Mosser, R I Morimoto
Affiliations

Molecular chaperones as HSF1-specific transcriptional repressors

Y Shi et al. Genes Dev. .

Abstract

The rapid yet transient transcriptional activation of heat shock genes is mediated by the reversible conversion of HSF1 from an inert negatively regulated monomer to a transcriptionally active DNA-binding trimer. During attenuation of the heat shock response, transcription of heat shock genes returns to basal levels and HSF1 reverts to an inert monomer. These events coincide with elevated levels of Hsp70 and other heat shock proteins (molecular chaperones). Here, we show that the molecular chaperone Hsp70 and the cochaperone Hdj1 interact directly with the transactivation domain of HSF1 and repress heat shock gene transcription. Overexpression of either chaperone represses the transcriptional activity of a transfected GAL4-HSF1 activation domain fusion protein and endogenous HSF1. As neither the activation of HSF1 DNA binding nor inducible phosphorylation of HSF1 was affected, the primary autoregulatory role of Hsp70 is to negatively regulate HSF1 transcriptional activity. These results reveal that the repression of heat shock gene transcription, which occurs during attenuation, is due to the association of Hsp70 with the HSF1 transactivation domain, thus providing a plausible explanation for the role of molecular chaperones in at least one key step in the autoregulation of the heat shock response.

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Figures

Figure 1
Figure 1
Hsp70 binds to the HSF1 activation domain in vitro and in vivo. (A) Identification of proteins binding to the HSF1 activation domain. GST alone, HSF1 activation domain (amino acids 395–503) GST fusion protein (GST–AD), or deletion mutant of HSF1 activation domain (amino acids 451–503) GST fusion protein (GST–ΔAD) were incubated with 35S-labeled HeLa whole cell extracts. Bound proteins were analyzed by SDS-PAGE and fluorography. The sizes of the molecular weight markers (MW) are indicated. (B) Hsp70 is among the HSF1 activation domain interactive proteins. Proteins bound to GST, GST–AD, or GST–ΔAD were analyzed by Western blot analysis with Hsp70-specific antibody 3A3. HeLa whole cell extract (WCE) input was included as a positive control. (C) Hsp70 interacts with the HSF1 activation domain as determined by immunoprecipitation assay. COS-7 cells were transfected with the β-actin hsp70 construct (S.P. Murphy, unpubl.) alone (−) or together with the GAL4–HSF1 activation domain (GAL4–AD) construct (+). Cell lysates were immunoprecipitated with Hsp70-specific antibody 3A3 and immunoblotted with HSF1-specific polyclonal antibody. Endogenous HSF1 and transfected GAL4–AD are indicated (right). An aliquot of the cell lysates (WCE) was analyzed directly by Western blot as a control. (D) Increased Hsp70 is associated with HSF1 during attenuation or recovery from heat shock. HeLa cells were heat-shocked at 42°C for 0, 15, 60, and 240 min, or at 42°C for 120 min and then allowed to recover at 37°C for 240 min (R). Cell lysates were immunoprecipitated with HSF1-specific polyclonal antibody and immunoblotted with Hsp70-specific monoclonal antibody C92 or with HSF1-specific monoclonal antibody 4B4.
Figure 2
Figure 2
Hsp70 and Hdj1 interact directly with the HSF1 activation domain. (A) Reconstitution of the interaction of Hsp70 and the HSF1 activation domain in vitro. Purified recombinant Hsp70, Hsc70, and the Hsp70 AAAA mutant were incubated with purified GST or GST–HSF1 activation domain fusion protein (GST–AD) on glutathione beads. The proteins bound to GST (lanes 5–7) or GST–AD (lanes 9–11) were analyzed by SDS-PAGE and Coomassie blue staining. Hsp70, Hsc70, and the Hsp70 AAAA mutant are included in lanes 1, 2, and 3, respectively; GST is in lane 4; GST–AD is in lane 8; molecular mass markers (MW) are in lane 12. (B) Reconstitution of the interaction of Hdj1 and the HSF1 activation domain in vitro. Purified recombinant Hdj1 and Hsp90 were incubated with GST or GST–AD on glutathione beads. The proteins bound to GST are in lanes 4 and 5; proteins bound to GST–AD in lanes 7 and 8; Hdj1 in lane 1; Hsp90 in lane 2; GST in lane 3; GST–AD in lane 6; and MW in lane 9. (C) The effect of nucleotides on the interaction of Hsp70 or Hdj1 with the HSF1 activation domain. Hsp70 or Hdj1 was incubated with GST–HSF1 activation domain in the absence of nucleotide (lanes 3,7), in the presence of 1 mm ATP (lanes 4,8), or 1 mm ATP-γs (lanes 5,9). Hsp70 input was included in lane 1; GST–AD in lane 2; Hdj1 in lane 6; MW in lane 10 (sizes at right).
Figure 3
Figure 3
The carboxyl-terminal substrate binding domain of Hsp70 interacts with the activation domain of HSF1. (A) A schematic diagram of full-length human Hsp70 (amino acids 1–640), deletion mutant Hsp70 N (amino acids 1–436 and 618–640), and Hsp70 C (amino acids 386–640) is shown. The ATP-binding domain (amino acids 1–386) is indicated as a hatched box; the substrate binding domain (amino acids 386–640) as a solid box. The epitopes for anti-Hsp70 monoclonal antibodies 3A3 or 5A5 are indicated. (B) The HSF1 activation domain interacts with the substrate binding domain of Hsp70. Purified full-length recombinant Hsp70, Hsp70 N (with a degradation product), and Hsp70 C (with some full-length Hsp70) were incubated with purified GST (lanes 5–7) or GST–HSF1 activation domain (GST–AD) (lanes 9–11), the bound proteins were washed extensively and analyzed by SDS-PAGE and immunoblotted with either 3A3 (top) or 5A5 (bottom) antibody (Ab). Inputs of full-length Hsp70, Hsp70 N, and Hsp70 C are shown in lanes 1, 2, and 3 individually; GST in lane 4; GST–AD in lane 8.
Figure 4
Figure 4
Mapping the Hsp70 binding site within the HSF1 activation domain. (A) Schematic diagram of wild-type and deletion mutants of HSF1 activation domain. Constructs I (wild type), and II–V (deletion mutants) are indicated as the regions of HSF1 activation domain fused to GST. The boundaries of each construct and the levels of Hsp70-binding activity are indicated. (B) A potential Hsp70 binding site is located from amino acid residue 425 to 439 of the HSF1 activation domain. HSF1–GST fusion proteins were incubated with HeLa whole cell extracts (WCE). The presence of Hsp70 as bound protein was detected by Western blot analysis with Hsp70-specific antibody 3A3. Purified recombinant Hsp70 and an aliquot of HeLa whole cell extracts were included as positive controls. (C) SDS-PAGE of each purified GST fusion protein visualized by Coomassie blue staining. Molecular weight markers (MW) are included.
Figure 5
Figure 5
Analysis of HSF1 transcriptional activity in cells transiently overexpressing Hsp70. (A) Coexpression of the HSF1 activation domain and Hsp70. (i) The transcriptional activity of GAL4–HSF1 activation domain in the presence of β-actin hsp70 plasmid DNA (0, 1, 5, and 10 μg) was measured as relative CAT activity from the reporter plasmid G5BCAT. The CAT activity was quantified with a PhosphorImager, standardized by cotransfected internal control β-gal activity, and plotted against the amount of transfected β-actin hsp70 plasmid DNA. (ii) The DNA-binding activity of GAL4–HSF1. Only the top part of the gel corresponding to the GAL4–HSF1 DNA binding complex is shown. The gel shift assay was done in probe excess. (iii) The levels of overexpressed Hsp70 were detected by Western blot analysis with Hsp70-specific monoclonal antibody 4G4. (B) Coexpression of the HSF1 activation domain and the Hsp70 AAAA mutant. (i) The transcriptional activity of GAL4–HSF1 activation domain in the presence of β-actin hsp70 AAAA plasmid DNA (0, 1, 5, and 10 μg). (ii) The levels of overexpressed Hsp70 AAAA mutant. A similar ECL exposure was obtained to that in A (iii). (C) Coexpression of the VP16 activation domain and Hsp70. (i) The transcriptional activity of GAL4–VP16 activation domain in the presence of β-actin hsp70 plasmid DNA (0, 1, 5, and 10 μg). (ii) The levels of overexpressed Hsp70. A similar ECL exposure was obtained to that in A (iii).
Figure 6
Figure 6
Analysis of HSF1 transcriptional activity in cells transiently overexpressing Hdj1. The GAL4–HSF1 activation domain (A) or the GAL4–VP16 activation domain (B) was transfected into COS-7 cells with increasing amount of β-actin hdj1 plasmid DNA (0, 1, 5, and 10 μg). (i) The transcriptional activity of GAL4–activation domain fusion was measured as relative CAT activity and plotted against the amount of transfected β-actin hdj1 plasmid DNA as in Fig. 5. (ii) The levels of overexpressed Hdj1 were examined by Western blot analysis with Hdj1-specific antibody. A similar exposure for ECL was obtained for the Western blots shown in A and B.
Figure 7
Figure 7
Analysis of HSF1 transcriptional activity in tetracycline-regulated Hsp70-overexpressing cells. (A) Transcriptional activity of the GAL4–HSF1. PETA70 cells transfected with the GAL4–HSF1 activation domain construct were maintained in media with or without anhydrotetracycline (tet or −tet) for 48 hr. GAL4–HSF1 transcriptional activity was measured as relative CAT activity, standardized by β-gal activity, and plotted accordingly. (B) DNA-binding activity of GAL4–HSF1. The gel shift assay was performed in probe excess. Both the GAL4–HSF1 DNA-binding complex (top, indicated by arrow) and excess free probe (bottom, indicated by asterisk) are shown. (C) Hsp70 level induced by withdrawal of anhydrotetracycline (−tet). Western blot analysis was performed with Hsp70-specific antibody 4G4.
Figure 8
Figure 8
Analysis of heat shock gene transcription, HSF1 DNA-binding, and inducible phosphorylation in Hsp70-overexpressing cells. PETA70 cells were maintained in media with or without anhydrotetracycline (tet or −tet) for 48 hr before 42°C heat shock treatment for 0, 15, and 60 min. (A) The transcription rate of hsp70 and hsp90α genes in control (tet) or Hsp70-overexpressed (−tet) cells was measured by run-on transcription analysis. (B) The DNA-binding activity of endogenous HSF1. (C) Antibody supershift of HSF1 DNA-binding activity in the absence (−) or presence (+) of 5 mm exogenous ATP. Only the top part of the gel corresponding to the HSF1 DNA-binding complex is shown. (D) The inducible phosphorylation state of HSF1 was examined by Western blot analysis with HSF1-specific polyclonal antibody. (C) Non-heat-shocked control; (HS) 60-min heat shock. (E) Hsp70 level upon anhydrotetracycline withdrawal (−tet) was measured by Western blot analysis with Hsp70-specific antibody 4G4.
Figure 9
Figure 9
Analysis of the transcription rate of hsp70 and hsp90α genes in Hsp70 AAAA mutant-overexpressing cells. (A) Schematic diagram of wild-type Hsp70 and the AAAA mutant. The binding activity to HSF1 activation domain (Fig. 2A) and the mutated residues are indicated. (B) The transcription rate of the hsp90α gene was measured by run-on transcription analysis. PERTA70–AAAA cells were maintained in media with or without doxycycline (+AAAA or −AAAA) for 24 hr before 42°C heat shock treatment for 0, 15, and 60 min. (C) DNA-binding activity as measured by gel mobility shift assay. (D) Antibody supershift of HSF1 DNA-binding activity. Only the top part of the gel corresponding to the HSF1 DNA-binding complex is shown. (E) The level of Hsp70 AAAA mutant induced by doxycycline for 24 hr was measured by Western blot analysis with Hsp70-specific antibody 4G4 and compared to the level of wild-type Hsp70 induced by withdrawal of anhydrotetracycline from the media for 48 hr.
Figure 10
Figure 10
Analysis of heat shock gene transcription in cells expressing different levels of Hsp70. Hsp70 was induced to various levels by withdrawal of anhydrotetracycline from media for 0, 8, 12, 24, and 48 hr. (A) The transcription rate of the hsp90α gene upon 1-hr heat shock treatment at 42°C was examined by run-on transcription analysis, quantified by a PhosphorImager, normalized to the signal obtained for pBR322 and gapdh genes, and plotted against time of anhydrotetracycline withdrawal (hr) as a solid line. HSF1 DNA-binding activity following the same treatment was measured by gel shift assay, quantified with a PhosphorImager, and plotted as a broken line. The levels of induced Hsp70 at different time points were measured by Western blot analysis, quantified with densitometry, and plotted as bars. (B) Hsp70 levels induced by anhydrotetracycline withdrawal were compared with that accumulated during 4-hr heat shock treatment at 41°C (intermediate heat shock temperature for PETA70 cells ranges from 39°C to 42°C, with the highest Hsp70 accumulation at 41°C, data not shown). Western blot analysis was performed with Hsp70-specific antibody 4G4. The relative Hsp70 levels were quantified by densitometry.
Figure 11
Figure 11
Model for regulation of the heat shock transcriptional response by Hsp70 and Hdj1. Upon heat shock, HSF1 is rapidly converted to a transcriptionally active trimer, which binds to HSEs upstream of heat shock genes. This leads to the elevated synthesis of heat shock proteins, such as Hsp70 and Hdj1. Hsp70 and Hdj1 bind to the HSF1 activation domain, repressing its transcriptional activity, and leading to attenuation of the heat shock response.
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