Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response
- Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Germany
- Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Spain
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, United States
- Centre for Genomic Regulation (CRG), Genome Biology Programme, Spain
- Institut für Biochemie und Molekularbiologie, Universität Bonn, Germany
Figures
Figure 1 with 2 supplements
Attenuation of the Hsf1-mediated heat shock response is defective in S. cerevisiae apj1Δ cells.
(A, B) S. cerevisiae wt and apj1Δ cells were grown at 30°C till logarithmic growth phase and shifted to 38°C. After 10 min, cycloheximide (CHX) was added to inhibit protein synthesis (B). Total cell extracts were prepared and levels of the heat shock protein Btn2 were determined by western blot analysis at the indicated time points after heat shock. Zwf1 levels were determined as loading control. (C) Changes in expression levels of selected Hsf1-target genes were determined by ribosome profiling in indicated yeast strains grown at 30°C and subjected to heat shock at 38°C for 0, 10, or 60 min. Levels of translated mRNAs were normalized to respective levels determined in wild-type cells prior to heat shock and shown in a log2-fold change-scale (log2FC) with the standard error (n=2). (D) Changes in expression levels of all 46 Hsf1 targets were determined and normalized to respective levels determined in wild-type cells prior to heat shock. Significance was determined by Wilcox test (**p<0.01; ***p<0.001).
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Figure 1—source data 1
Original western blots with bands shown in Figure 1 highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig1-data1-v1.zip
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Figure 1—source data 2
Original western blots of Figure 1.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig1-data2-v1.zip
Figure 1—figure supplement 1
Attenuation of the Hsf1-mediated heat shock response is defective in S. cerevisiae apj1Δ cells.
(A/B) S. cerevisiae wt and apj1Δ cells harboring an Hsf1-controlled unstable GFP reporter were grown at 30°C till logarithmic growth phase and shifted to 38°C. After 10 min, cycloheximide (CHX) was added to inhibit protein synthesis (B). Total cell extracts were prepared and levels of the heat shock protein Btn2 were determined by western blot analysis at the indicated time points after heat shock. Zwf1 levels were determined as loading control. (C/D) Levels of translated mRNAs of Hsf1-target genes were determined prior and post heat shock (30–38°C) by ribosome profiling in apj1Δ, sis1-4xcga, and apj1Δ sis1-4xcga cells and were normalized to respective mRNA levels determined in wild-type cells prior to heat shock. Normalized expression levels (log2-scale) of Hsf1 targets were compared between JDP mutants and wild-type (wt) cells after 10 and 60 min post heat shock (C). Alternatively, induction factors of mRNA expression (ratio mRNA levels at 10 min vs 0 min post heat shock) and attenuation factors (ratio mRNA levels at 10 min vs 60 min post heat shock) were compared for each strain separately (D).
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Figure 1—figure supplement 1—source data 1
Original western blots with bands shown in Figure 1—figure supplement 1 highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original western blots shown in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Expression levels of yeast J-domain proteins (JDPs).
(A) Mean of normalized gene counts of JDPs Apj1, Sis1, and Ydj1 in ribosome profiling libraries of wild-type cells prior and 10 min and 60 min post heat shock (30–38°C). Normalization was done with DeSeq2, standard deviations are shown (n=2). (B) Log2FC induction of JDPs Apj1, Sis1, and Ydj1 in wild-type cells after 10 min of heat shock (30–38°C) was determined by ribosome profiling and DeSeq2. Standard deviations are shown (n=2).
Figure 2
Persistent activation of Hsf1 in apj1Δ cells upon heat shock.
(A) S. cerevisiae wt and apj1Δ cells expressing Hsf1-GFP were grown at 30°C and heat shocked to 38°C. Cellular localizations of Hsf1-GFP were determined at indicated time points and the proportions of cells showing two or more nuclear Hsf1-GFP foci were determined (n>221 for wt, n>133 for apj1∆). (B) S. cerevisiae wt and apj1Δ cells expressing LacI-GFP and harboring HSP12 and HSP104 gene loci linked to 128 and 256 repeats of the lacO operator sequence, respectively, were grown at 30°C and heat shocked to 38°C. The percentage cells showing one or two LacI-GFP foci, reporting on coalescence of HSP104 and HSP12 gene loci, were determined at indicated time points (n>82 for wt, n>35 for apj1∆). Scale bars are 2 μm.
Figure 3 with 1 supplement
Chromatin occupancy of Hsf1 and Apj1 at Heat Shock Response genes is anti-correlated.
(A) ChIP experiments using S. cerevisiae cells grown at 30°C and expressing GFP or GFP-Apj1* (GFP-Apj1-34AAA37). Cells were shifted to 42°C for 30 min, crosslinked, and processed for ChIP. Raw read counts of inputs and IPs for the heat shock gene SSA4 (HSP70) are depicted. (B) ChIP enrichment (vs TOS1 as control) of GFP-Apj1* and GFP for the indicated Hsf1 targets and the control TOS1 are shown (n=3). Significance was determined by unpaired two-tailed t-test (*p<0.05; **p<0.01). (C) Top 18 binding peaks of GFP-Apj1* ChIP experiments were subjected to sequence analysis using MEME and searched for binding sites of transcription factors (using TOMTOM), revealing the Hsf1 target HSE (heat shock element). (D) Hsf1 and Apj1 occupancies at UAS regions of Hsf1-dependent heat shock gene loci. Occupancies were determined at the indicated time points after heat shock (30–39°C) by a ChIP assay. The percentage of input was calculated, and the mean values were plotted with SD (n=2). Statistical significance was determined relative to T=0 min sample by an unpaired two-tailed t-test. ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001. (E) Hsf1 and Apj1 occupancies of UAS regions of the native BUD3 gene and of BUD3 that was placed under Hsf1 control (BUD3-HSE). The latter was accomplished by fusion of the HSP82 promoter (containing three HSEs) with the minimal promoter region of BUD3 (Chowdhary et al., 2019). Occupancies were determined at the indicated time points after heat shock (30–39°C) as above. The percentage of input was calculated, and the mean values were plotted with SD (n=2).
Figure 3—figure supplement 1
Apj1 promotes the displacement of DNA-bound Hsf1 from HSE.
(A) Levels and phosphorylation status of Hsf1 are unaltered in apj1∆ cells. S. cerevisiae wt and apj1∆ cells expressing Hsf1-FLAG3-V5 under control of its native promoter were grown at 30°C till logarithmic growth phase and shifted to 38°C. Total cell extracts were prepared at the indicated time points after heat shock and levels of Hsf1 were determined by western blot analysis. Zwf1 levels were determined as loading control. Phosphorylated Hsf1 is upshifted and indicated by ‘*’. (B) Hsf1 and Apj1 occupancies at UAS regions of Hsf1-dependent heat shock gene loci. Occupancies were determined at the indicated time points after heat shock (30–39°C) by a ChIP assay. The percentage of input was calculated, and the mean values were plotted with SD based on two biological replicates. (C) Hsf1 and Apj1 occupancies at ARS504, a non-transcribed region, that is considered a negative control. The percentage of input was calculated, and the mean values were plotted with SD based on two biological replicates.
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Figure 3—figure supplement 1—source data 1
Original western blots with bands shown in Figure 3—figure supplement 1A highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original western blots of Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig3-figsupp1-data2-v1.zip
Figure 4 with 2 supplements
Hsf1 binding to UAS regions of heat shock genes is prolonged in apj1Δ cells.
Hsf1 occupancies were determined in S. cerevisiae wt and apj1Δ cells at the indicated time points after heat shock (30–38°C) by a ChIP assay. The percentage of input was calculated, and the mean values were plotted with SD (n=4). Statistical significance (relative to T=0 min sample) was determined by one-way ANOVA: ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001.
Figure 4—figure supplement 1
Hsf1 binding to UAS regions of heat shock genes is prolonged in apj1Δ cells.
(A) Hsf1 occupancies were determined in S. cerevisiae wt and apj1Δ cells at the indicated time points after heat shock (30–38°C) by a ChIP assay. The percentage of input was calculated, and the mean values were plotted with SD based on two biological replicates. (B) Hsf1 occupancies at ARS504, a non-transcribed region, which is considered negative control for Hsf1, were determined in S. cerevisiae wt and apj1Δ cells. The percentage of input was calculated, and the mean values were plotted with SD based on four biological replicates. Statistical significance (relative to T=0 min sample) was done by one-way ANOVA: ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001.
Figure 4—figure supplement 2
Apj1 occupancies at UAS of Hsf1-dependent heat shock genes are specific.
Ydj1 (A) and Sis1 (B) occupancies at indicated heat shock gene loci were determined in S. cerevisiae wt and apj1Δ cells at the indicated time points after heat shock (30–38°C) by a ChIP assay. The percentage of input was calculated, and the mean values were plotted with SD based on two biological replicates.
Figure 5 with 2 supplements
Loss of Apj1 and Ydj1 triggers almost full Hsf1 activation.
(A) Changes in expression levels of selected Hsf1-target genes were determined by ribosome profiling in indicated yeast strains grown at 25°C. Levels of translated mRNAs in mutant strains were normalized to respective levels determined in wild-type cells and are shown in a log2-scale with the standard error (n=2–3). (B) Indicated yeast strains were grown at 25°C and then heat shocked at 35°C for 30 min. Total cell extracts were prepared and levels of the Hsf1 targets Btn2, Hsp42, and Apj1 were determined by western blot analysis. Levels of histone H3 are provided as loading control. (C) S. cerevisiae wt and apj1Δ ydj1Δ cells expressing Hsf1-GFP were grown at 30°C and heat shocked to 38°C. Cellular localizations of Hsf1-GFP were determined at indicated time points and the proportions of cells showing nuclear Hsf1-GFP foci were determined (n>221 for wt, n>203 for apj1∆). (D) S. cerevisiae wt and apj1Δ ydj1Δ cells expressing LacI-GFP and harboring HSP12 and HSP104 gene loci linked to 128 and 256 repeats of the lacO operator sequence, respectively, were grown at 30°C and heat shocked to 38°C. The percentage of cells showing one or two LacI-GFP foci, reporting on coalescence of HSP104 and HSP12 gene loci, was determined at indicated time points (n>82 for wt, n>169 for apj1∆ydj1∆).
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Figure 5—source data 1
Original western blots with bands shown in Figure 5B highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig5-data1-v1.zip
Figure 5—figure supplement 1
Loss of Apj1 and Ydj1 triggers Hsf1 activation.
(A) Indicated yeast strains were grown at 25°C and total cell extracts were prepared. Levels of Sis1 and Ydj1 chaperones were determined by western blot analysis. Levels of histone H3 are provided as loading control. (B) Volcano plots showing the log2-fold change and the -log10 p-value of all expressed genes in the apj1Δ, sis-4xcga, ydj1-4xcga, apj1Δ sis1-4xcga, and apj1Δ ydj1-4xcga strains compared to wild-type. Genes that have an expression level change of |log2FC|>1 and a p-value<0.05 are depicted in black. Hsf1-target genes are indicated in red and labeled if they pass the thresholds described before (n=2–3) (C) mRNA expression levels of Hsf1-target genes, determined by ribosome profiling in the wt strain after 10 min of HS from 30 to 38°C and in sis1-4xcga and apj1Δ ydj1-4xcga cells grown at 25°C were normalized to the wt grown at 25°C (x- and y-axis, respectively). Data was analyzed with the DeSeq2 package (n=2–3).
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Figure 5—figure supplement 1—source data 1
Original western blots with bands shown in Figure 5—figure supplement 1A highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original western blots of Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Loss of Apj1 and Ydj1 triggers Hsf1 activation.
(A) Changes in expression levels of selected Hsf1-target genes were determined by RNA sequencing in indicated yeast strains grown at 25°C. Levels of mRNAs in mutant strains were normalized to respective levels determined in wild-type cells and are shown in a log2-scale with the standard error (n=2). (B) Volcano plots showing the log2-fold change and the -log10 p-value of all expressed genes based on transcriptome analysis in the apj1Δ, sis-4xcga, ydj1-4xcga, apj1Δ sis1-4xcga, and apj1Δ ydj1-4xcga strains compared to wild-type. Genes that have an expression level change of |log2FC|>1 and a p-value<0.05 are depicted in black. Hsf1-target genes are indicated in red and labeled if they pass the thresholds described before (n=2).
Figure 6 with 1 supplement
High Hsf1 activity rescues growth of apj1 ydj1 mutant cells.
(A/B) Serial dilutions of indicated yeast strains were spotted on YPD plates and incubated at indicated temperatures for 3 days. (C) S. cerevisiae wt, apj1Δ, ydj1Δ, and apj1Δ ydj1Δ cells overexpressing SIS1 (TDH3:Sis1) from plasmid or harboring an empty vector (EV) were grown at 25°C and levels of the Hsf1 targets Btn2 and Hsp42 were determined by western blot analysis. Levels of histone H3 were determined as loading control. (D) Serial dilutions of indicated yeast strains overexpressing Sis1 (TDH3::SIS1) from plasmid or harboring an empty vector (EV) were spotted on SC-Leu plates and incubated at indicated temperatures for 3 days.
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Figure 6—source data 1
Original western blots with bands shown in Figure 6C highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig6-data1-v1.zip
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Figure 6—source data 2
Original western blots of Figure 6C.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig6-data2-v1.zip
Figure 6—figure supplement 1
Loss of Apj1 and Ydj1 triggers Hsf1 activation and restores proteostasis.
(A/B) Serial dilutions of indicated yeast strains were spotted on YPD plates and incubated at indicated temperatures for 3 days. apj1H34Q harbors a mutated J-domain ‘HPD’ motif and cannot interact with Hsp70 (Ssa1-4). (C) Indicated yeast strains were grown at 25°C and total cell extracts were prepared. Levels of Btn2, Hsp104, Hsp42, and Apj1 chaperones were determined by western blot analysis. Levels of histone H3 are provided as loading control. (D) S. cerevisiae wt, apj1Δ, ydj1Δ, and apj1Δ ydj1Δ cells expressing Sis1-GFP were grown at 25°C and heat shocked to 35°C. Cellular localizations of Sis1-GFP were determined and quantified (n>38 for wt, n>90 for apj1∆, n>63 for apj1∆ydj1∆). Scale bar is 2 μm.
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Figure 6—figure supplement 1—source data 1
Original western blots with bands shown in Figure 6—figure supplement 1C highlighted.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original western blots of Figure 6—figure supplement 1C.
- https://cdn.elifesciences.org/articles/107157/elife-107157-fig6-figsupp1-data2-v1.zip
Figure 7
Regulation of Hsf1 activity via diverse J-domain proteins (JDPs).
Hsp70 is targeted to Hsf1 in non-stressed cells by diverse J-domain proteins, including Ydj1, Sis1, and Apj1, to repress heat shock gene expression. Stress conditions trigger protein misfolding and aggregation. Binding of JDPs/Hsp70 to misfolded and aggregated proteins liberates Hsf1 to bind to heat shock elements (HSE) located upstream of heat shock genes, triggering their expression. Apj1 re-targets Hsp70 to HSE-bound Hsf1 during the attenuation phase, triggering Hsf1 dissociation and reducing heat shock gene expression. The HSR ultimately returns to the repressed state.
Author response image 1
Serial dilutions of indicated yeast strains were spotted on YPD plates without and with 1 M sorbitol and incubated at indicated temperatures for 2 days.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/107157/elife-107157-mdarchecklist1-v1.pdf
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Supplementary file 1
Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp1-v1.docx
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Supplementary file 2
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp2-v1.docx
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Supplementary file 3
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp3-v1.docx
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Supplementary file 4
Sequences of RT-PCR primer used in this study.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp4-v1.docx
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Supplementary file 5
RiboSeq data showing time course of heat shock response.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp5-v1.xlsx
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Supplementary file 6
RiboSeq data of wt and JDP mutant strains at 25°C.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp6-v1.xlsx
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Supplementary file 7
RNAseq data of wt and mutant strains at 25°C.
- https://cdn.elifesciences.org/articles/107157/elife-107157-supp7-v1.xlsx
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Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response
eLife 14:RP107157.
https://doi.org/10.7554/eLife.107157.3
