Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes
- Department of Biochemistry and Molecular Biology Louisiana State University Health Sciences Center, United States
Figures
Figure 1
Thermal and chemical stresses used in this study elicit distinct proteotoxic responses.
(A) Growth curve of strain W303-1B grown in liquid culture (YPDA). Mid-log phase cultures were diluted to OD600=0.4 and shifted to different conditions: no stress (NS, 25°C), heat shock (HS, 39°C), or ethanol stress (ES, 8.5% v/v, 25°C). OD600 was monitored over time. Means and SD are shown. N=2. (B) Viability assay of W303-1B cells following exposure to heat shock (25° to 39°C upshift for the indicated time) or ethanol stress (8.5% v/v ethanol for the indicated time at 25°C). An aliquot was taken from each condition at the indicated stress timepoints and diluted in rich media. Cells were spread on YPDA plates and grown at 30°C for 3 days. Colony forming units (CFUs) were determined using ImageJ/FIJI. Plotted are percentages of CFUs of stressed cells normalized to those of the 0 min control. Graphs depict means + SD. N=2. (C) Experimental strategy for imaging Hsp104 foci. Cells were attached to a concanavalin A (ConA)-coated surface, followed by heat shock or ethanol stress treatment (see Materials and Methods). Synthetic complete media (SDC) was supplemented with ethanol to a final concentration of 8.5% for ES samples. Scale bar: 2 µm. (D) Both heat shock and ethanol stress induce formation of Hsp104 foci. DPY1561 haploid cells were attached to a VAHEAT substrate using Concanavalin A and subjected to an instantaneous heat shock (25° to 39°C) or to ethanol stress (25°C, 8.5% v/v). The 0 min control was kept at 25°C without stress. Hsp104-mTagBFP2 foci were visualized by confocal microscopy. Shown are maximal projections of 11 z-planes, taken with an interplanar distance of 0.5 µm. Scale bar: 2 µm. (E) Cells subjected to the above treatments were assayed for Hsp104 puncta number and volume. Violin plots summarizing this analysis are depicted. An average of 200 cells per timepoint per condition was quantified using Imaris image analysis software (v.10.0.1). For this analysis, we made the assumption that the diffuse Hsp104 clusters seen in HS cells are comparable to the compact Hsp104 foci in ES cells. N=2. Significance was determined by Mann Whitney test, stress vs. no stress (0 min). ***, p<0.001; ****, p<0.0001; ns, not significant.
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Figure 1—source data 1
Spreadsheet tabulates the number of Hsp104 foci per cell and the volume of individual Hsp104 foci (Figure 1E).
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Figure 2
Heat shock and ethanol stress elicit distinct patterns of Sis1 subcellular relocalization.
(A) Live cell confocal microscopy of the diploid strain LRY033 expressing Sis1-mKate, Hsf1-mNeonGreen, and Hsp104-mTagBFP2. Cells were treated as in Figure 1D. 11 z-planes were captured with an interplanar distance of 0.5 µm. Shown is a representative plane for each timepoint. (B) Subcellular localization analysis of Sis1, Hsf1, and Hsp104 in cells subjected to no stress (25°C), heat shock (at 39° or 42°C), or ethanol stress (at 5% or 8.5% v/v [25°C]) for 10 min. Cells from strain LRY033 were treated as described in Figure 1D. A representative plane is shown for each condition. Line profiles are plotted for each channel on the right. Arrows were drawn to bisect the nucleus. Scale bar: 2 µm.
Figure 3 with 3 supplements
Ethanol stress transcriptionally induces Heat Shock Response (HSR) genes but with markedly slower kinetics than thermal stress.
(A) RNA abundance of Hsf1-dependent HSR genes was determined by Reverse Transcription-qPCR in strain W303-1B. Heat shock was performed at 39°C; ethanol stress was done using 8.5% (v/v) ethanol at 25°C. Insets display transcript abundance using a zoomed-in scale. Depicted are means + SD. N=2, qPCR = 4. Statistical analysis: T-test, one-tailed, no stress vs. stress conditions. *, p<0.05; **, p<0.01, ***; p<0.001. (B) As in (A), but the Hsf1-, Msn2-dual regulated genes HSP12 and HSP26 were evaluated. *, p<0.05; ***, p<0.001; ****, p<0.0001.
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Figure 3—source data 1
Spreadsheet contains the RT-qPCR data plotted for HSR RNA analysis (Figure 3).
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Figure 3—figure supplement 1
Ethanol stress transcriptionally induces HSR genes.
(A) RNA abundance of Hsf1-dependent genes was determined as in Figure 3. Cells were exposed to 8.5% (v/v) ethanol at 25°C for the indicated times. Depicted are means + SD. N=2, qPCR=4. (B) As in (A), but the Hsf1-, Msn2-dual regulated genes HSP12 and HSP26 were evaluated. (C) Quantification of SCR1 RNA levels used for normalization of transcripts, analyzed by RT-qPCR. N=2, qPCR=4. A one-way ANOVA test (GraphPad Prism 8.0) was performed. ns (not significant), p>0.05.
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Figure 3—figure supplement 1—source data 1
Spreadsheet contains mRNA levels for ethanol stressed cells (Figure 3—figure supplement 1A, B), as well as SCR1 RNA levels for heat shock and ethanol stress (Figure 3—figure supplement 1C).
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Figure 3—figure supplement 2
In response to chronic heat stress HSR mRNA levels gradually attenuate, whereas in response to chronic ethanol stress they remain constant.
Abundance of HSR mRNAs was determined by RT-qPCR. The experiment was conducted as described in Figure 3 except longer time points were evaluated. Depicted are means + SD. N=2, qPCR=4.
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Figure 3—figure supplement 2—source data 1
Spreadsheet contains mRNA levels for HSR genes under chronic heat shock and ethanol stress (4 hr; Figure 3—figure supplement 2).
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Figure 3—figure supplement 3
Induced HSR protein production is evident early during heat shock while it is delayed during ethanol stress.
(A) Analysis of chaperone levels by Immunoblot. Strain W303-1B was exposed to constant heat shock or ethanol stress conditions for different timepoints. Levels of histone H3, Hsp104, and Btn2 were monitored using antibodies against endogenous proteins. Histone H3 serves as the loading control. (B) Quantification of Hsp104 and Btn2 protein levels. Samples were treated as described in (A). Protein levels were normalized to histone H3 and 0 min timepoints. Shown are means + SD. N=2.
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Figure 3—figure supplement 3—source data 1
Raw files for Hsp104, Btn2, and histone H3 blots (Figure 3—figure supplement 3A).
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Figure 3—figure supplement 3—source data 2
Labeled blots for Hsp104, Btn2, and histone H3 protein quantification (Figure 3—figure supplement 3A).
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Figure 3—figure supplement 3—source data 3
Spreadsheet tabulates Hsp104 and Btn2 protein levels normalized to histone H3 (Figure 3—figure supplement 3B).
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Figure 4 with 2 supplements
Hsf1 and Pol II recruitment to HSR genes is delayed in ethanol stressed cells, while histone occupancy transiently increases.
(A) Map of a representative HSR gene depicting locations of primers used for chromatin immunoprecipitation (ChIP) analysis. Heat shock: shades of red and pink. Ethanol stress: shades of blue. (B) ChIP analysis of Hsf1, Pol II (Rpb1) and histone H3 occupancy to the enhancer (UAS), promoter and coding regions of the indicated genes. Mid-log cultures of strain BY4741 were subjected to the indicated times of heat shock (39°C) or ethanol stress (8.5% v/v, 25°C). Time points evaluated for all three factors: 0-, 2.5-, 10-, 20-, and 60 min. Antibodies raised against full-length Hsf1, CTD of Rbp1 or the globular domain of histone H3 were used (see Materials and methods). ChIP signals were normalized to input. Shown are means + SD. N=2, qPCR = 4.
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Figure 4—source data 1
Spreadsheet tabulates Hsf1, Pol II, and histone H3 occupancy.
Chromatin immunoprecipitation (ChIP) data for HSR genes in cells exposed to heat shock or ethanol stress (Figure 4B).
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Figure 4—figure supplement 1
Pol II occupancy of the indicated HSR genes vs. their mRNA levels in cells subjected to either heat shock or ethanol stress.
(A) Pol II ChIP and RT-qPCR data of the indicated HSR genes in cells exposed to HS are co-plotted to allow comparison of the occupancy of Pol II (over ORF) versus the corresponding transcript levels. Data derived from Figures 3A and 4B. (B) As above, except cells were exposed to 8.5% (v/v) ethanol.
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Figure 4—figure supplement 1—source data 1
Spreadsheet tabulates Pol II occupancy vs. HSR mRNA levels in cells exposed to heat shock or ethanol stress (Figure 4—figure supplement 1).
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Figure 4—figure supplement 2
Ethanol and thermal stresses induce chromatin compaction.
(A) ChIP analysis of histone H3 occupancy at inducible (PGM2), constitutive (ACT1, TUB1), and inactive/silenced loci (ARS504, HMLα1, YFR057W). Experiment was carried out and data quantified as in Figure 4B. Shown are means + SD. N=2, qPCR=4. (B) Live cell confocal fluorescence microscopy of the diploid strain DBY1447. Histone H2A-mCherry was used to measure chromatin volume in cells cultivated in SDC medium at 25°C and then subjected to thermal (39°C) or ethanol stress (8.5% v/v) for the times indicated. VAHEAT device was used for heat shock (see Materials and methods). Imaging was done across 11 z-planes with 0.5 µm of interplanar distance. Representative single plane images are shown. Scale bar: 2 µm. (C) Violin plots depicting H2A-mCherry volume measurements of DBY1447 cells treated as in (B). An average of 100 cells per timepoint, per condition, were evaluated. H2A-mCherry volume was determined using 3D Objects Counter in ImageJ/Fiji (v. 1.54 f). Statistical significance between no stress (0 min) and stress (2.5–180 min) samples was determined using Mann Whitney U Test. N=2. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.
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Figure 4—figure supplement 2—source data 1
Histone H3 ChIP analysis of different genomic loci, under HS or ES (Figure 4—figure supplement 2A).
Histone H2A-mCherry volume measurements from cells exposed to HS or ES (Figure 4—figure supplement 2C).
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Figure 5 with 2 supplements
Ethanol stress induces intergenic interactions between HSR genes that are comparable to those induced by acute thermal stress.
(A) Intrachromosomal (cis) interactions between HSR genes were analyzed by Taq I-3C. W303-1B cells were instantaneously shifted from 30° to 39°C for 2.5 min (HS) or exposed to 8.5% v/v ethanol at 30°C for 10 or 20 min (ES). No stress samples were kept at 30°C. Location of Taq I coordinates are provided in Figure 5—figure supplement 1. F (forward) primers are positioned near the indicated Taq I restriction site. 3C signals were normalized to the 3C signal derived from using a naked genomic DNA template. Graphs depict means + SD; N=2; qPCR=4. (B) Interchromosomal (trans) interactions between HSR genes were detected as in (A).
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Figure 5—source data 1
Spreadsheet tabulates 3C data of cells under no stress, 2.5 min heat shock, and 10- or 20 min ethanol stress.Intrachromosomal and interchromosomal interactions are analyzed (Figure 5A and B).
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Figure 5—figure supplement 1
Physical maps of genes used in this study.
Taq I restriction sites are numbered relative to the ATG (+1) codon. Coding regions are represented by grey boxes. Locations of Hsf1 binding sites (Heat Shock Elements [HSEs]) are shown as triple black vertical lines. Stress Response Elements (STREs) (Msn2 binding sites) are shown as green vertical lines (shown only for select genes). The primers used to analyze 3C interactions are represented as arrows. TSS, Transcription Start Site. TTS, Transcription Termination Site.
Figure 5—figure supplement 2
Intragenic interaction frequency induced by ethanol stress is comparable to that induced by thermal stress, despite modest transcriptional output.
Comparison of HSP104 intragenic interactions (left) with HSP104 mRNA levels (right). Samples from strain W303-1B were exposed to thermal (HS, 39°C) or ethanol stress (ES, 8.5%) for the indicated times. Intragenic interactions were analyzed by Taq I-3C; mRNA data are from Figure 3A. Locations of Taq I restriction sites are in Figure 5—figure supplement 1. Depicted are means + SD. N=2, qPCR=4. Taq I-3C analysis of intragenic interactions of the indicated Hsf1 target genes. Analysis and symbols as in (A).
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Figure 5—figure supplement 2—source data 1
Spreadsheet tabulates intragenic interactions in HSP104 under HS and ES conditions, compared to HSP104 mRNA levels under the same conditions (Figure 5—figure supplement 2A).
Also tabulated are intragenic interactions at SSA4, HSP82, MDJ1, and HSP12 under ES or HS (Figure 5—figure supplement 2B).
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Figure 6
Ethanol-induced HSR gene interactions are detectable by 2.5 min but typically dissipate within 60 min.
(A) Taq I-3C analysis of intergenic interactions occurring during ethanol stress was conducted as described in Figure 5. All samples were kept at 25°C. Plotted are means + SD. N=2, qPCR=4. (B) As in (A), but for intragenic interactions.
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Figure 6—source data 1
Spreadsheet tabulates 3C data of cells subjected to chronic ethanol stress (Figure 6).
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Figure 7 with 1 supplement
HSR gene transcription and coalescence are strongly correlated in heat-shocked but not ethanol-stressed cells.
(A) HSP12 and HSP104 are flanked by LacO arrays in the heterozygous diploid strain VPY705. In addition, HSP104 has a 24xMS2 loop array integrated within its 5’-UTR. MCP-mCherry binds to the nascent chimeric HSP104 transcript and is visualized as a red dot adjacent to the gene which appears as a green dot. (B) Live cell confocal fluorescence microscopy of strain VPY705 heat-shocked at 39°C using a VAHEAT device or exposed to 8.5% ethanol at 25°C for the indicated times. An Olympus spinning disk confocal microscope system was used for imaging. Scale bar: 2 µm. (C) Quantification of VPY705 cells treated as above and scored for the coalescence of HSP104-HSP12 and the presence of chimeric MS2x24-HSP104 mRNA. Cells were scored positive for coalescence only when a single green dot could be visualized in the nucleus across the 11 z-planes. Transcription was scored as positive only when a red dot above background could be seen near the large green dot (HSP104). Approximately 40 cells were scored per timepoint, per condition. Graphs represent means + SD. N=2. (D) Single cell analysis of HSP12-HSP104 coalescence (green) and HSP104 transcription (blue) at discrete timepoints over a heat shock time course. Each row in the transcription analysis corresponds to the same cell in the coalescence analysis. Blue gradient represents the intensity of mCherry signal (HSP104 transcript) in each cell, as quantified by ImageJ/Fiji (v. 1.54f). (E) As in (D) but for ethanol stress. (F) Pearson correlation coefficient analysis showing the correlation (r) between percent of cells positive for transcription and percent of cells positive for coalescence under HS and ES conditions (derived from Figure 7—figure supplement 1D). Each plotted value corresponds to a different stress timepoint.
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Figure 7—source data 1
Spreadsheet tabulates the percentage of cells with HSP104-HSP12 coalescence and HSP104 mRNA foci upon ES or HS treatment (Figure 7C and F).
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Figure 7—figure supplement 1
HSR gene coalescence and transcription are temporally uncoupled in ethanol stressed cells.
Relative location of HSP104 and TMA10 on Chr. XII in the diploid strain ASK727. One allele of HSP104 is flanked by an integrated LacO256 array and one allele of TMA10 is flanked by an integrated TetO200 array. As indicated, these labeled alleles are located on the same chromosome (Chowdhary et al., 2019). ASK727 also expresses GFP-LacI and TetR-mCherry (N- and C-terminal fusion, respectively) to allow visualization of the two genes as a green and red dot, respectively. Live cell widefield fluorescence microscopy of ASK727. Cells were immobilized onto ConA-coated coverslips and exposed to either heat shock (25°C to 38°C upshift) or ethanol stress (8.5% v/v; 25°C) for the indicated times (see Materials and methods). 11 z-planes with 0.5 µm interplanar distance were captured for each condition. A representative z-plane is shown per condition. Scale bar: 2 µm. (B) ASK727 cells, treated as in B, were scored for colocalization of HSP104 and TMA10 upon exposure to either heat shock or ethanol stress. A cell was scored as positive when the highest intensity signal from both genes overlapped in the same z-plane. An average of 70 cells were evaluated per condition, per timepoint. Displayed are means + SD. N=2. A one-tailed t-test was performed to assess significance. *, p<0.05. Note: we interpret HSP104-TMA10 coalescence observed at T=0 min to principally reflect coincidental overlap given absence of 3C signal under the no stress condition. A similar consideration applies to the HSP104-HSP12 gene pair analyzed below. (C) VPY705 cells were subjected to heat shock (39°C; VAHEAT device) or ethanol stress (8.5%) for the indicated times and imaged using an Olympus Spinning Disk Microscope System. Imaging was acquired over 11 z-planes, with 0.5 µm of distance between planes. Left: Percentage of cells showing coalescence between GFP-labeled alleles of HSP104 and HSP12 (Note that the theoretical ceiling for coalescence of these tagged alleles is 25% given their heterozygous state). Right: Percentage of cells in the population displaying an mCherry dot (MCP-mCherry bound HSP104 transcripts) colocalizing with the GFP-labeled HSP104 gene. An average of 40 cells per timepoint per condition was scored. Graphs depict means + SD. N=2.
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Figure 7—figure supplement 1—source data 1
Spreadsheet tabulates the percentage of cells with HSP104-TMA10 coalescence under HS or ES conditions (Figure 7—figure supplement 1C).
Also tabulated is the percentage of cells exhibiting HSP104-HSP12 coalescence and HSP104 mRNA foci upon ES or HS treatment (Figure 7—figure supplement 1D).
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Figure 8 with 1 supplement
Ethanol stress induces rapid formation of long-lasting Hsf1 condensates.
(A) Hsf1 is a transcription factor bearing N- and C-terminal domains with high disorder tendency (determined by IUPRED2). NTA, N-terminal activator; DBD, DNA binding domain; 3-mer, trimerization domain; CE2, conserved element 2 (Hsp70 binding site); CTA, C-terminal activator. (B) Hsf1-GFP condensates form in response to ethanol stress. Diploid cells expressing Hsf1-GFP (ASK741) were grown in synthetic complete medium supplemented with adenine (SDC +Ade) and mounted onto ConA-coated coverslips. Live cell widefield microscopy was performed on cells exposed to either heat shock (38°C) or ethanol stress (8.5% v/v) or left untreated (25°C). A representative plane is shown for each condition out of 11 z-planes imaged (interplanar distance of 0.5 µm). Scale bar: 2 µm. (C) ASK741 cells were subjected to a 38°C heat shock for the indicated times and scored for the presence of Hsf1 condensates. A cell was scored as positive if it contained at least one clearly defined puncta. Approximately 200 cells were evaluated per timepoint. A one-tailed t-test was used to assess significance (stress versus no stress condition). N=2. **, p<0.01; ***, p<0.001.(D) ASK741 cells were exposed to 8.5% v/v ethanol for the indicated times and the presence of Hsf1 condensates were scored from a total of 200 cells per timepoint. Significance was determined as in (C).
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Figure 8—source data 1
Spreadsheet tabulates the percentage of cells containing Hsf1-GFP puncta under heat shock or ethanol stress (Figure 8C and D).
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Figure 8—figure supplement 1
Hsf1-mNeonGreen rapidly forms condensates in response to both thermal and ethanol stress.
(A) Visualization of Hsf1-mNeonGreen (LRY040 haploid strain) exposed to heat shock (39°C) or ethanol stress (8.5% or 5% v/v, 25°C). Control (0 min) was kept at 25°C. Single plane is shown, from an 11 z-planes stack taken with 0.5 µm of interplanar distance. Dashed box represents zoom-in of a representative cell for that time point (continuous box). Scale bar: 2 µm (B) Cells treated as in (A) were scored for the presence of Hsf1-mNeonGreen foci using Imaris software (v.10.0.0; see Materials and methods). Depicted is the percentage of cells in the population displaying ≥1 Hsf1 puncta. An average of 150 cells were scored per condition, per timepoint. N=2. Shown are means + SD.
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Figure 8—figure supplement 1—source data 1
Spreadsheet tabulates the percentage of cells containing Hsf1-mNeonGreen puncta under heat shock, 8.5% v/v ethanol stress or 5% v/v ethanol stress (Figure 8—figure supplement 1B).
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Figure 9 with 1 supplement
Hsf1 and Pol II are critically required for HSR gene interactions in response to either heat shock or ethanol stress.
(A) Experimental strategy. Degron-tagged cells were treated with 1 mM IAA at 25°C for 30–40 min prior to exposure to either heat shock (HS, 39°C for 2.5 min) or ethanol stress (ES, 8.5% v/v ethanol for 10 min) followed by HCHO crosslinking and 3C analysis. (B) Strains LRY016 (W303-1B; OsTIR1), LRY100 (LRY016; Hsf1-mAID), and LRY102 (LRY016; Rpb1-mAID) were subjected to the above protocol and physical interactions between the indicated chromosomal loci were detected by Taq I-3C as in Figure 5. Representative interchromosomal interactions are shown. Graphs represent means + SD. Statistical significance between the indicated interaction frequencies was determined using a one-tailed t-test. *, p<0.05; **, p<0.01; ns, not significant. A no stress sample, maintained at 30°C for 10 min following IAA treatment and then crosslinked, was handled in parallel. No signal above background was detected for any pairwise test.
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Figure 9—source data 1
Spreadsheet tabulates 3C analysis of cells pretreated with 1 mM auxin followed by 2.5 min HS or 10 min ES (Figure 9B).
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Figure 9—figure supplement 1
Hsf1 and Rpb1 are efficiently degraded in degron-tagged strains following addition of auxin.
(A) Analysis of protein degradation. Strains LRY100 and LRY102 (Hsf1-mAID and Rpb1-mAID, respectively) were grown in YPDA to mid-log phase and subjected to 1 mM IAA treatment for different lengths of time. Cells were harvested and processed by Western blot. cMyc antibody was used to visualize mAID tagged proteins, endogenous Pgk1 serves as the loading control. Quantification of mAID tagged protein levels is shown on the right. Normalization was done to Pgk1 and 0 min samples. Shown are means + SD. N=2. (B) Growth curve assay of LRY100 and LRY102 cells exposed to 1 mM IAA. Cells were pre-grown in YPDA to mid-log phase, then diluted with equivalent volume of YPDA +IAA in ethanol (vehicle) for a final concentration of 1 mM IAA and 1.7% ethanol or just 1.7% ethanol (vehicle-treated control). Samples were incubated with shaking at 25°C, aliquots were removed to monitor OD600 at the indicated times. Plots depict means + SD. N=2. (C) Spot dilution analysis of cells from the indicated strains. Cells were pre-grown to early log phase, diluted to OD600=0.5 in autoclaved distilled water, then serially diluted 1:3. A 6x8 applicator was used to spot cells on YPDA or YPDA +1 mM IAA plates. Incubation was done at the indicated temperatures for 3 days.
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Figure 9—figure supplement 1—source data 1
Raw files for immunoblots of Hsf1-mAID-9xMyc, Rpb1-mAID-9xMyc and the corresponding Pgk1 as loading control in cells treated with 1 mM auxin at 25°C (Figure 9—figure supplement 1A).
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Figure 9—figure supplement 1—source data 2
Labeled blots for Hsf1-mAID-9xMyc, Rpb1-mAID-9xMyc, and the corresponding Pgk1 as loading control in cells treated with 1 mM auxin (IAA) at 25°C (Figure 9—figure supplement 1A).
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Figure 9—figure supplement 1—source data 3
Spreadsheet tabulates relative Hsf1-mAID-9xMyc and Rpb1-mAID-9xMyc protein levels, normalized to Pgk1 and 0 min of treatment with 1 mM auxin (Figure 9—figure supplement 1A).
Also tabulated is the optical density (OD600) of cells in liquid culture at 25°C +/-1 mM auxin. Cells bear Hsf1-mAID-9xMyc or Rpb1-mAID-9xMyc (Figure 9—figure supplement 1B).
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Figure 10
Central findings of this study.
(A) Kinetics of the yeast HSR to thermal and chemical stresses. (B) Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in a distinct fashion in response to thermal vs. chemical stress. Heat Shock (pink arrows): S. cerevisiae cells exposed to heat shock (30° to 39°C upshift) undergo protein misfolding (proteotoxicity), leading to chaperone titration and activation of Hsf1 accompanied by formation of Hsf1 condensates (1). Rapid target search is hypothesized to occur via multivalent interactions between Hsf1’s activation domains and chromatin-bound proteins (Brodsky et al., 2020) (2). Subsequently, Hsf1 cooperatively binds HSEs and induces the 3D reorganization of Heat Shock Responsive (HSR) genes (3). Simultaneously, RNA Pol II is recruited to HSR genes and transcription is induced (4). Productive transcription (pink shading) ensues, followed by dissolution of HSR gene interactions (5). Rapid export of HSR mRNAs (Zander et al., 2016) facilitates production of chaperone and cytoprotective proteins, which aid in restoration of proteostasis and disassembly of Hsf1 condensates (Chowdhary et al., 2022) (6). Thickness of arrows (for both HS and ES) symbolizes rapidity and/or magnitude of the subsequent step. Ethanol Stress (blue arrows): Exposure to 8.5% (v/v) ethanol induces proteotoxicity, titration of chaperones, and subsequent activation of Hsf1, triggering formation of condensates (1, 2). Condensates may aid Hsf1 target search by facilitating the multivalent interactions described above (2). Hsf1 then binds HSEs, inducing HSR gene repositioning (3). Subsequently, Pol II is recruited and transcription is induced (4). Ethanol stress-induced interactions dissipate well before maximal HSR gene transcriptional induction is achieved (5). The weak transcriptional HSR gene output, coupled with suppressed HSR mRNA export under ES (Izawa et al., 2008), results in a low level of chaperone synthesis (6) that fails to resolve proteotoxicity. This likely contributes to the persistence of Hsf1 condensates.
Figure 11
Acetal formation reaction (McMurry, 2012).
Tables
Table 1
Kinetics of select nuclear phenomena in response to heat shock and ethanol stress.
| Parameter | Heat Shock (39°C) | Ethanol Stress (8.5% v/v) |
|---|---|---|
| Hsf1 binding to HSEs | Rapid, peak at 2.5 min | Slight delay, peak at 10 min |
| Pol II recruitment to HSR genes | Rapid, peak at 2.5 min | Severely delayed, detection starts at 10 min |
| Histone H3 occupancy at HSR genes | Rapid depletion | Transient increase, followed by gradual depletion |
| Genome compaction* | Transient | Sustained |
| HSR gene transcription | Rapid | Delayed |
| HSR gene coalescence | Rapid, transient | Slightly delayed, transient |
| Hsf1 condensates | Rapidly induced, transient, well defined | Rapidly induced, stable, poorly defined |
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*
As inferred from enhanced H3 ChIP signal at non-Hsf1 regulated loci.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (S. cerevisiae) | BY4741 | Research Genetics | ||
| Cell line (S. cerevisiae) | W303-1B | Rodney Rothstein | ||
| Recombinant DNA reagent | pFA6a-link-ymNeonGreen-SpHis5 plasmid | Addgene | Cat# 125704 RRID:Addgene_125704 | PMID:30783202 |
| Recombinant DNA reagent | pGZ154 plasmid | C.K. Govind, University of Oakland | ||
| Recombinant DNA reagent | pHyg-AID*–9myc | Addgene | Cat# 99518 RRID:Addgene_99518 | PMID:23836714 |
| Chemical compound, drug | Indole-3-acetic acid (IAA) | Sigma-Aldrich | Cat# I3750 | |
| Chemical compound, drug | Sodium azide | Sigma-Aldrich | Cat# S2002 | |
| Chemical compound, drug | Ethanol | Decon Labs | Cat# 04-355-223 | |
| Chemical compound, drug | Phenylmethylsulfonyl fluoride (PMSF) | Sigma-Aldrich | Cat# P7626 | |
| chemical compound, drug | Phenol:Chloroform:Isoamyl Alcohol mixture | Sigma-Aldrich | Cat# 77617 | |
| Chemical compound, drug | Formaldehyde | Fisher Scientific | Cat# 14-650-250 | |
| Chemical compound, drug | Glycine | Bio-Rad | Cat# 1610724 | |
| chemical compound, drug | Protein A-Sepharose beads | GE Healthcare (Cytiva) | Cat# 17096303 | |
| Commercial assay or kit | High-Capacity cDNA Reverse Transcription Kit | Applied Biosystems | Cat# 4368814 | |
| Commercial assay or kit | iTaq Universal SYBR Green Supermix | BioRad Laboratories | Cat# 1725125 | |
| Antibody | Anti-Btn2 (rabbit polyclonal) | Bernd Bukau, University of Heidelberg | WB (1:5000) | |
| Antibody | Anti-cMyc (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-40 RRID:AB_627268 | WB (1:1000) |
| Antibody | Anti-Histone H3 (rabbit polyclonal) | Abcam | Cat# ab1719 | WB (1:1000) ChIP (1 µL/rxn) |
| Antibody | Anti-Hsf1 (rabbit polyclonal) | PMID:8943356 | ChIP (1.5 µL/rxn); Gross Lab | |
| Antibody | Anti-Hsp104 (rabbit polyclonal) | Enzo Life Sciences | Cat# ADI-SPA-1040-F RRID:AB_11181448 | WB (1:1000) |
| Antibody | Anti-Pgk1 (mouse monoclonal) | ThermoFisher Scientific | Cat# 459250 RRID:AB_2532235 | WB (1:10,000) |
| Antibody | Anti-Rpb1 (rabbit polyclonal) | PMID:16199876 | ChIP (1.5 µL/rxn); Gross Lab | |
| Antibody | Anti-Mouse, Horseradish peroxidase conjugated (goat) | Santa Cruz Biotechnology | Cat# sc-2005 RRID:AB_631736 | WB (1:5000) |
| Peptide, recombinant protein | Concanavalin A | Sigma-Aldrich | Cat# C2010 | |
| Peptide, recombinant protein | TaqI restriction enzyme | New England Biolabs | Cat# R0149L | |
| Commercial assay or kit | Quick Ligation Kit | New England Biolabs | Cat# M2200L | |
| Software, algorithm | FIJI/ImageJ | https://imagej.net/software/fiji/ | ||
| Software, algorithm | Imaris | https://imaris.oxinst.com/ | ||
| Software, algorithm | IUPRED2 | https://iupred2a.elte.hu/ | ||
| Other | VAHEAT device | Interherence GmbH | Temperature controller for live cell imaging |
Additional files
-
Supplementary file 1
Supplemental tables.
(a) Yeast Strains (b) Plasmids (c) Primers used for Strain Construction (d) Primers used for RT-qPCR (e) Primers used for ChIP (f) Primers used for Taq I-3C.
- https://cdn.elifesciences.org/articles/92464/elife-92464-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/92464/elife-92464-mdarchecklist1-v1.docx
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Downloads (link to download the article as PDF)
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Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes
eLife 12:RP92464.
https://doi.org/10.7554/eLife.92464.4
