Figures and data
Ethanol stress induces extensive protein aggregation
(A) Growth curve of strain W303-1B grown in liquid culture (YPDA). Mid-log phase cultures were diluted to A600 =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). A600 was monitored over time. Means and SD are shown. N=2.
(B) Viability assay of W303-1B cells following exposure to heat shock or ethanol stress. An aliquot was taken from each condition at the listed 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 stressed cells normalized to 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. LRY033 cells were maintained at 25°C (no stress) or exposed to either 39°C heat shock or 8.5% ethanol stress. Hsp104-BFP foci were visualized by confocal microscopy. Shown are maximal projections of 11 z-planes, taken with 0.5 µm of interplanar distance. Scale bar: 2 µm.
(E) Cells subjected to the above treatments were assayed for Hsp104 puncta. An average of 40 cells per timepoint, per condition, was quantified using Imaris software (v.10.0.0). Significance was determined by Mann Whitney test. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not significant.
Ethanol stress transcriptionally induces Hsf1-dependent genes but with markedly slower kinetics than thermal stress
(A) RNA abundance of Heat Shock Response (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. Depicted are means + SD. N=2, qPCR=4. Statistical analysis: T-test, one-tailed, unequal variance, no stress vs stress conditions. *, p<0.05; **, p<0.01.
(B) As in (A), but the Hsf1-, Msn2-dual regulated gene HSP12 was evaluated. ***, p<0.001.
Hsf1 and Pol II recruitment is delayed while histone occupancy transiently increases at HSR genes in ethanol stressed cells
(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 at different loci within the indicated HSR 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). 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.
Ethanol stress induces intergenic interactions between HSR genes that are as frequent as those induced by acute thermal stress
(A) Intrachromosomal (cis) interactions between HSR genes were analyzed by TaqI-3C. W303-1B cells were instantaneously shifted from 30° to 39°C for 2.5 min (heat shock (HS)) or exposed to 8.5% v/v ethanol at 25°C for 10 or 20 min (ES). No stress samples were kept at 25°C. Location of TaqI coordinates are provided in Figure 4–figure supplement 1. F (forward) primers are positioned near the indicated TaqI 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 performed as in (A).
Ethanol-induced HSR gene interactions are detectable by 2.5 min but typically dissipate within 60 min
(A) TaqI-3C analysis of intergenic interactions occurring during ethanol stress was conducted as described in Figure 4. All samples were kept at 25°C. Locations of TaqI restriction sites are provided in Figure 4–figure supplement 1. Data are plotted as means + SD. N=2, qPCR=4.
(B) As in (A), but for intragenic interactions (crumpling).
HSR genes coalesce in response to ethanol stress at a frequency comparable to that elicited by heat shock
(A) Relative location of HSP104 and TMA10 on Chr. XII in the diploid strain ASK727. As indicated, one allele of HSP104 is flanked by an integrated LacO256 array and one allele of TMA10 is flanked by a TetO200 array. ASK727 also expresses GFP-LacI and TetR-mCherry to allow visualization of the two genes as a green and red dot, respectively.
(B) 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 (25°C, 8.5%) for the indicated times (see Materials & 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.
(C) 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. One-tailed t-test was performed to assess significance. *, p<0.01. 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.
(D) Chromosomal location of HSP12 and HSP104 and the LacO arrays flanking each in the diploid strain VPY705. HSP104 has, in addition, a 24xMS2 loop array integrated within the 5’-UTR downstream of the endogenous promoter. Upon transcriptional activation of HSP104, MCP-mCherry binds to the nascent chimeric transcript and is visualized as a red dot adjacent to the gene (green dot).
(E) Live cell confocal fluorescence microscopy of strain VPY705 treated as in (B) for the indicated times but using an Olympus spinning disk confocal microscope system for imaging and a VAHEAT device for heat shock (39°C, see Materials & Methods). Scale bar: 2 µm.
(F) Quantification of VPY705 cells treated as above and scored for the coalescence of HSP104-HSP12 and the presence of chimeric MS2×24-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 signal above background could be seen for a red dot near the large green dot (HSP104). Approximately 40 cells were scored per timepoint, per condition. Graphs represent means + SD. N=2.
Ethanol stress induces rapid formation of long-lasting Hsf1 condensates
(A) Hsf1 is a transcription factor with high disorder tendency. The domain map for Hsf1 is shown at the top. Tendency for disorder was determined using IUPRED2.
(B) Condensate formation for Hsf1-GFP occurs in response to ethanol stress. Cells from the diploid strain ASK741 bearing Hsf1-GFP were grown in synthetic complete medium supplemented with adenine (SDC+Ade) and mounted onto ConA-coated coverslips (see Materials & Methods). Live cell widefield microscopy was performed for cells exposed to heat shock (HS, 38°C) or ethanol stress (ES, 8.5% v/v) or left untreated (25°C). A representative plane is shown for each condition out of 11 z-planes taken (interplanar distance of 0.5 µm). The red box indicates a zoomed image derived from the green boxed area. Scale bar: 2 µm.
(C) Cells from strain ASK741 were subjected to a 38°C heat shock for the indicated times and scored for the presence of Hsf1 condensates. A cell was deemed positive if it contained at least one clearly defined puncta. Approximately 200 cells were evaluated per field, per timepoint. A one-tailed t-test was used to assess significance of stress versus no stress condition. N=2. **, p<0.01; ***, p<0.001.
(D) Cells from strain ASK741 were exposed to 8.5% v/v ethanol for the indicated times and presence of Hsf1 condensates were scored from a total of 200 cells per field, per timepoint. Significance was determined as in (C).
Hsf1 and Pol II are required for HSR gene interactions in response to both heat shock and ethanol stress
(A) Experimental strategy. Degron-tagged cells were treated with 1 mM indole acetic acid (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. No Stress (NS) samples were maintained at 30°C for 10 min following IAA treatment and then crosslinked.
(B) Strains LRY016 (OsTIR1), LRY100 (OsTIR1, Hsf1-mAID) and LRY102 (OsTIR1, Rpb1-mAID) were subjected to the above protocol and physical interactions between the indicated chromosomal loci were detected by TaqI-3C (see Figure 4–figure supplement 1 for location of TaqI restriction sites). Shown is an example of intrachromosomal interactions. Graphs represent means + SD. One-tailed T-test. *, p<0.05.
(C) As in (B), but for interchromosomal interactions. One tailed-T-test. **, p<0.01.
Comparison of kinetics of heat shock and ethanol stress on HSR genes
Heat Shock Responsive genes undergo rapid 3D genome repositioning following exposure to ethanol stress despite their delayed and muted transcription
Working model depicting the response of Hsf1-regulated genes in Saccharomyces cerevisiae under either heat shock (left) or ethanol stress (right).
Heat shock rapidly induces Hsf1 occupancy of Heat Shock Elements (HSEs) upstream of HSR genes, leading to fast and robust Pol II recruitment, histone eviction and high levels of HSR gene transcription. These parameters are kinetically linked to the coalescence of HSR genes and the presence of Hsf1 transcriptional condensates. Transcription at HSR genes decreases once the cell acclimates to the high temperature; this is accompanied by dissolution of condensates and relaxation of genome structure. All typically attenuate within 60 min.
Ethanol stress induces an initial chromatin compaction, which Hsf1 overcomes to bind HSEs, albeit with a slight delay (represented using …). Hsf1 then recruits Pol II, but transcriptional output is markedly lower than under heat shock and this may be due to reduced productive elongation of Pol II through HSR coding regions. Despite the low transcriptional activity, HSR genes physically interact. Formation of Hsf1 condensates is kinetically linked to HSR intergenic interactions yet condensate presence is insufficient to maintain HSR interactions as the latter dissipate between 20 – 60 min. Hsf1 condensates persist for >2.5 h.
