DOI: 10.5061/dryad.b2rbnzsm6
title: Induction of Sis1 promotes fitness but not feedback in the heat shock response

description: The heat shock response (HSR) controls expression of molecular chaperones
 to maintain protein homeostasis. Previously, we proposed a
 feedback loop model of the HSR in which heat-denatured proteins sequester
 the chaperone Hsp70 to activate the HSR, and subsequent induction
 of Hsp70 deactivates the HSR. However, recent work has implicated
 newly synthesized proteins (NSPs) – rather than unfolded mature
 proteins – and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their
 contributions to HSR dynamics have not been determined. Here we
 generate a new mathematical model that incorporates NSPs and Sis1
 into the HSR activation mechanism, and we perform genetic
 decoupling and pulse-labeling experiments to demonstrate that
 Sis1 induction is dispensable for HSR deactivation. Rather than
 providing negative feedback to the HSR, transcriptional regulation of Sis1
 by Hsf1 promotes fitness by coordinating stress granules and
 carbon metabolism. These results support an overall model in
 which NSPs signal the HSR by sequestering Sis1 and Hsp70,
 while induction of Hsp70 – but not Sis1 – attenuates the
 response.
description: <strong>Strain
 construction</strong>                         
 Yeast strains and plasmids used in this study are
 listed in Table S1. All strains are derived from the W303 parent strain.
 CRISPR-mediated promoter swapping was performed to create the
 1xSup35pr-Sis1 (1701) and 1xSup35pr-Sis1-P2A-mscarlet (1661) strains.
 CRISPR-Cas9 mediated precise, scarless replacement of the native Sis1
 promoter with the 600-bp Sup35 promoter. To construct the final
 non-inducible Sis1 line (2xSup35pr-Sis1, 1761), we incorporated a second
 copy of Sup35pr-Sis1 at the tryp locus. In
 all other cloned lines, genes were tagged at the endogenous locus. Cells
 were transformed with double-stranded DNA fragments containing ~20 bp
 homologous flanking regions. This method takes advantage of
 homology-directed repair mechanisms in <em>S.cerevisiae</em>,
 as described previously (Longtine et al.,
 1998). <strong>Cell Growth</strong>
 For
 heat shock time courses followed by flow cytometry, cells were cultured in
 1xSDC media overnight at room temperature (synthetic media with dextrose
 and complete amino acids). Before RT-PCR or cell growth assays, cells were
 cultured in yeast extract peptone dextrose (YPD) media shaking at 30ºC
 overnight. Cells were subjected to heat shock at 39°C unless otherwise
 specified. <strong>HSE-YFP reporter heat shock assays               
    </strong>                        
 Three biological replicates of each strain were
 serially diluted five times (1:5) in 1xSDC and grown overnight at room
 temperature. In the morning, cells had reached logarithmic phase, and 750
 µL of each replicate was transferred to a PCR tube and shaken for one hour
 at 30ºC to aerate. Then, cells were exposed to heat shock at 39ºC. At the
 pre-determined time points, (0,5,10,15,30,60,90,120,180,240 minutes), 50
 µl of cell culture was transferred to a well of a 96-well plate,
 containing 1xSDC and a final concentration of 50 mg/mL cycloheximide to
 stop translation. After the time course, cells were incubated at 30ºC for
 one hour to allow fluorescent reporter maturation before measurement. All
 experiments were performed using C1000 Touch Thermal Cycler (Bio-Rad).
 Cell fluorescence was measured by flow cytometry and results were analyzed
 as described below.  <strong>Anchor-Away
 Assay</strong> Cells were grown overnight as described above.
 Rapamycin was added to a final concentration of 10 µM, and the time course
 was started immediately. During the time course, cells were maintained at
 30ºC shaking. At the predetermined time points (0, 15, 30, 45, 60, 75, 90
 minutes), 50 µL of cells were transferred to a 96-well plate identically
 to the heat shock assay described above. <strong>Translation Inhibition Assay (Rapamycin
 Pre-treatment)</strong> Cells were grown overnight, and in the morning,
 shaken at 30ºC for 1 hour. Rapamycin was added to a final concentration of
 10 µM, and cells
 were left shaking at 30ºC for 5 minutes. Then, the heat shock time course
 was performed at 39ºC as described above. <strong>Flow
 cytometry</strong> HSE-YFP and mscarlet reporter levels in heat shock
 time course and rapamycin treatment assays were measured at the University
 of Chicago Cytometry and Antibody Technology Facility. These measurements
 were performed using the 488-525 FITC fluorescence filter (HSE-YFP) and
 561-PE Dazzle (mscarlet) on the BD Fortessa High Throughput Flow
 Cytometer. The raw fluorescence values were normalized by side scatter in
 FlowJo. Then, the median fluorescence value was calculated. Each data
 point represents the average of three biological
 replicates. <strong>Dilution series spot growth assays          
 </strong> Yeast strains were grown overnight shaking at 30ºC
 in YPD. In the morning, they were diluted to and final optical density
 (OD) of 1 and serially diluted 1:10 in water. Each diluted yeast culture
 was spotted onto 1xYPD plates. Images were taken after two days of growth
 at 30º or 37ºC. <strong>Heat Shock Quantitative Growth
 Assay</strong> Yeast strains were grown overnight at 30ºC in
 1xYPD. In the morning, they were diluted to OD= 0.1 in 1xYPD. SpectroStar
 Nano microplate reader was used to measure cell density every 20 minutes.
 Each data point represents the mean of three biological
 replicates. <strong>Glucose Starvation Quantitative Growth
 Assay</strong> Yeast strains were grown for 24 hours in 2% glucose
 YEP media then diluted to OD=0.1 and grown overnight again. In the
 morning, cells were diluted to OD=0.1 in 1xYEP containing 2% glycerol, 2%
 EtOH and growth was monitored every 20 minutes at
 30ºC. <strong>RNA quantification and
 analysis</strong> ​​RT-qPCR was performed as previously
 described in (Unable to find
 information for 12149321), except for the following: cells
 were grown at 30°C in YPD
 (yeast extract-peptone-dextrose) to a mid-log density
 (OD<sub>600</sub> = 0.8). Rapamycin was added to
 a final concentration of 10 ug/mL for 5 min. At this point, a portion of
 the culture was maintained at 30°C (0 min HS), and the
 remainder was subjected to heat shock at 39°C for 5, 15, 60, and 120
 min. Cells were flash-frozen immediately after heat shock and then
 harvested. Total RNA was extracted using hot acid phenol extraction
 method, followed by ethanol precipitation(Solís et al., 2016). To determine fold change mRNA levels, mean mRNA levels of heat-shocked samples were normalized to the NHS sample(Pfaffl, 2001). mVenus forward primer: caacattgaagatggtggtgttc mVenus reverse primer: ctttggataaggcagattgatagg <strong>RNA-Seq Analysis</strong> RNA read counts for 39 Hsf1 target genes were collected in a recent paper and reanalyzed here(Triandafillou et al., 2020). GSE accession number 152916 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152916) Experimental methods are described in detail in the original paper. In brief, cells were exposed to heat shock at 42ºC for 20 minutes. Cells exposed to heat shock and cycloheximide were treated with 200 ug/mL cycloheximide (CHX) simultaneously upon HS. Each data point represents the average read count for a single Hsf1 target gene (two biological replicates). Translation dependence was calculated as 1-(CHX+HS/HS). <strong>Halo-Tagging</strong> To image newly synthesized proteins, we incubated cells with blocker (20uM, 7-bromoheptanol) for 5 minutes, washed two times with 2xSDC media, and then incubated with 0.4 uM of Halo JF646dye at 39ºC, to start HS treatment simultaneously. Under this treatment, only protein induced during heat shock was visualizable. Lattice light sheet microscopy was used to visualize 5–20 cells every 2.5 minutes throughout the 30-minute heat shock.  <strong>Lattice Light Sheet Microscopy and Quantification</strong> Lattice light-sheet imaging was performed at the University of Chicago Integrated Light Microscopy Core (Intelligent Imaging Innovations) and run in SlideBook 6.0 software. Captured images were deconvoluted using Graphics processing unit–based Richardson-Lucy deconvolution with measured PSFs via Brian Northan’s “Ops” implementation (https://github.com/imagej/ops-experiments). 3D reconstructions and videos were assembled using ClearVolume(Royer et al., 2015). The mean Halo intensity at the ring around the Nsr1-marked nucleolus was divided by the mean intensity over the total cell.