Figure 4. Phosphorylation is dispensable for Hsf1 function but tunes the gain of its transcriptional activity.

(A) ³²P incorporated into wild type Hsf1-3xFLAG-V5 and Hsf1^∆po4-3xFLAG-V5 during 30 min of heat shock (upper panel). Hsf1-3xFLAG-V5 and Hsf1^∆po4-3xFLAG-V5 were affinity purified by anti-FLAG IP, resolved by SDS-PAGE and phosphor-imaged. Western blot of total lysate from wild type Hsf1-FLAG-V5 and Hsf1^∆po4-FLAG-V5 cells was probed with an anti-FLAG antibody; Hsf1^∆po4-FLAG migrates faster than wild type Hsf1-FLAG (lower panel). Schematics of the domain architecture and color code for wild type Hsf1, Hsf1^∆po4 and Hsf1^PO4*. (B) Wild type, Hsf1^∆po4 and Hsf1^PO4* cells were monitored for growth by dilution series spot assays. Cells were incubated at the indicated temperatures for two days. (C) Wild type, Hsf1^∆po4 and Hsf1^PO4* cells expressing the HSE-YFP reporter were assayed for Hsf1 transcriptional activity in control and heat shock conditions by flow cytometry. Bars are the average of median YFP values for three biological replicates, and error bars are the standard deviation. See Materials and methods for assay and analysis details. (D) Genome-wide mRNA levels were quantified in basal conditions in wild type and Hsf1^∆po4 cells by RNA-seq. Within each sample, relative expression levels for each mRNA (gray dots) are plotted as fragments per kilobase per million mapped reads (FPKM). Hsf1-dependent genes (HDGs) are highlighted in purple. Source data are included as Figure 4—source data 2. (E) Fold changes of each mRNA in heat shock conditions compared to basal conditions were calculated for wild type and Hsf1^∆po4 cells and plotted against each other. Hsf1-dependent genes (HDGs) are highlighted in purple. Source data are included as Figure 4—source data 2. (F) Genome-wide mRNA levels were quantified in basal conditions in wild type and Hsf1^PO4* cells by RNA-seq (gray dots). Hsf1-dependent genes (HDGs) are highlighted in orange. Source data are included as Figure 4—source data 2. (G) Fold changes of each mRNA in heat shock conditions compared to basal conditions were calculated for wild type and Hsf1^PO4* cells and plotted against each other. Hsf1-dependent genes (HDGs) are highlighted in orange. Source data are included as Figure 4—source data 2. (H) Schematic of mutants with different numbers of aspartate (D) residues. 33, 49 or 82 D residues were introduced in the CTA in the ∆po4 background. (I) Mutants depicted in (H) expressing the HSE-YFP reporter were assayed for Hsf1 transcriptional activity in control and heat shock conditions by flow cytometry as above.

DOI: http://dx.doi.org/10.7554/eLife.18638.013

Figure 4—figure supplement 1. Mutational analysis of Hsf1 phosphorylation sites reveals a single essential serine and allows for generation of a phospho-mimetic.

(A) Hsf1 mutants lacking or mimicking single or clustered phosphorylation sites were assayed for activity in basal and heat shock conditions by measuring the HSE-YFP reporter by flow cytometry. The average of the median of the YFP distribution of three replicates of each mutant is plotted and the error bars represent the standard deviation. The ∆NTA and ∆CTA mutants are known to be hyperactive and impaired, respectively (Sorger, 1990), and serve as positive controls for altered activity. (B) Electrophoretic mobility shift assay showing that recombinant full-length wild type Hsf1 efficiently binds to and shifts HSE-containing DNA. The shift can be reverted with competition with excess unlabeled HSE. However, mutations S225A, which removes the hydroxyl group, and S225D, which partially mimics a phosphate group, reduce DNA binding and thus diminish the shift. The same amount of total Hsf1 was loaded in each lane, and the percent of labeled HSE shifted was quantified. (C) S225 is the only essential serine in Hsf1. Mutation of S225 to alanine renders cells inviable (top right plate). Restoration of S225 as the only serine in a mutant with all the other 152 S/T residues mutated to alanine rescues growth. hsf1∆ cells bearing a URA3-marked copy of wild type HSF1 on a plasmid (pRS316-HSF1) and transformed with the indicated Hsf1 mutant were streaked on 5-FOA plates and incubated at 30°C for two days. (D) Fluorescent microscopy images of wild type Hsf1, Hsf1^∆po4 and Hsf1^PO4* under basal conditions tagged at their C-termini with YFP showing that all localize to the nucleus. (E) ChIP-seq data (reads per million mapped reads, RPM) for Hsf1 and hsf1^∆PO4 under basal conditions plotted along the first 150 kb of chromosome XII.

DOI: http://dx.doi.org/10.7554/eLife.18638.016