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. 2016 Nov 10;5:e18638. doi: 10.7554/eLife.18638

Figure 2. Prediction and validation of synthetic perturbations to the Hsf1-Hsp70 feedback loop.

(A) Cartoon schematic of activation by overexpressing full length Hsf1. Hsf1 can be expressed at many different levels by titrating the concentration of estradiol in the media (See Figure 2—figure supplement 1 and Materials and methods). The Hsf1 domain architecture is displayed below. The DNA binding domain (DBD) is between N- and C-terminal activation domains (NTA and CTA). (B) Cartoon schematic of activation via overexpression of the Hsf1 decoy. The decoy domain architecture is displayed below. (C) Simulation of the HSE-YFP reporter as a function of the expression level of full length Hsf1 or the decoy. (D) Experimental measurement of the HSE-YFP reporter by flow cytometry in cells expressing full length Hsf1, the decoy or mKate alone across a dose response of estradiol. Cells were monitored following growth in the presence of the indicated concentrations of estradiol for 18 hr. Data points 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.

DOI: http://dx.doi.org/10.7554/eLife.18638.007

Figure 2—source data 1. Table of peptide counts from proteins identified in decoy IP/MS experiments with decoy-3xFLAG-V5 and Hsf1-3xFLAG-V5 as bait.
DOI: http://dx.doi.org/10.7554/eLife.18638.008
elife-18638-fig2-data1.xlsx (41.4KB, xlsx)
DOI: 10.7554/eLife.18638.008

Figure 2.

Figure 2—figure supplement 1. Overexpression of a decoy of Hsf1 activates endogenous Hsf1.

Figure 2—figure supplement 1.

(A) Anti-FLAG western blot of cells expressing Hsf1-FLAG-V5 under the control of an estradiol-inducible promoter. Cells were incubated for four hours across a two-fold dilution series of estradiol (from 512 nM down to 1 nM) and the Hsf1 expression level was compared to expression from the endogenous promoter. (B) Cells expressing YFP-Ubc9ts were induced to express the mKate2-labeled decoy for four hours with 512 nM estradiol and were either left at 25°C or shifted to 39°C for 15 min and imaged by spinning disc confocal microscopy. YFP-Ubc9ts forms aggregates during heat shock, while the decoy remains apparently soluble. (C) Schematic of decoy constructs for domain analysis. (D) Measurement of the HSE-YFP reporter as a function of estradiol for the constructs depicted in (C).
DOI: http://dx.doi.org/10.7554/eLife.18638.009