Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis

Abstract

All rights reserved. I am especially thankful to my mom, dad and brother, all of whom were critical for getting me to grad school in the first place. I'm also forever thankful that they were always there to support and listen to me through my many struggles, even when the problems I was describing were unrelatable or unintelligibly described. know it would have been significantly less silly, ludicrous and off-putting to everyone else ix on the periphery if Patrick weren't down the hall from me, but I'm so glad I didn't have to live in that counterfactual universe. We have spent countless hours talking about science, politics, our personal lives, and many other things that I hope never get repeated, and I have enjoyed it all so very much.I have also been extremely fortunate for my academic advisors Vlad Denic and Edo Airoldi, along with my mentor and friend David Pincus. The three of them put an incredible amount of time into helping and guiding me as I grew as a scientist. When I reflect on the progress I've made since I started graduate school, I know that it would not have been possible without them constantly pushing me to be better. While it wasn't always fun or easy, I am so thankful for the time, energy and effort they put into me personally. They all went so far beyond what could reasonably be expected from an advisor, and even though all three were at challenging early stages of their own careers, they made an exceptional effort to help me as I started my own. I will always be thankful and grateful to Edo, Vlad and Dave for molding me into the scientist that I am today. In order to adapt when stress causes the folding environment to deteriorate, cells need a mechanism to adjust the availability of chaperones according to need. One simple mechanism employed by some bacteria to regulate expression of chaperones during heat shock involves RNA secondary structure that occludes the ribosome binding site at low 2 temperatures, but melts at high temperature de-repressing translation during thermal stress (Cimdins et al., 2014). However, rather than regulating each chaperone gene individually, eukaryotic cells have evolved a highly conserved feedback mechanism that coordinates up-regulation of many proteostasis factors in response to stress. The first indication of this mechanism were made by Ferruccio Ritossa in 1962 when he noticed a novel pattern of "puffs" on the polytene salivary gland chromosomes of Drosophila larvae that were subjected to a serendipitous heat shock when a co-worker increased their incubator's temperature (Ritossa, 1996). Subsequent controlled experiments revealed that shifting larvae from 25°C to 30°C caused puffs that were stable at the lower temperature to rapidly dissipate and induced the appearance of novel, reproducible puffs (Ritossa, 1962). It was soon revealed that the temperature stress caused by the actions of an unwitting co-worker has spurred the first observation of the dramatic changes in gene expression that comprise the heat shock response.It was subsequently revealed tha...

Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

“…Fluorescence microscopy was therefore applied to more precisely determine the dynamics of Hsf1 nuclear depletion. In contrast to cessation of growth (5 h: Fig A), nuclear depletion of Hsf1 is much faster, with nuclear GFP signal dropping to background levels within 15 min (Fig D–F), in agreement with Hsf1 nuclear depletion in a different genetic background (Solís et al , ). To establish whether any residual Hsf1 remains bound to target promoters after 15 min, at levels undetectable by fluorescence microscopy, chromatin immunoprecipitation (ChIP) was performed on two Hsf1 bound promoters (MacIsaac et al , ): BTN2 , which is TFIID‐dominated, and SSA1, which is SAGA‐dominated (Rhee & Pugh, ).…”

“…These are enriched for the Gene Ontology (GO) term protein folding ( P < 2 × 10 −24 ), in agreement with the established role of Hsf1 in regulating the expression of protein chaperones, also under non‐heat‐shock conditions (Gross et al , ; Solís et al , ). The 21 targets are a subset of the promoters previously reported to be bound by Hsf1 under standard and heat‐shock conditions (Hahn et al , ) and correspond almost exactly with Hsf1 direct targets also recently defined using anchor‐away (Solís et al , ). Most importantly, half of these targets are dominated by SAGA (11/21) and the other half by TFIID (10/21) (Rhee & Pugh, ).…”

“…Integration of the mRNA synthesis and Hsf1 binding data leads to identification of 21 direct targets (Materials and Methods). These are enriched for the Gene Ontology (GO) term protein folding (P < 2 × 10 À24 ), in agreement with the established role of Hsf1 in regulating the expression of protein chaperones, also under non-heat-shock conditions (Gross et al, 1990;Solís et al, 2016). The 21 targets are a subset of the promoters previously reported to be bound by Hsf1 under standard and heat-shock conditions (Hahn et al, 2004) and correspond almost exactly with Hsf1 direct targets also recently defined using anchoraway (Solís et al, 2016).…”

“…This agrees with the previously established essential role of Hsf1 under standard growth conditions (Wiederrecht et al , ). Given that it is the lack of expression of target genes that causes HSF1 gene deletion lethality (Solís et al , ), growth cessation is an indirect measure of Hsf1 loss‐of‐function dynamics. Fluorescence microscopy was therefore applied to more precisely determine the dynamics of Hsf1 nuclear depletion.…”

“…Hsf1 is required for the induction of heat-shock proteins but also binds some promoters under standard growth conditions (Hahn et al, 2004). This is likely the reason why HSF1 is essential for cell viability even in the absence of a heat shock (Wiederrecht et al, 1988), a proposal that has recently been confirmed (Solís et al, 2016). Previous studies have mostly used temperature-sensitive HSF1 mutant alleles to study Hsf1 function (Smith & Yaffe, 1991;Zarzov et al, 1997;Imazu & Sakurai, 2005).…”