Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response

Reviewer #1 (Public review):

Summary:

In this study, the authors present a thorough mechanistic study of the J-domain protein Apj1 in Saccharomyces cerevisiae, establishing it as a key repressor of Hsf1 during the attenuation phase of the heat shock response (HSR). The authors integrate genetic, transcriptomic (ribosome profiling), biochemical (ChIP, Western), and imaging data to dissect how Apj1, Ydj1, and Sis1 modulate Hsf1 activity under stress and non-stress conditions. The work proposes a model where Apj1 specifically promotes displacement of Hsf1 from DNA-bound heat shock elements, linking nuclear PQC to transcriptional control.

Strengths:

Overall, the work is highly novel - this is the first detailed functional dissection of Apj1 in Hsf1 attenuation. It fills an important gap in our understanding of how Hsf1 activity is fine-tuned after stress induction, with implications for broader eukaryotic systems. I really appreciate the use of innovative techniques, including ribosome profiling and time-resolved localization of proteins (and tagged loci) to probe the Hsf1 mechanism. The overall proposed mechanism is compelling and clear - the discussion proposes a phased control model for Hsf1 by distinct JDPs, with Apj1 acting post-activation, while Sis1 and Ydj1 suppress basal activity.

The manuscript is well-written and will be exciting for the proteostasis field and beyond.

Reviewer #2 (Public review):

Despite over 50 years of investigation, our understanding of how the ubiquitous heat shock response, governed by the transcription factor HSF1, was regulated was minimal. In recent years, a coordinated yet simple negative feedback circuit has been elucidated in high detail that centers on the chaperone Hsp70 as a direct-binding inhibitor of HSF1 transcriptional activation. However, roles for the obligatory Hsp70 J-domain partner co-chaperones are currently poorly understood. The present study applies several orthogonal techniques to the question and uncovers an unexpected role for the nuclear JDP Apj1 in attenuation of the heat shock response (HSR) via removal of Hsf1 from HSEs in heat shock gene promoter regions. Interestingly, Apj1 appears to play no role in initiating repression of Hsf1, as null mutants do not exhibit constitutive derepression of the HSR. This role is likely filled by the general nucleo/cytoplasmic JDP Ydj1, as previously reported. These results enhance understanding of HSR regulation and underscore the pivotal role that chaperones play in controlling pro-survival gene expression.

Overall, the work is exceptionally well done and controlled, and the results are properly and appropriately interpreted. Several of the approaches, while powerful, are somewhat indirect (i.e., following gene expression via ribosomal profiling) but ultimately provide a compelling answer to the main question being asked. However, at the end of the day, there is really only one major finding here: Apj1 regulates Hsf1 attenuation via Hsp70. That finding is strongly supported by the experimental data but lacks the one piece of mechanistic evidence found in other recent papers - differential binding of Ssa1/2 to Hsf1 at either the N- or C-terminal binding sites.

Reviewer #3 (Public review):

Summary:

The heat shock response (HSR) is an inducible transcriptional program that has provided paradigmatic insight into how stress cues feed information into the control of gene expression. The recent elucidation that the chaperone Hsp70 controls the DNA binding activity of the central HSR transcription factor Hsf1 by direct binding has spurred the question of how such a general chaperone obtains specificity. This study has addressed the next logical question: how J-domain proteins execute this task in budding yeast, the leading cell model for studying the HSR. While an involvement and in part overlapping function of general class A and B J-domain proteins, Ydj1 and Sis1 are indicated by the genetic analysis, a highly specific role for the class A Apj1 in displacing Hsf1 from the promoters is found, unveiling specificity in the system.

Strengths

The central strong point of the paper is the identification of class A J-domain protein Apj1 as a specific regulator of the attenuation of the HSR by removing Hsf1 from HSEs at the promoters. The genetic evidence and the ChIP data strongly support this claim. This identification of a specific role for a lowly expressed nuclear J-domain protein changes how the wiring of the HSR should be viewed. It also raises important questions regarding the model of chaperone titration, the concept that a chaperone with limited availability is involved in a tug of war involving competing interactions with misfolded protein substrates and regulatory interactions with Hsf1. Perhaps Apj1, with its low levels and interactions with misfolded and aggregated proteins in the nucleus, is the titrated Hsp70 (co)chaperone that determines the extent of the HSR? This would mean that Apj1 is at the nexus of the chaperone titration mechanism. Although Apj1 is not a highly conserved J domain protein among eukaryotes the strength of the study is that is provides a conceptual framework for what may be required for chaperone titration in other eukaryotes: One or more nuclear J-domain proteins with low nuclear levels that has an affinity for Hsf1 and that can become limiting due to interactions with misfolded Hsp70 proteins. The provides a pathway for how these may be identified using, for example, ChIP-seq.

Weaknesses

A built-in challenge when studying the mechanism of the HSR is the general role of the Hsp70 chaperone system and its J domain proteins. Indeed, a weakness of the study is that it is unclear which of the phenotypic effects have to do with directly recruiting Hsp70 to Hsf1 dependent on a J domain protein and what instead is an indirect effect of protein misfolding caused by the mutation. This interpretation problem is clearly and appropriately dealt with in the manuscript text and in experiments, but is of such fundamental nature that it cannot easily be fully ruled out. One way forward is a reconstituted biochemical system that monitors how Hsf1 DNA binding is affected by the Hsp70 system, misfolded proteins, and the various J domain proteins. Yet this approach is clearly beyond the scope of this study.

Author response:

Reviewer 1:

We thank the reviewer for his/her very positive comments.

Reviewer 2:

We thank the reviewer for his/her positive evaluation. We plan to add RNAseq data of yeast wild-type and JDP mutant strains as more direct readout for the role of Apj1 in controlling Hsf1 activity. We agree with the reviewer that our study includes one major finding: the central role of Apj1 in controlling the attenuation phase of the heat shock response. In accordance with the reviewer we consider this finding highly relevant and interesting for a broad readership. We agree that additional studies are now necessary to mechanistically dissect how the diverse JDPs support Hsp70 in controlling Hsf1 activity. We believe that such analysis should be part of an independent study but we will indicate this aspect as part of an outlook in the discussion section of a revised manuscript.

Reviewer 3:

We thank the reviewer for his/her suggestions. We agree that it is sometimes difficult to distinguish direct effects of JDP mutants on heat shock regulation from indirect ones, which can result from the accumulation of misfolded proteins that titrate Hsp70 capacity. We also agree that an in vitro reconstitution of Hsf1 displacement from DNA by Apj1/Hsp70 will be important, also to dissect Apj1 function mechanistically. We will add this point as outlook to the revised manuscript.