Lack of Hikeshi activates HSF1 activity under normal conditions and disturbs the heat-shock response

Hikeshi mediates proper nucleocytoplasmic distribution of HSP70, which is important for regulating of HSF1 activity and nuclear proteostasis, and adaptive response to heat shock.


1)
would benefit from a DAPI stain or other positive control for nuclear localization. The claim of nuclear localization in 1C in response to MG132 is less convincing and the results should be modified to reflect minor localization relative to heat shock. 2) Fig. 2D -there is some variability in HSR derepression resulting from CRISPR knockout vs. siRNA depletion of Hikeshi -did the authors quantify Hikeshi levels in the latter experiments by western blot? It is important to do so when using shRNA or siRNA approaches. 3) Fig. 3E -did the authors consider titrating "regular" Hsp70 without the NLS attached? This could help reveal if Hsp70 is capable of functionally repressing HSF1 in an Hikeshi null background. Given the results of Fig. 1D showing significant Hsp70 localizztion in Hikeshi mutants, additional pathway(s) must be involved. 4) Fig. 4D -The legend references Fluc protein stability in wild type or knockout cells, but the Y axis of the plot is labeled "activity". Which was measured? GFP signal or luciferase activity? Only the former reflects protein stability. The latter is a more complicated property that reflects possible changes in stability and folding. Given the minor differences shown in Fig. 4B, these considerations are important. 5) What is happening in Fig. 6C -why are heat shock genes downregulated in the knockout lines after heat shock? This result seems contrary to the previous results. Additionally, Fig. 6G should be moved to supplementary information and/or summarized in a more easily interpretable manner. 6) No citations describing the major advancements in understanding Hsp70 regulation of HSF1 in yeast via nuclear Hsp70 are included. Given that very little has been done in this area in vertebrate cells and much has been revealed in yeast, this is an unacceptable oversight. The following references should be included as part of the discussion regarding the Hsp70/Hsf1 regulatory circuit: about HSF1, but does not attempt to modulate HSF1 (eg. by siRNA) to try to confirm observations. Many of the results are not only overinterpreted, but also lack confirmation of the "screening" results for differential expression by qPCR, and there are no details regarding how many replicates are used, how many separate experiments were performed etc. Overall, this study is too preliminary to be considered for publication in its present form.  Fig EV1 c and d needs to be validated by qPCR to give confidence that the results are consistent and significant. In Figure 3 it is not clear what the replicate size is for RNA sequencing (why is there no confirmation of the differential expression presented in Fig 3c by qPCR). In short, every figure needs clear information regarding the number of replicates, number of experiments -the impression, without this, is that everything was performed only once ? And further, confirmation of "screening" results by qPCR seems essential to give weight to the observational data. 4. The authors overstate/overinterpret their results on many occasions, and fail to perform qPCR to confirm conclusions of differential expression. Eg.
a. The title is misleading -it should read "Lack of Hikeshi reduces HSF1 activity and impacts the heat shock response" b. On page 7 "depletion of Hikeshi affects the expression of various genes and particularly induces upregulation of mRNA expression transcribed from HSF1" should read "...particularly induces upregulation of expression of known HSF1 targets" because this specific data they are discussing (Fig 2) only shows that specific genes are upregulated by Hikeshi KO (and appear to just happen to be known targets of HSF1 -where are the references ?). It would be important for the authors to perform HSF1 knockdown in Hikeshi-KO to confirm the upregulation of these specific genes is HSF1 dependent. c. The same issue arises in the discussion on page 13 of Fig 2b where the authors state "predictably, many of these Hsp70related genes were upregulated by HSF1". This is not a correct description of the result, which did not validate the role of HSF1 in the upregulation of these genes. The authors really need to perform HSF1 knockdown in Hikeshi-KO to confirm the upregulation of these specific genes is indeed HSF1 dependent. d. The abstract is misleading when it says "depletion of Hikeshi induces a reduction in nuclear HSP70 and upregulation of the mRNA expression of genes regulated by HSF1 under nonstressed conditions". The authors have not confirmed HSF1 upregulated these genes in their experimental system, with the results essentially indirect (ie. reduced activation of the heat shock element promoter in Hikeshi KO cells). At best the authors could argue that there may be a correlation between lack of Hikeshi, and lower HSF1 activity, but if the authors performed HSF1 knockdown in Hikeshi-KO to confirm the upregulation of these specific genes, they would be able to make claims that the effects are HSF1 dependent. e. On page 8, "Therefore, dysfunction of Hikeshi, which leads to a decrease in nuclear Hsp70, causes dysregulation of HSF1 transcriptional activity" should read "Therefore, lack of Hikeshi, which leads to a decrease in nuclear Hsp70..." f. Figure 5 legend title states "Hikeshi suppresses nuclear polyQ-induced apoptosis"; in fact, Hikeshi downregulates OR reduces OR contributes to suppression of nuclear polyQ-induced apoptosis because we can see that caspase activity is still increased in WT cells, albeit at a lower level than in KO cells (Fig 5b). Similarly page 10 "..KO cells significantly suppressed the apoptosis induction..." 5. On page 11, the authors conclude that "the heat shock response was sustained during recovery from heat shock" in the Hikeshi-KO cells because more genes that are heat shock responsive were at their peak expression levels 3 h after stress in the Hikeshi KO cell compared to WT cells. It seems much more likely that the transcriptional response to heat shock was delayed, with peak expression of heat shock responsive genes occurring 3 hours after heat stress ? 6. The authors suggest Hikeshi imports Hsp70 under non stressed conditions but have not shown binding interaction under non stressed conditions. 7. The authors should acknowledge that given that many heat shock responsive genes show delayed upregulation rather than inhibited upregulation in the Hikeshi-KO cells (Fig 6d). Can the authors be sure there is not another factor compensating for loss of Hikeshi to affect transcription of these genes ? 1. Figure 1a should go into the supplementary figures. The whole paragraph "Hikeshi orthologs are widely distributed in eukaryote" shiould be removed from results and put into introduction or discussion. 2. A number of spelling mistakes should be amended including those in legend Figure 3 and Figure 4 3. On page 8 the authors state Fluc is frequently used to monitor chaperone activity of Hsp70. The authors need to supply some references here. 4. The authors' claim on P. 7 that "many of these genes that are upregulated in Hikeshi-KO under nonstressed conditions are known to be regulated by .... HSF-1" needs appropriate literature citations (or the text should be deleted). 5. The authors go backwards and forwards between heat shock response and heat stress response -are these the same ? What is meant by the different terms ? Can the authors be clear ?
In short, the current paper requires a complete overhaul in terms not only of the writing, but also requires a major body of confirmatory experimentation, including HSF1 knockdown experiments. Only in this way, can the study be considered solid or rigorous enough for Life Science Alliance.
Reviewer #3 (Comments to the Authors (Required)): Comments on «Nuclear HSP70, supplied by Hikeshi, controls HSF1 activity and affects the heat stress response" In the article entitled «Nuclear HSP70, supplied by Hikeshi, controls HSF1 activity and affects the heat stress response", Kose et al. investigate the function of Hikeshi in non-stressed as well as heat stressed cells in regulating gene expression and proteostasis. With this work, they follow up on their previous papers, in which they showed that Hikeshi functions as a nuclear import factor for HSP70 upon heat stress. Here they now show that Hikeshi is also required to maintain a steady state level of nuclear HSP70 in unstressed cells. Lack of Hikeshi leads to changes in gene expression, notable higher expression of several HSF1 target genes. This effect could be rescued by HSP70 targeted to the nucleus, suggesting that the misregulation is due to the failure of Hikeshi to import HSP70. The authors further show a positive effect of nuclear localized HSP70 on preventing aggregate formation of a polyQ protein, stabilization of an unstable Luciferase reporter and suppressing gene misregulation observed in Hikeshi ko cells. Overall, this paper presents a number of interesting obvervations but some additional control experiments and some changes in the presentation of the data are needed before publication. Major paper conclusions: 1. "Knockout of the Hikeshi gene induces a reduction in nuclear HSP70 under nonstressed conditions" • Conclusion well supported by Figure 1. 2. "Knockout of the Hikeshi gene induces upregulated expression of the HSF1-regulated gene under nonstressed conditions" • Conclusion partially supported by data presented in Figure 2. A detailed description of how the analysis of the RNAseq data was performed is missing. Were there replicates? What are the statistical test used to select the upregulated genes? I am no expert in mRNAseq but it seems that the information given here is insufficient and I cannot judge whether the analysis done is appropriate. As a example, the text states for figure 2D that "Consistently, siRNA-mediated depletion of Hikeshi also showed that most of these HSF1-regulated genes, except for HSPA1L, were upregulated under nonstressed conditions", however for BAG3 this increase does not look significant. 3. "Nuclear HSP70 regulates the transcriptional activity of HSF1" • Conclusion largely supported by data presented in Figure 3. As control for Figure 3B,E and 4C, co-transfection of a Hsc70 plasmid with NES should be shown to exclude indirect effects of overexpression not related to nuclear localization. 4. "Nuclear HSP70 functions in the protein stability of nuclear luciferase monitoring proteins." • Conclusion largely supported by data presented in Figure 4. As control for Figure 4C, co-transfection of a Hsc70 plasmid with NES should be shown to exclude indirect effects of overexpression not related to nuclear localization. 5. "Hikeshi suppresses nuclear polyQ-induced apoptosis." • Conclusion largely supported by Figure 5: Is the effect of polyQ also alleviated if NLS-HSP70 is expressed? This would again exclude indirect effects. 6. "Heat shock response is impaired in the Hikeshi-KO cells." • This is not entirely convincing. Figure 6C: if in unstressed conditions the levels are different, than fold change heat stress versus normal growth will be different even if the absolute expression level is identical. Is absolute induction reduced in HikeshiKO? Figure 6G would suggest that the levels are actually similar in heat stress. • Figure 6D,E: very difficult to read this chart! The concept of "delayed genes" is rather difficult to understand for me and it is also unclear, what the delayed peak actually means. I guess it suggests that there is continued transcription that leads to accumulation of the mRNA beyond the acute heat shock period, but this does not really mean a delay in expression but rather a delay in shut-off. I'm not sure how to improve this section, but maybe the authors can come up with a more intuitive way of presenting their message. • 6G: In the discussion it says that in HS most cells in KO cells were lower expressed, but this is obvious only in the recovery, not in the HS conditions. This does not clearly fit with the argumentation that the peak is later, or does it? Levels are similar in HS but reduced in recovery would actually suggest faster shut off or decay?
Minor comments 1. "supplied by Hikeshi" is an unusual formulation that I found difficult to understand in the title. I would suggest to change this to e.g. "imported by" or "Hikeshi-imported nuclear HPS70" 2. The introduction would profit from a few additional citations: o Exogenously expressed classical nuclear localization signal (NLS)-tagged HSP70, which is carried into the nucleus by the importin-α/β pathway, significantly suppresses cell death of Hikeshi-depleted HeLa cells caused by heat stress. o Its transcriptional activity of HSF1 is regulated by various posttranslational modifications, including phosphorylation. 3. p5: "that Hikeshi was acquired only after the emergence ...." I'm not an expert here, but I doubt that acquisition of Hikeshi after the emergence of eukaryotic cells alone is enough evidence to implicate a function connected to the nucleus. 4. Section title p6: "upregulated expression of the HSF1-regulated gene" should be "upregulated expression of HSF1-regulated genes" 5. Were the cell lines tested for mycoplasma? In the DAPI stain in Figure 5 there are some small dots visible around the cells.  The claim of nuclear localization in 1C in response to MG132 is less convincing and the results should be modified to reflect minor localization relative to heat shock.
We added images of DAPI staining in new Figs 1A and 1B.
To describe differences between the effect of MG132 and heat stress for nuclear accumulation of HSP70, we have rewritten the text (Results: "Knockout of the Hikeshi gene induces a reduction in nuclear HSP70 under nonstressed conditions", p5, paragraph 2) as follows.
"In accordance with previous reports (Kose et al, 2012; Rahman et al, 2017), heat shock induced nuclear import of HSP70 was strongly inhibited in Hikeshi-KO cells ( Fig 1A). The addition of MG132, a proteasome inhibitor well known to induce cellular proteotoxic stress, also induced nuclear accumulation of HSP70, but to lesser extent than those of heat shock. The addition of MG132 also induced significant nucleolar accumulation of HSP70, which was also seen in cells under heat shock conditions. These MG132 induced nuclear/nucleolar accumulation of HSP70 was strongly inhibited in the Hikeshi-KO cells ( Fig 1B). The effect of Hikeshi KO on heat shock and MG132 induced nuclear/nucleolar accumulation of HSP70 was similarly seen in HeLa cells and hTERT-RPE1 cells. "

2) Fig. 2D -there is some variability in HSR derepression resulting from CRISPR knockout vs.
siRNA depletion of Hikeshi -did the authors quantify Hikeshi levels in the latter experiments by western blot? It is important to do so when using shRNA or siRNA approaches.
We quantified protein levels of Hikeshi by western blotting, and the results is shown in new Fig   S2C. The protein expression levels of Hikeshi were reduced to ~10% by siRNA treatment.
Quantitative real-time PCR analysis showed that mRNA expression levels of Hikeshi were reduced to about 5 % by siRNA treatment. In addition to this, we described a variability in HSR derepression resulting from CRISPR knockout vs. siRNA depletion of Hikeshi in Text (Results: "Knockout of the Hikeshi gene induces a reduction in nuclear HSP70 under nonstressed conditions", p7, paragraph 1) as follows.
"siRNA-mediated depletion of Hikeshi, by which protein expression levels of Hikeshi were reduced to ~10%, also showed that most of these 9 genes were upregulated under nonstressed conditions, with some exceptions (Fig S2C). We found some variability in upregulation levels of these 9 genes resulting from Hikeshi-KO cells and siHikeshi-treated cells. Unlike RNA-seq results with Hikeshi-KO cells (Fig 2C), in siHikeshi-treated cells, HSPA1L was not upregulated in RNA-seq experiment, BAG3 was not significantly upregulated in both RNA-seq and qPCR experiments, and DEDD2 was not upregulated in qPCR experiment ( Fig S2C). We presume these discrepancies due to protein levels of Hikeshi remaining in siHikeshi-treated cells." regulates the transcriptional activity of HSF1", p9, paragraph 2) as follows.
"The increased luciferase activities in the Hikeshi-KO cells were strongly suppressed by cotransfection with the NLS-Hsc70-expressing plasmid in a dose-dependent manner ( Fig 3E).
Cotransfection of non-tagged or nuclear export signal (NES)-tagged Hsc70 expressing plasmid did not suppressed the luciferase activity ( Fig S4C). These results show that HSP70 that localized in the nucleus inhibited transcriptional activation through the HSE promoter." The enhanced effect on luciferase activities of non-tagged or NES-tagged Hsc70 may indicate that cytoplasmic luciferases that expressed through the HSE promoter are stabilized by chaperone function of cytoplasmic Hsc70. Fig. 4D -The legend references Fluc protein stability in wild type or knockout cells, but the Y axis of the plot is labeled "activity". Which was measured? GFP signal or luciferase activity?

4)
Only the former reflects protein stability. The latter is a more complicated property that reflects possible changes in stability and folding. Given the minor differences shown in Fig. 4B, these considerations are important.
We measured luciferase activities after inhibition of the newly-synthesized luciferase proteins by treatment with cycloheximide, as described in Text (Results: Hikeshi-mediated nuclear-localized HSP70 functions in nuclear protein stability, p 9, paragraph 3). The Y axis of the plot is "luciferase activity" instead of GFP signal. As Reviewer's comment, we agree that luciferase activity is influenced by both protein stability and re-folding activity of chaperones.
Nevertheless, we consider that we can claim that Fluc protein "stability" is affected by nuclear Hsp70, because luciferase activity of nuclear-localized Fluc-SM, a single mutant (SM) protein that are more unstable than wild-type Fluc, was more influenced than nuclear-localized Fluc-WT in the Hikeshi-KO cells (Fig 4B). It is likely that absence of nuclear Hsp70s influence protein stability. We added words to describe this in Text (Results: Hikeshi-mediated nuclear-localized HSP70 functions in nuclear protein stability, p 9, paragraph 3 to p10, paragraph 1) as below.
"Notably, these effects were more significant with the NLS-Fluc-R188Q single mutant (SM), which tends to be more unstable than wild-type Fluc (Gupta et al, 2011). Although luciferase activity is influenced by both protein stability and re-folding activity of chaperones, our data showing that luciferase activity of nuclear-localized Fluc-SM was more influenced than that of nuclear-localized Fluc-WT in the Hikeshi-KO cells may suggest that the function of nuclear Hsp70 confers with protein stability." To avoid confusion, we changed "protein stability" to "protein activity" of legend title in Figure 4. Fig. 6C -why are heat shock genes downregulated in the knockout lines after heat shock? This result seems contrary to the previous results. Additionally, Fig. 6G should be moved to supplementary information and/or summarized in a more easily interpretable manner.

5) What is happening in
I am sorry for this confusion. HSF1-regulated genes are weakly upregulated in Hikeshi KO cells compared with WT cells under normal conditions (previous and present Fig 2C), while their heat-shock-responsive expressions (fold change, from nonstressed to heat shock conditions) are suppressed in Hikeshi KO cells compared to WT cells (previous Fig 6C). In accordance with the reviewer's suggestions, and to avoid confusions, we reorganized Figure 6, including new data of qPCR, and completely rewrote this session in Text (Results: Heat shock response is impaired in the Hikeshi-KO cells; p 11, paragraph 2 to p12, paragraph 3).
In new Fig 6, we show that the expression levels of heat-shock-responsive (HSR) genes (defined as genes whose expressions are induced for more than 2-fold at heat shock conditions relative to nonstressed conditions in WT cells) are upregulated in response to heat shock even in Hikeshi-KO cells but to lesser extent than WT cells (new Fig 6A). 9 HSF1  Thank you for these suggestions. We cited all these manuscripts in "Results" and "Discussion" regarding the HSP70/HSF1 regulatory circuit (p 8, paragraph 1; p14, paragraph 2; p15, paragraph 1). We rewrote the sentences in Text (Discussion: p 14, paragraph 2) as below.

Reviewer #2 (Comments to the Authors (Required)):
This study from an eminent laboratory is disappointing in many key aspects, the most important being that it does not directly address in any way the question of mechanism i.e. the study largely uses Hikeshi knockout cells and tries to draw conclusions about HSF1, but does not attempt to modulate HSF1 (eg. by siRNA) to try to confirm observations. Many of the results are not only overinterpreted, but also lack confirmation of the "screening" results for differential expression by qPCR, and there are no details regarding how many replicates are used, how many separate experiments were performed etc. Overall, this study is too preliminary to be considered for publication in its present form. Fig 1d but  To validate results of RNA-seq data, we performed quantitative real-time PCR analyses and added these results in new Figures (2C, 2D, S2C, S3C, S3D, S4A, S4B, and S5). Fig 2c and 2d (and Fig EV1 c and d) are representative of the RNA seq results -how many replicates were performed for RNA seq in Figure 2 and Fig EV1? As per point #2, expression data in Fig 2c and 2d and Fig EV1 c and d needs to be validated by qPCR to give confidence that the results are consistent and significant. In Figure 3 it is not clear what the replicate size is for RNA sequencing (why is there no confirmation of the differential expression presented in Fig   3c by qPCR). In short, every figure needs clear information regarding the number of replicates, number of experiments -the impression, without this, is that everything was performed only once ? And further, confirmation of "screening" results by qPCR seems essential to give weight to the observational data.

A trend in the entire paper is the lack of detail regarding replicates eg. the expression data in
We agree with these comments. We performed one RNA-seq experiment for each cell and picked up genes whose expression level change were common to two different knockout cells lines (Hikeshi KO #1 and Hikeshi KO #2 for HeLa cell, Hikeshi KO #3 and Hikeshi KO #4 for hTERT-RPE1 cell) as shown in Fig 2, Fig S2, Fig 3,  Statistical significance was determined using unpaired t-test (NS; no significance, p > 0.05).
qPCR results confirmed that the expression levels of all 9 genes, which we selected as HSF1-regulatged genes (in RNA seq data), were significantly upregulated (in new Fig 2C). In accordance with the reviewer's comments, we further performed HSF1 knockdown experiments in WT and Hikeshi-KO cells to confirm whether these selected 9 genes are regulated by HSF1.
qPCR results showed that mRNA expression levels of these genes were downregulated by HSF1 depletion, confirming that all these genes are indeed regulated by HSF1. We added these results in new Fig 2D and  On the other hand, in qPCR experiments, we obtained different results from RNA-seq data in some genes, which are described in Text (p 7p 8). For example, qPCR analysis showed that HSPA1L, but not DEDD2, was significantly upregulated in siHikeshi-treated HeLa cells, whereas RNA-seq analysis showed that DEDD2, but not HSPA1L, was upregulated (new Fig S2C). Meanwhile, RNA-seq analysis showed that mRNA expression of HSPA1L was suppressed in HeLa cells stably-expressing NLS-Hsc70, whereas qPCR analysis showed that mRNA expression of HSPA1L was not significantly suppressed in one of the two clonal cells stably-expressing NLS-Hsc70 and HeLa cells transiently-expressing NLS-Hsc70 (new Figs   S4A, B). mRNA expression levels of HSPA1L, which is known to be highly expressed in the testis, under normal conditions in HeLa and RPE1 cells is very low (RNA-seq data in new Fig   S5). This may make it difficult to assess HSPA1L expression with qPCR technique.
In summary, we conclude that results of qPCR basically support our RNA-seq data, although there are only a few differences, We have rewritten our manuscript to include these qPCR results. "predictably, many of these Hsp70-related genes were upregulated by HSF1". This is not a correct description of the result, which did not validate the role of HSF1 in the upregulation of these genes. The authors really need to perform HSF1 knockdown in Hikeshi-KO to confirm the upregulation of these specific genes is indeed HSF1 dependent.

d. The abstract is misleading when it says "depletion of Hikeshi induces a reduction in nuclear HSP70 and upregulation of the mRNA expression of genes regulated by HSF1 under nonstressed conditions". The authors have not confirmed HSF1 upregulated these genes in their experimental system, with the results essentially indirect (ie. reduced activation of the heat shock element promoter in Hikeshi KO cells). At best the authors could argue that there may be a correlation between lack of Hikeshi, and lower HSF1 activity, but if the authors performed
HSF1 knockdown in Hikeshi-KO to confirm the upregulation of these specific genes, they would be able to make claims that the effects are HSF1 dependent.
We performed HSF1 knockdown in WT and Hikeshi KO cells, and performed qPCR experiments of 9 genes that we claimed as HSF1-regulated genes. The qPCR results showed that mRNA expression levels of all 9 genes were downregulated by HSF1 depletion, indicating that mRNA expression of 9 genes are regulated by HSF1. The results are shown in new Fig 2D, and described in the Text (Results: Knockout of the Hikeshi gene induces upregulated expression of the HSF1-regulated gene under nonstressed conditions; p 7, paragraph 1). We also cited the following papers for describing HSF1 target genes (p 6, paragraph 2; p 7, paragraph 1; e. On page 8, "Therefore, dysfunction of Hikeshi, which leads to a decrease in nuclear Hsp70, causes dysregulation of HSF1 transcriptional activity" should read "Therefore, lack of Hikeshi, which leads to a decrease in nuclear Hsp70..." We rewrote "dysfunction of Hikeshi" to "lack of Hikeshi". (p 9, paragraph 2). (Fig 5b). Similarly page 10 "..KO cells significantly suppressed the apoptosis induction..."

Hikeshi downregulates OR reduces OR contributes to suppression of nuclear polyQ-induced apoptosis because we can see that caspase activity is still increased in WT cells, albeit at a lower level than in KO cells
We rewrote "suppress" to "reduce" in legend of Figure 5, and in p11 paragraph1 "Coexpression of Hikeshi (Fig 5C) or NLS-Hsc70 (Fig 5D) in the Hikeshi-KO cells significantly reduced the apoptosis induction caused by nuclear polyQ81 proteins" 5. On page 11, the authors conclude that "the heat shock response was sustained during recovery from heat shock" in the Hikeshi-KO cells because more genes that are heat shock responsive were at their peak expression levels 3 h after stress in the Hikeshi KO cell compared to WT cells. It seems much more likely that the transcriptional response to heat shock was delayed, with peak expression of heat shock responsive genes occurring 3 hours after heat stress ?
We have reanalyzed the experimental data in Fig 6. As pointed out by this reviewer, the result showed that the transcriptional response to heat shock was delayed in the Hikeshi-KO cells, and then mRNA expressions of these genes continued to increase during 3 hours after heat shock.
We therefore agree with reviewer's comment. We described results shown in new paragraph 2) including our comments "These results suggest that in the Hikeshi-KO cells, upregulation of HSR genes in response to heat shock is weakened and is sustained at HS and beyond (up to 3 hrs after recovery to normal temperature after HS)"

The authors suggest Hikeshi imports Hsp70 under non stressed conditions but have not shown binding interaction under non stressed conditions.
We previously showed that Hikeshi interacts efficiently with ATP-from HSP70 in the pulldown assay without raising temperature and mediates nuclear import of HSP70 in an in vitro transport assay without raising the temperature (Kose et al, 2012). We have stated these previous observations in the Discussion (page 13, paragraph 2). 7. The authors should acknowledge that given that many heat shock responsive genes show delayed upregulation rather than inhibited upregulation in the Hikeshi-KO cells (Fig 6d). Can the authors be sure there is not another factor compensating for loss of Hikeshi to affect transcription of these genes ?
As described above (Q5), we have removed previous Fig 6D and completely reorganized the According to reviewer's suggestion, we moved previous Fig 1A to new supplementary Fig S1, and previous first session of Results was described in the Introduction of revised manuscript (page 4, paragraph 3). Figure 3 and Thank you for this. We corrected spelling mistakes including those in legend of Figure 3 and

On page 8 the authors state Fluc is frequently used to monitor chaperone activity of Hsp70.
The authors need to supply some references here.
We added the following citations for use of Fluc to monitor chaperone activity of Hsp70 in Text

The authors go backwards and forwards between heat shock response and heat stress response -are these the same ? What is meant by the different terms ? Can the authors be clear ?
We meant heat shock as "acute heat stress", therefore, we unified to heat stress to heat shock in the revised manuscript.
In short, the current paper requires a complete overhaul in terms not only of the writing, but also requires a major body of confirmatory experimentation, including HSF1 knockdown experiments. Only in this way, can the study be considered solid or rigorous enough for Life Science Alliance.
We have confirmed major body of confirmatory experiment as described above, and revised writing as suggested by this reviewer.

Reviewer #3 (Comments to the Authors (Required)):
Comments on «Nuclear HSP70, supplied by Hikeshi, controls HSF1 activity and affects the heat  Figure 2. A detailed description of how the analysis of the RNAseq data was performed is missing. Were there replicates? What are the statistical test used to select the upregulated genes? I am no expert in mRNAseq but it seems that the information given here is insufficient and I cannot judge whether the analysis done is appropriate. As a example, the text states for figure 2D that "Consistently, siRNA-mediated depletion of Hikeshi also showed that most of these HSF1-regulated genes, except for HSPA1L, were upregulated under nonstressed conditions", however for BAG3 this increase does not look significant.
We performed one RNA-seq experiment for each cell and picked up genes whose expression levels change were similar in two different knockout cell lines. In the revised manuscript, to validate results of RNA-seq, we performed qPCR analyses (n=3 biologically independent samples) and added these results in new Figs (2C&D, 6C, S2C, S3C&D, S4A&B, S6B).
As pointed out by reviewer, qPCR analysis showed that BAG3 (and DEDD2) was not significantly upregulated in siHikeshi-treated HeLa cells, whereas significant upregulation of HSPA1L was confirmed by qPCR, but not previous RNA-seq. We presume some variability in upregulation levels of these genes resulting from Hikeshi-KO cells and siHikeshi-treated cells due to protein levels of Hikeshi remaining in siHikeshi-treated cells (We quantified protein levels of Hikeshi by western blotting, new Fig S2C). We described about it in Text (p 7, paragraphs 1) and removed previous Fig 2D to supplemental Fig S2C. Taken together, we think that qPCR results basically support our RNA-seq data, although there are only a few differences. We have updated our manuscript to include qPCR results in Text (Three sessions of Results: page 6-7, page 8, and page 12).

"Nuclear HSP70 regulates the transcriptional activity of HSF1"
• Conclusion largely supported by data presented in Figure 3. As control for Figure 3B,E and 4C, co-transfection of a Hsc70 plasmid with NES should be shown to exclude indirect effects of overexpression not related to nuclear localization. 4. "Nuclear HSP70 functions in the protein stability of nuclear luciferase monitoring proteins." • Conclusion largely supported by data presented in Figure 4. As control for Figure 4C, co-transfection of a Hsc70 plasmid with NES should be shown to exclude indirect effects of overexpression not related to nuclear localization.
As suggested by this reviewer, we performed the reporter assay using NES-tagged Hsc70.
Contrary to the inhibitory effect of NLS-Hsc70, expression of NES-tagged (and non-tagged) Hsc70 did not inhibit but rather enhanced luciferase activities in the Hikeshi-KO cells in a dose-dependent manner. We think, this result indicates that cytoplasmic luciferases expressed through the HSE promoter are stabilized by chaperone function of cytoplasmically-expressed Hsc70. We showed these results in new Fig S4C and   According to suggestions, we performed the experiment using NLS-tagged Hsc70. Expression of NLS-Hsc70, as well as Hikeshi, suppressed nuclear polyQ-induced apoptosis. We added these results in new Fig 5D, and described in Text (p11, paragraph 1).

"Heat shock response is impaired in the Hikeshi-KO cells."
• This is not entirely convincing. Figure 6C: if in unstressed conditions the levels are different, than fold change heat stress versus normal growth will be different even if the absolute expression level is identical. Is absolute induction reduced in HikeshiKO? Figure 6G would suggest that the levels are actually similar in heat stress.
• Figure 6D,E: very difficult to read this chart! The concept of "delayed genes" is rather difficult to understand for me and it is also unclear, what the delayed peak actually means. I guess it suggests that there is continued transcription that leads to accumulation of the mRNA beyond the acute heat shock period, but this does not really mean a delay in expression but rather a delay in shut-off. I'm not sure how to improve this section, but maybe the authors can come up with a more intuitive way of presenting their message.
We agree with these comments. Therefore, we completely reorganized Figure 6 including new data of qPCR. We removed previous Fig 6A,  genes at HS were lower in the Hikeshi-KO cells compared with that in WT cells (Fig 6A right   panel). HSF1-regulated genes showed similar tendency (Fig 6B and C). Therefore, we concluded that heat-shock-responsive upregulation of these HSR and HSF1-regulated genes was weakened in Hikeshi-KO cells.
As for mRNA expressions beyond HS, mRNA expressions of HSR and HSF1-regulated genes in WT cells peaked at R1.5h, and then decreased at R3h. On the other hand, mRNA expressions of their genes in two Hikeshi-KO cells gradually increased from non-HS to R3h (left panels in Fig 6A-C). Consequentially, absolute mRNA expression of these genes in Hikeshi-KO cells tend to increase gradually beyond HS at higher levels than that in WT cells (right panels in Fig 6A-C). Taken together, consistent with the reviewer's comment, we concluded that upregulation of these gene in response to heat shock was delayed and sustained (and delayed shut-off) beyond the acute heat shock period in Hikeshi-KO cells.
• 6G: In the discussion it says that in HS most cells in KO cells were lower expressed, but this is obvious only in the recovery, not in the HS conditions. This does not clearly fit with the argumentation that the peak is later, or does it? Levels are similar in HS but reduced in recovery would actually suggest faster shut off or decay?
We thank the reviewer for pointing out this. We moved previous Fig 6G to Fig S7). We rewrote the Text (Discussion: p15, paragraph 1) as follows.
"In addition, the expression levels of many heat shock protein (HSP) genes including HSF1-regulated genes in Hikeshi-KO cells were higher than those in WT cells under nonstressed conditions, while the expression levels of the same genes in Hikeshi-KO cells were lower than those in WT cells at R1.5h and R3h (Fig S7)." Further, mRNA expression pattern of the HSF1-regulated genes we analyzed and the other HSP genes, which are not highly upregulated at HS (fold change < 2), seems to be different. We need further experiments to understand the mechanism of transcriptional regulation of these HSP genes in detail.

Minor comments
1. "supplied by Hikeshi" is an unusual formulation that I found difficult to understand in the title. I would suggest to change this to e.g. "imported by" or "Hikeshi-imported nuclear HPS70" We changed the title as follow: We changed section title to "Knockout of the Hikeshi gene induces upregulated expression of HSF1-regulated genes under nonstressed conditions" (page 6). Figure 5 there are some small dots visible around the cells.

Were the cell lines tested for mycoplasma? In the DAPI stain in
We checked contamination of mycoplasma using MycoStrip (Mycoplasma Detection Kit, InvivoGen), and then confirmed that these cells were not contaminated with mycoplasma. We often saw such a DAPI staining when we transfected plasmid DNA into culture cells using FuGENE HD transfection reagent (Promega). We think that the appearance of such dots is due to experimental method using non-liposomal transfection reagents. Transfection reagent (FuGENE HD) we used in transfection is described in Materials and Methods. Thank you for submitting your revised manuscript entitled "Lack of Hikeshi activates HSF1 activity under normal conditions and disturbs the heat shock response". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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