HSF1-dependent and -independent regulation of the mammalian in vivo heat shock response and its impairment in Huntington's disease mouse models

The heat shock response (HSR) is a mechanism to cope with proteotoxic stress by inducing the expression of molecular chaperones and other heat shock response genes. The HSR is evolutionarily well conserved and has been widely studied in bacteria, cell lines and lower eukaryotic model organisms. However, mechanistic insights into the HSR in higher eukaryotes, in particular in mammals, are limited. We have developed an in vivo heat shock protocol to analyze the HSR in mice and dissected heat shock factor 1 (HSF1)-dependent and -independent pathways. Whilst the induction of proteostasis-related genes was dependent on HSF1, the regulation of circadian function related genes, indicating that the circadian clock oscillators have been reset, was independent of its presence. Furthermore, we demonstrate that the in vivo HSR is impaired in mouse models of Huntington’s disease but we were unable to corroborate the general repression of transcription that follows a heat shock in lower eukaryotes.

. Schematic depicting the comparisons of treatment and genotype in this study.
(A) Comparison of the 'absolute' values of the expression of gene of interest (GOI). Left panel: The 'absolute' levels are standardized to housekeeping genes, and groups are all normalized to the same value (usually control/vehicle treated wild type). This allows a comparison of treatment effects (levels of II against I and IV against III), as well as analysis of effects due to genotype/treatment differences (right panel: V: un-treated condition = differences purely due to genotype; VI: treated conditions = differences due to genotype/treatment). This kind of comparison is used in Figure 2B  Transcript induction of heat shock response genes and Hsf1 at 4 hours after heat shock (HS) or HSP90 inhibition (HSP990) in tibialis anterior muscle (A), liver (B) and cortex (C) of R6/2 and wild type mice at 12 week of age. Data are mean ± SEM relative to the levels of control or vehicle treated wild type animals; n ≥ 6; two-way ANOVA with Tukey post hoc test. Treatment: *p < 0.05, **p < 0.01, ***p < 0.001; genotype/treatment: # p < 0.05, ## p < 0.01, ### p < 0.001. Figure S4. The heat shock response is impaired in the R6/2 HD mouse model at the protein level.
Heat shock protein induction (HSP90, HSP70, HSP40, HSP25) at 24 hours after heat shock (HS) or HSP90 inhibition (HSP990) of R6/2 and wild type mice at 12 week of age. Data are mean ± SEM relative to the levels of control or vehicle treated wild type animals; n ≥ 6; two-way ANOVA with Tukey post hoc test. Treatment: *p < 0.05, **p < 0.01, ***p < 0.001; genotype/treatment: # p < 0.05, ## p < 0.01, ### p < 0.001. TUBA1a/b was used as a loading control.  (A) Scatter plot showing the significant log 2 fold transcriptome wide changes at 4 hours after heat shock (HS) in quadriceps femoris muscle of R6/2 and wild type mice at 12 week of age. Lanes 1 and 2 represent the significantly regulated genes through treatment with HS in R6/2 or wild type mice. Lanes 3 to 5 show the common (lane 3) and distinct (lanes 4 and 5) responses to treatment. Lane 6 compares HS treated R6/2 or wild type mice. Here, we corrected for differences due to the genotype by subtracting the log 2 fold changes of significantly different genes (genotype) from their log 2 induction value (HS). Only genes with a resulting fold change of ≥ 1.25 were considered for further analysis. (B) Chromatin mark predictions for genes shown in (A). Only significantly enriched chromatin marks (p < 0.001) were considered. We used the combined score, which is the product of the p-value with the z-score of the deviation from the expected rank, as a measure for prediction quality. Together, the average (y-axis) and the sum (circle diameter) of the combined scores are a good indicator of the confidence of the chromatin mark predictions. Figure S6. Comparison of the transcriptional response to heat shock and HSP990 in wild type mice.
(A) Scatter plot showing the significant log 2 fold transcriptome wide changes at 4 hours after HSP90 inhibition (HSP990) and heat shock treatment (HS) in quadriceps femoris muscle of wild type mice at 12 week of age. We corrected for differences due to the effects of anesthesia (control) and vehicle treatment by subtracting the log 2 fold changes of significantly different genes (vehicle vs. control) from their log 2 induction value (HS or HSP990). Only genes with a resulting fold change of ≥ 1.25 were considered for further analysis. (B) Chromatin mark predictions for genes shown in (A). Only significantly enriched chromatin marks (p < 0.001) were considered. We used the combined score, which is the product of the p-value with the z-score of the deviation from the expected rank, as a measure for prediction quality. Together, the average (y-axis) and the sum (circle diameter) of the combined scores are a good indicator of the confidence in the chromatin mark predictions. (C) Transcription factor network of responses to heat shock (HS) and HSP90 inhibition (HSP990) in wild type mice. 719 dysregulated genes were significantly higher induced by HS and 747 genes by HSP990 treatment. To predict upstream regulators, we created gene lists for these significantly regulated (up and down combined) genes for each condition and used the ENCODE transcription factor ChIP-seq database (2015) to identify the significantly enriched transcription factors (n ≤ 10 with a combined score of ≥ 5). Circle diameter is an indicator of the confidence of the predictions. Figure S7. Differential systemic response to HSP90 inhibition in Hsf1 knockout compared to wild type mice.
(A) Scatter plot showing the significant log 2 fold transcriptome wide changes at 4 hours after HSP90 inhibition (HSP990) in quadriceps femoris muscle of Hsf1 knockout (Hsf1 -/-) and wild type mice at 10-12 week of age. Lanes 1 and 2 represent the significantly regulated genes through heat shock in Hsf1 knockout and wild type mice. Lanes 3 to 5 show the common (lane 3) and distinct (lanes 4 and 5) responses to treatment. Lane 6 compares HSP990 treated Hsf1 knockout and wild type. Here, we corrected for differences due to the genotype by subtracting the log 2 fold changes of significantly different genes (genotype) from their log 2 induction value (HSP990). Only genes with a resulting fold change of ≥ 1.25 were considered for further analysis. (B) Chromatin mark predictions for genes shown in (A). Only significantly enriched chromatin marks (p < 0.001) were considered. We used the combined score, which is the product of the p-value with the z-score of the deviation from the expected rank, as a measure for prediction quality. Together, the average (y-axis) and the sum (circle diameter) of the combined scores are a good indicator of the confidence in the chromatin mark predictions. (C) Venn diagram and transcription factor network of common and distinct responses to HSP90 inhibition in Hsf1 knockout and wild type mice. Data correspond to lanes 3, 4 and 5 in (A) and (B). To predict upstream regulators, we created gene lists for significantly regulated (up and down combined) genes for each condition and used the ENCODE transcription factor ChIP-seq database (2015) to identify the significantly enriched transcription factors (n ≤ 10 with a combined score of ≥ 5). RNA polymerase II hits were filtered out. Circle diameter is an indicator of the confidence of the predictions. Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown. Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown. Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown.  Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown. 1 Can be higher induced by HSP990 than by heat shock, or not changed by HSP990, but repressed through heat shock. 2 Can be higher induced by heat shock than by HSP990, or not changed by heat shock, but repressed through HSP990. Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown. Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown. Upstream regulators were predicted using the ChIP-x Enrichment Analysis (1: ChEA) and ENCODE transcription factor ChIP-seq database 2015 (2: ENCODE). Chromatin marks were predicted using the ENCODE histone modifications database 2015. Only the top non-redundant significantly enriched terms followed by their combined score are shown.