What this is
- (HSF-1) is essential for activating the () in the nematode Caenorhabditis elegans.
- HSF-1 regulates the expression of several genes in response to heat and ER stress.
- This study explores the role of HSF-1 in enhancing cellular tolerance to ER stress, suggesting it may be a critical factor in maintaining proteostasis.
Essence
- HSF-1 is crucial for the induction of genes during heat and ER stress in C. elegans, contributing to cellular stress tolerance.
Key takeaways
- HSF-1 is required for the activation of genes upon heat shock and tunicamycin-induced ER stress in C. elegans.
- Silencing HSF-1 significantly reduces the expression of genes, indicating its vital role in the cellular response to stress.
- Experiments show that HSF-1 activity enhances survival during ER stress, suggesting its potential as a target for improving stress resilience.
Caveats
- The study primarily focuses on C. elegans, so findings may not fully translate to other organisms, including humans.
- Variability in survival outcomes across trials suggests that additional factors may influence the relationship between HSF-1 and ER stress tolerance.
Definitions
- Unfolded Protein Response (UPR): A cellular stress response activated by the accumulation of unfolded proteins in the endoplasmic reticulum, aimed at restoring proteostasis.
- Heat Shock Factor-1 (HSF-1): A transcription factor that regulates the expression of heat shock proteins in response to stress, playing a key role in cellular protection.
AI simplified
Introduction
Proteotoxic stress induces conserved cell protection mechanisms such as the heat shock response (HSR) and the unfolded protein response of the endoplasmic reticulum (UPRER) to maintain proteostasis1. During the HSR, the guardian of cytoplasmic proteostasis, genes encoding heat shock proteins (HSPs) are upregulated by the heat shock transcription factor HSF12–4. HSF1 act through the heat shock responsive element (HSE), which is a highly conserved 5’ regulatory cis-element consisting of at least three consecutive inverted repeats: TTCnnGAAnnTTC, (“n” denotes any nucleotide), found in the proximal promoter regions of genes regulated by HSFs5. The upregulated molecular chaperones restore the native spatial structure of cytoplasmic proteins or prevent their aggregation and target them for degradation6.
The ER safeguards proteostasis through mechanisms that are distinct from those operating in the cytoplasm. In the ER, newly synthesized membrane or secretory proteins undergo several posttranslational modifications. The proper translation and folding of these proteins is ensured by complex quality control mechanisms. Glucose starvation, inhibition of protein glycosylation, disturbance of Ca2+ homeostasis and proteotoxic stresses including heat shock cause the accumulation of unfolded proteins in the ER (ER stress) and results in the activation of a set of pathways called the unfolded protein response of the ER (UPRER)7. The UPRER is a complex stress response involving the upregulation of genes that encode ER-resident chaperones, the acceleration of lipid synthesis and protein secretion, and the inhibition of protein synthesis. These processes help restore proteostasis in the ER8. The importance of this cellular stress response is underlined by the fact that UPRER dysfunction (deficiency or hyperactivity) has been implicated in aging and the incidence of various age-associated diseases, such as neurodegenerative disorders, diabetes, heart disease and cancer9.
As in vertebrates, the UPRER in nematodes can be activated by three parallel intracellular signaling pathways10–12. Upon ER stress the transcription factor XBP-1 (X-box-binding protein 1) is activated by IRE-1 (inositol-requiring protein 1). In parallel, PEK-1, the orthologue of PERK (protein kinase RNA-like endoplasmic reticulum kinase), activates the transcription factor ATF-4 (activating transcription factor 4). The third pathway relies on the activity of ATF-6, the orthologue of activating transcription factor 6. The transcription factors XBP-1, ATF-4 and ATF-6 activate ERAD (Endoplasmic Reticulum Associated Degradation) components, genes regulating lipid synthesis, and upregulate ER resident chaperones, such as the Ig-binding protein BiP ortholog HSP-4.
The basic molecular machineries of the HSR and UPRER have been elucidated, but how the UPRER interacts with other cellular stress response pathways to maintain cellular proteostasis remain a subject of ongoing research. High temperatures affect all compartments of the cell leading to the formation of unfolded proteins in the cytosol as well as in the ER1,13. It is therefore plausible that the function of compartment-specific stress responses, such as the UPRER and HSR, may co-operate to overcome proteotoxicity. A handful of studies has investigated the relationship between HSF1 and UPRER in yeast, invertebrates and human cell lines so far13–18. Nevertheless, the present study is pioneering in the elucidation of this relationship in a multicellular animal species, the nematode Caenorhabditis elegans. Here, we show that upon heat and ER stresses HSF-1 is required for the activation of UPRER in C. elegans, and this contributes to the survival of the animal during ER stress. We also show that UPRER genes are upregulated by HSF1 upon heat- and tunicamycin-induced ER stress in human cell lines. These findings suggest a novel regulatory link between the cytoplasmic HSR and UPRER which is mediated by HSF1.
Results
Several UPRgenes are upregulated by HSF-1 upon heat shock in ER C. elegans
To test whether HSF-1 induces the expression of UPRER genes upon heat shock, we compared two sets of HSF-1 regulated genes19,20 with a published gene set containing differentially expressed genes (DEGs) that were upregulated by tunicamycin-induced ER stress, and this was dependent on the activity of UPRER regulators ATF-6, IRE-1 or PEK-121. Our analysis revealed that several genes encoding key chaperones associated with the UPRER are induced upon heat shock in an HSF-1 dependent manner (Figs. 1a, b, S1a, b and Table S1). These include orthologues of HSPA5 (BiP), hsp-3 and hsp-4, genes encoding protein disulfide isomerases (pdi-1 and pdi-2) and the glutathione transferase gene gst-1. Two of these genes, hsp-4 and gst-1, were present in both overlaps. Of note, we identified some of these genes as being bound by HSF-1, using the Gene Transcription Regulation Database22, such as gst-1, hsp-3, hsp-4, cdr-4, and pdi-6. Moreover, we identified putative HSEs in the promoter (2000 bp upstream) of HSF-1 target genes induced by tunicamycin treatment, using FIMO (Find Individual Motif Occurrences) from the MEME Suite23. These genes are listed in Supplementary Table S1. Some of these HSEs were conserved in closely related Caenorhabditis species, including hsp-4, pdi-2, pdi-6, sdz-35, and cdr-4, which suggests that HSF-1 may directly regulate the expression of certain UPRER genes in C. elegans. HSP-4, the ortholog of the Hsp70 family protein HSPA5 (BiP) is a key chaperone of the ER12 and used widely to monitor the activity of UPRER in C. elegans24,25. We analyzed the heat shock-induced expression of an hsp-4p::gfp reporter (zcIs4) in wild type animals and also when HSF-1 was depleted by RNAi. Intriguingly, silencing of hsf-1 significantly attenuated the activation of the reporter upon heat shock (35 °C for one hour), followed by 3, 6, or 24 h of recovery at 20 °C (Figs. 1c, d and S1c, d). Next, we depleted HSF-1 in somatic tissues, using an auxin-inducible degron (AID) system26 and confirmed that heat shock-induced expression of hsp-4p::gfp reporter requires HSF-1 activity (Fig. S1e, f). Altogether, these results indicate that HSF-1 activity significantly contributes to the induction of UPRER genes upon heat shock in C. elegans.
In human cell lines, the HSF1-regulated chaperone Hsp90 (HSPC1) has been implicated in controlling the UPRER by stabilizing the cytosolic domain of IRE127. Therefore, it is possible that the expression of hsp-4p::gfp reporter is compromised in hsf-1(RNAi) animals because decreased HSP-70 and/or HSP-90 protein levels result in IRE-1 destabilization. To address this question, we tested the effect of hsp-90 silencing on the expression of hsp-4p::gfp. We found that the expression of hsp-4p::gfp is enhanced, especially in the spermatheca and intestine in hsp-90(RNAi) animals (Fig. 1e, f). This result suggests that IRE1 is not destabilized by HSP-90. Moreover, since HSP-90 is a known negative regulator of HSF-1 activity28, this result supports that knockdown of hsp-90 enhances hsp-4 expression through HSF-1.
HSF-1 is required for the induction of UPRgenes upon heat shock. () Several genes upregulated by HSF-1 following heat stress are also upregulated upon tunicamycin induced ER stress (RF: representation factor). () Diagram showing the relative levels of UPRgenes upregulated by HSF-1 following heat stress (ns: not significant). () Representative fluorescent images showing that the expression of UPRreporteris induced by HSF-1 upon heat shock. () Quantification of thereporter expression in wild-type andanimals under normal conditions and upon heat shock (35 °C). Three replicates of at least 30 animals per strain/trial were analyzed. Data points represent the fluorescent intensity of individual animals, p values were determined using two-way ANOVA with Tukey’s multiple comparisons test; * =< 0.05, ** =< 0.01, *** =< 0.001; error bars represent ± SD.) () Representative fluorescent images showing that the silencing ofinduces the expression of UPRreporter. () Quantification of thereporter expression in wild-type and(RNAi) animals under normal conditions and upon heat shock (35 °C). Three replicates of at least 30 animals per strain/trial were analyzed. Data points represent the fluorescent intensity of individual animals, p values were determined using two-way ANOVA with Tukey’s multiple comparisons test; * =< 0.05, ** =< 0.01, *** =< 0.001; error bars represent ± SD.) Source data supporting panels a and b are provided in Supplementary Table. ER ER ER ER a b c d e f hsp-4p::gfp hsp-4p::gfp hsf-1(RNAi) p p p hsp-90 hsp-4p::gfp hsp-4p::gfp hsp-90 p p p S1 Source data supporting panels d and f are provided in Supplementary Table. S2
HSF-1 is required for the activation ofchaperone upon tunicamycin-induced ER stress hsp-4/BiP
To investigate the possible role of HSF-1 in the regulation of the ER stress response, we used tunicamycin (TM) to induce proteotoxic stress specifically in the ER24. We found that both 6 and 24 h following TM treatment the expression of hsp-4p::gfp is activated robustly in the wild-type background, while in hsf-1(RNAi) animals the induction of the reporter is significantly weaker at multiple time points (Fig. 2a, b). Interestingly, hsp-4p::gfp expression increased significantly between 6 and 24 h after tunicamycin treatment in the wild-type background. However, hsf-1 silencing prevented further increases in reporter expression 6 h after TM treatment. This suggests that HSF-1 enhances the activation of hsp-4 upon TM-induced ER stress. To confirm these results, we tested the activity of the hsp-4p::gfp reporter when ER stress is induced by the RNAi knockdown of tag-335, encoding an enzyme required for the N-linked glycosylation of ER resident proteins24. Depleting HSF-1 activity using auxin-inducible degradation, we found that similarly to the TM treatment, the proper induction of the hsp-4p::gfp reporter upon ER stress induced by the silencing of tag-335 requires HSF-1 activity (Supplementary Fig. S2a, b).
To support the role of HSF-1 activity in the induction of hsp-4p::gfp upon TM treatment, we tested the effect of silencing hsp-90 on the induction of the hsp-4p::gfp reporter upon TM treatment. Similar to our observations in heat-shocked animals, we found that reporter expression increases in untreated (DMSO) and after 24 h of TM treatment when hsp-90 is silenced (Fig. 2c, d).
To determine whether HSF-1 can activate hsp-4 independently of the IRE-1/XBP-1 branch of the UPRER in C. elegans, we assessed hsp-4 induction in response to heat shock and tunicamycin treatment in an xbp-1 loss-of-function mutant background (Supplementary Fig. S2c, d). Consistent with previous findings21, our results showed that hsp-4 activation is abolished in the absence of XBP-1 (Supplementary Fig. S2c, d), confirming that XBP-1 activity is crucial for the induction of hsp-4 under both stress conditions.
HSF-1 functions to alleviate ER-stress in. () Silencingreducesinduction under ER stress. Representative fluorescence images ofexpression in control RNAi and(RNAi) animals under control conditions (DMSO) or upon tunicamycin treatment. () Quantification of thereporter expression in control andanimals under normal conditions and upon-tunicamycin induced ER stress. Three replicates of at least 30 animals per strain/trial were analyzed. Data points represent the fluorescent intensity of individual animals, p values were determined using two-way ANOVA with Tukey’s multiple comparisons test; * =< 0.05, ** =< 0.01, *** =< 0.001; error bars represent ± SD). () Representative fluorescent images showing that the silencing ofinduces the expression of UPRreporter. () Quantification of thereporter expression in wild-type andanimals under normal conditions and upon tunicamycin treatment. Three replicates of at least 30 animals per strain/trial were analyzed. Data points represent the fluorescent intensity of individual animals, p values were determined using two-way ANOVA with Tukey’s multiple comparisons test; * =< 0.05, ** =< 0.01, *** =< 0.001; error bars represent ± SD.) () HSF-1 is required for survival under ER stress. Survival curves of wild-type control (EV),,control (EV),animals treated with tunicamycin (50 µg/ml). () Hormetic heat shock-induced ER stress resistance requires HSF-1 activity. Survival curves of RNAi control (EV) and(RNAi) animals in control conditions and after hermetic heat shock treated with tunicamycin (50 µg/ml). In the case ofand, three replicates of at least 30 animals per strain/trial were analyzed. Comparison of survival curves were performed using Log-rank (Mantel-Cox) test (** =< 0.01, *** =< 0.001). Source data supporting panels b and d are provided in Supplementary Table. Source data supporting panels e and f are provided in Supplementary Table. Caenorhabditis elegans hsf-1 hsp-4 hsp-4p::gfp hsf-1 hsp-4p::gfp hsf-1(RNAi) p p p hsp-90 hsp-4p::gfp hsp-4p::gfp hsp-90(RNAi) p p p hsf-1(RNAi) ire-1(ok799) ire-1(ok799); hsf-1(RNAi) hsf-1 p p a b c d e f e f ER S2 S3
HSF-1 alleviates ER-stress in C. elegans
Since the transcriptional activation of hsp-4 upon ER stress was weaker in HSF-1 depleted animals (Fig. 1c, d), we were curious whether HSF-1 activity contributes to the survival of nematodes during ER stress. To address this question, we performed ER stress tolerance assays. According to our results, the survival rate of hsf-1(RNAi) and ire-1(ok799) mutant nematodes was significantly lower than that of wild-type animals (Fig. 2e, and Supplementary Fig. S3a). Interestingly, the tunicamycin tolerance of ire-1 (ok799); hsf-1(RNAi) animals was not significantly different from that of ire-1(ok799) mutant animals, suggesting that HSF-1 and IRE-1 function in the same pathway to regulate ER stress tolerance (Fig. 3e and Supplementary Fig. S3 a). Similarly, hsf-1(sy441) mutants exhibited reduced tolerance to tunicamycin (TM) compared to wild-type animals. However, the difference in survival between tunicamycin-treated ire-1(ok799) single mutants and ire-1(ok799); hsf-1(sy441) double mutants was inconsistent across the three trials (Supplementary Fig. S4).
To investigate whether HSF-1 actively enhances tolerance to ER stress, we applied a hormetic heat-shock to wild-type and hsf-1(RNAi) animals for one hour at 35 °C on the first day of adulthood. After 16 h of recovery at 20 °C, control and heat-shocked animals were placed onto plates containing tunicamycin. Results showed that hormetic heat shock enhances the tolerance of control animals to ER stress, but weakens their resistance to tunicamycin when HSF-1 is silenced (Fig. 2f and Supplementary Fig. S3b). These results suggest that the beneficial effect of hormetic heat shock on survival depends on HSF-1 activity.
![HSF1 is required for the proper induction of UPRgenes in human cell lines. () and () Venn diagrams showing that genes upregulated upon tunicamycin treatmentsignificantly overlap (RF: 1.86;< 2.35e−06 and RF: 1.75;< 3.83e−13 respectively) with genes directly activated by HSF1 in human cell lines. () Table showing a selected list of HSF1 target genes that are also upregulated by tunicamycin treatment based on two separate published RNAseq experiments. () Activation ofsplicing and induction of() mRNA levels depends on HSF1 upon heat shock in HT-1080 cell line. () Activation ofsplicing and induction of() mRNA levels depends on HSF1 upon tunicamycin induced ER stress in HT-1080 cell line. Data of three experiments were plotted with error bars indicating the SD. p values were determined by two-tailed, unpaired t-test; * =< 0.05, ** =< 0.01, *** =< 0.001, ns = not significant; SD = standard deviation. ER , [29] [30] [28] , [29] [30] a b c d e p p XBP1 HSPA5 BIP XBP1 HSPA5 BIP p p p Source data underlying panelsare provided in Supplementary TableSource data underlying panelsandare provided in Supplementary Table. a–c d e S5 S4](https://europepmc.org/articles/PMC13018630/bin/41598_2026_43060_Fig3_HTML.jpg.jpg "Click to view full size")
HSF1 is required for the proper induction of UPRgenes in human cell lines. () and () Venn diagrams showing that genes upregulated upon tunicamycin treatmentsignificantly overlap (RF: 1.86;< 2.35e−06 and RF: 1.75;< 3.83e−13 respectively) with genes directly activated by HSF1 in human cell lines. () Table showing a selected list of HSF1 target genes that are also upregulated by tunicamycin treatment based on two separate published RNAseq experiments. () Activation ofsplicing and induction of() mRNA levels depends on HSF1 upon heat shock in HT-1080 cell line. () Activation ofsplicing and induction of() mRNA levels depends on HSF1 upon tunicamycin induced ER stress in HT-1080 cell line. Data of three experiments were plotted with error bars indicating the SD. p values were determined by two-tailed, unpaired t-test; * =< 0.05, ** =< 0.01, *** =< 0.001, ns = not significant; SD = standard deviation. ER , [29] [30] [28] , [29] [30] a b c d e p p XBP1 HSPA5 BIP XBP1 HSPA5 BIP p p p Source data underlying panelsare provided in Supplementary TableSource data underlying panelsandare provided in Supplementary Table. a–c d e S5 S4
HSF1 controls the activation of the unfolded protein response in human cells under heat and tunicamycin-induced stress
To investigate whether the regulatory interaction between HSF1 and UPRER is evolutionarily conserved, we first examined whether tunicamycin-induced genes are direct targets of HSF1. Using HSF1base (https://hsf1base.org/↗)29 and transcriptomic data from human cell lines30,31, we found a significant overlap between genes upregulated by tunicamycin and known HSF1 target genes (Fig. 3a, b, and Supplementary Tables S5).
Among the 60 HSF1 target genes also induced by tunicamycin treatment30, genes involved in protein folding within the ER were significantly overrepresented (Supplementary Fig. S5). These genes encode ER chaperones such as CALR (Calreticulin), PDIA3 (Protein Disulfide Isomerase Family A Member 3), and SERP1 (Stress-Associated Endoplasmic Reticulum Protein 1), as well as key UPRER-associated transcription factors including CHOP (C/EBP Homologous Protein), XBP1 (X-box Binding Protein 1), and ATF3 (Activating Transcription Factor 3) (Fig. 3c).
To further investigate a possible role of HSF1 in UPRER regulation, we performed quantitative RT-PCR to measure mRNA levels of spliced XBP1 (XBP1s) and HSPA5 (BiP) following heat shock at 42 °C for 3h in HT-1080 and HEK-293T human cell lines, with or without treatment with KRIBB11, a well-characterized HSF1 inhibitor32. Effective inhibition of HSF1 was confirmed by the suppression of HSPA1 (HSP70) mRNA induction in KRIBB11-treated cells post–heat shock (Fig. 3d). In HT-1080 cells, heat shock led to a marked increase in XBP1s and HSPA5 mRNA levels, both of which were significantly reduced upon KRIBB11 treatment, indicating that HSF1 contributes to UPRER gene induction under heat stress (Fig. 3d). In contrast, in HEK-293T cells exposed to the same conditions, HSPA5 mRNA levels did not increase, whereas XBP1s expression was elevated in an HSF1-dependent manner. However, the induction of XBP1s was not statistically significant (two-way ANOVA test, p = 0.132) (Supplementary Fig. S6a). To strengthen these findings, we tested whether KRIBB11 also suppresses the heat-stress-induced expression of genes independent of HSF1. The Ras-related glycolysis inhibitor and calcium channel regulator gene (RRAD) has been shown to be robustly upregulated upon heat shock, independently of HSF1, in MCF7 breast adenocarcinoma cells33. We found that RRAD mRNA levels are moderately induced in control and KRIBB11-treated HT-1080 and HEK-293T cell lines upon heat shock (Supplementary Fig. S6 c). We next examined whether HSF1 is also required for tunicamycin-induced UPRER activation in human cell lines, as observed in C. elegans. In both HT-1080 and HEK-293T cells, tunicamycin treatment led to increased expression of XBP1s and HSPA5, which was significantly attenuated by HSF1 inhibition (Fig. 3e and Supplementary Fig. S6 b).We tested for the presence of a putative heat shock element (HSE) in the promoter region (2000 bp upstream) of HSF1 target genes induced by tunicamycin treatment using FIMO (Supplementary Table S5). However, we did not find well-defined HSE sequences in the promoters of XBP1, HSPA5, or other canonical UPR genes. Instead, putative HSEs were present in the promoters of several genes induced by ER stress or identified as modulators of ER stress signaling, such as ER membrane selenoprotein K (SELK)34, cellular-flice-like inhibitory protein (c-FLIP)35, cellular inhibitor of apoptosis protein 1 (cIAP1)36, growth differentiation factor 15 (GDF15)37, and leucine-rich repeat-containing protein 59 (LRRC59)38.
These results suggest that HSF1 contributes to the induction of UPRER genes in response to both heat shock and tunicamycin in human cell lines, raising the possibility of an evolutionarily conserved regulatory link between HSF1 and the UPRER.
Discussion
In this work, we showed that activity of HSF-1, the master regulator of the HSR, is required for the proper induction of the ER resident chaperone HSP-4/BiP upon heat stress and tunicamycin-induced ER stress in C. elegans (Figs. 1 and 2a, b). We observed that the silencing of hsp-90, a well-known inhibitor of HSF-1 activity, led to an enhancement of HSP-4/BiP expression in control, heat-shocked, and tunicamycin-treated C. elegans (Figs. 1e and f and 2c and d). We also found that reduced HSF-1 activity impairs basal tolerance to ER stress (Fig. 2e). Moreover, HSF-1 is essential for the enhanced ER stress resistance induced by hormetic heat shock (Fig. 2f). Our analysis of published transcriptomic data indicated that HSF1 target genes overlap with UPRER genes in human cells (Fig. 3a–c and Supplementary Fig. S5). We also found evidence that HSF1 affects the stress-induced activation of UPRER in two human cell lines (Fig. 3d, e and Supplementary Fig. S6).
A regulatory relationship between HSF1 and UPRER has been proposed earlier in several organisms, such as yeast, C. elegans, as well as in murine and human cell lines13,15–17,39. In C. elegans, it has been shown that heat stress leads to the upregulation of hsp-4 in one-day-old nematodes25,40. However, whether HSF-1 has a role in the activation of UPRER was not addressed in these studies. The impact of HSF-1 on ER stress tolerance was also reported by Howard et al.16. This research identified that HSF-1 is essential for the elevated tunicamycin tolerance triggered by the depletion of the eukaryotic translation initiation factor 4G (IFG-1/eIF4G). In nematodes, inhibition of eIF4G/IFG-1 results in decreased protein synthesis41, suggesting that in this context, HSF-1 promotes ER stress resistance through mechanisms other than chaperone induction, most likely by suppressing translation. In contrast, the findings that hormetic heat stress enhances tunicamycin tolerance (Fig. 2f) and induces the expression of hsp-4 and other UPRER genes in an HSF-1-dependent manner (Figs. 1 and 2) as well as the presence of putative HSEs in the promoters of several UPRER genes (e.g. hsp-4) indicate that HSF-1 can also promote ER stress resistance by directly or indirectly upregulating ER chaperones.
In human cell lines, HSP90 has been shown to stabilize the cytoplasmic domains of IRE1 and PERK, thereby attenuates the UPRER27. Since hsp-90 is a well-known target of HSF-1, it is conceivable that depleting HSF-1 may impair the proper induction of the UPRER upon heat- or tunicamycin-induced stress due to insufficient HSP-90 levels. However, our data showing that hsp-90 silencing alone leads to HSP4/BiP induction makes this possibility unlikely.
At the same time, HSP-90 is a negative regulator of HSF-1 activity. It stabilizes inactive HSF-1 monomers across several species42,43, which suggests that the increased hsp-4p::gfp expression in hsp-90(RNAi) animals is due to elevated HSF-1 activity. Nevertheless, this hypothesis needs to be demonstrated by testing whether the upregulation of hsp-4p::gfp depends on HSF-1, since tissue-specific downregulation of hsp-90 mRNA levels activates hsp-70 expression independently of HSF-1 through transcellular chaperone signaling44.
Together, these results suggest that in C. elegans HSF-1 supports ER stress resilience via multiple, context-dependent mechanisms by attenuating protein synthesis, and by activating chaperone gene expression during heat stress.
Some of our findings are also supported by a recent preprint45. The authors similarly report that HSF-1 is required for heat-induced UPRER activation. However, they observed that TM-induced hsp-4p::gfp expression remains unaffected upon hsf-1 knockdown by RNAi. This discrepancy may stem from methodological differences in how TM-induced reporter activation was assessed. Nevertheless, these observations further highlight the interplay between compartment-specific stress response pathways.
In yeast, constitutively active Hsf1 enhances survival under ER stress in both wild-type and UPR-deficient ire1Δ mutants through multiple pathways, including the induction of KAR2 (Karyogamy 2) the yeast ortholog of hsp-415–17. In contrast, our findings do not support a similar mechanism in C. elegans, as hsp-4 expression was not induced in xbp-1(-) mutant animals following either heat shock or tunicamycin treatment (Fig. S2c, d). These results suggest that, unlike in yeast, HSF-1 is unable to drive hsp-4 expression independently of the IRE-1/XBP-1 pathway in C. elegans.
A previous study reported that heat stress induces an atypical unfolded protein response in HEK293 human cell line13. The same study demonstrated that heat shock induces XBP1 mRNA splicing. However, the inactivation of HSF1 did not suppress this induction, but rather prolonged it. Additionally, the promoter of HSPA5 was activated in heat-shocked cells, yet the expression of dominant negative HSF1 (dnHSF1) did not abolish this induction, suggesting that heat shock-induced UPRER is not controlled by HSF1. These discrepancies can be attributed to several factors. First, the two studies employed divergent heat shock regimens (45 °C for 30 min followed by recovery versus 42 °C for 3 h without recovery at 37 °C). Second, the methods utilized to detect gene expression differed (reporter versus quantitative PCR [qPCR]). Third, the mode of HSF-1 inhibition differed between the studies (dnHSF1 versus KRIBB11). Moreover, a recent work highlights that the relationship between HSF1 and UPRER is dependent on the cellular context18. It was reported that in normal human lung cells, mild hypothermia induces HSF1, which in turn activates UPRER and promotes cell death, but this regulatory axis is lost in cancer cells, including lung adenocarcinoma. We observed that HT-1080 cancer cells were more sensitive to heat shock-induced ER stress than HEK293T cells, suggesting that the interaction between HSF1 and UPRER depends on the actual cellular context.
An intriguing question is how stress in the endoplasmic reticulum (ER) activates HSF1. In the case of heat shock-induced ER stress, HSF1 activation is expected, as elevated temperatures may cause proteotoxic stress within the ER as well as in cytoplasm. However, tunicamycin induces ER stress through a different mechanism, by inhibiting N-linked glycosylation, a critical step in glycoprotein synthesis, leading to the accumulation of misfolded proteins specifically within the ER46,47. During prolonged ER stress, misfolded or damaged proteins may be retro-translocated into the cytosol via the ER-associated degradation (ERAD) pathway, potentially overwhelming the proteasome48. This cytosolic proteotoxic burden can indirectly activate HSF1, which triggers the heat shock response. Consistent with this model, studies in yeast have shown that HSF1 overexpression enhances ER proteostasis by promoting ERAD32, and that the HSR facilitates the removal of damaged ER proteins15,17.
In summary, our results suggest that HSF-1 influences ER proteostasis through multiple mechanisms in C. elegans (Fig. 4): (1) by directly or indirectly inducing the expression of ER-resident chaperones such as HSP-4/BiP, and (2) by resolving cytosolic stress induced by the overwhelmed ERAD and ubiquitin proteasome system, and (3) inhibiting translation through a yet unknown mechanism decreasing the protein load of the ER. It must be noted, however, that although our findings shed light on the interactions between HSF1 and the UPRER, the exact mechanisms operating in different animal models await further clarification.
HSF-1 influences ER proteostasis through multiple mechanisms in. Upon proteotoxic stress, cytoplasmic chaperones such as HSP-90 are titrated away from HSF-1, which is then activated by trimerization and phosphorylation, then translocates to the nucleus, and activates the transcription of molecular chaperones to restore proteostasis. Similarly, upon ER stress, HSP-4/BIP releases IRE-1, which becomes active and splices out a retained intron from themRNA. The active form of XBP-1 transcription factor is then translated and activates the transcription of genes encoding ER-resident chaperones, such as HSP-4/BIP, which help restore protein homeostasis in the ER. Note that the PEK-1/PERK and the ATF-6/ATF6 branches of UPRactivation are not presented in the Figure. According to our model, HSF-1 may alleviate ER stress through multiple mechanisms: (1) by directly or indirectly promoting the expression of ER-resident chaperones, such as HSP-4/BiP; (2) by facilitating the resolution of cytosolic proteotoxic stress resulting from an overwhelmed ERAD pathway and ubiquitin–proteasome system; and (3) by reducing translational output via an as-yet unidentified mechanism, thereby decreasing the protein-folding load imposed on the ER. C. elegans xbp-1 ER
Methods
Caenorhabditis elegans strains and maintenance
Unless indicated, nematodes were maintained and propagated at 20 °C on nematode growth medium (NGM)-containing plates and fed with Escherichia coli OP50 bacteria. The following C. elegans strains were used in this study:
wild-type N2 Bristol isolate.
PS3551 hsf-1(sy441) I.
RB925 ire-1(ok799) II.
TTV833 hsf-1(sy441) I., ire-1 (ok799) II.
TJ375 gpIs1[hsp-16.2p::gfp].
SJ4005 zcIs4[hsp-4p::gfp].
JTL611 hsf-1(ljt3[hsf-1::degron::gfp]) I; ieSi57[eft-3p::TIR1::mRuby::unc-54 3’UTR + Cbr-unc-119(+)] II; unc-119(ed3) III.
TTV859 hsf-1(ljt3[hsf-1::degron::gfp]) I; ieSi57 [eft-3p::TIR1::mRuby::unc-54 3’UTR + Cbr-unc-119(+)] II; unc-119(ed3) III.; zcIs4[hsp-4p::gfp] V.
SJ17 xbp-1(zc12) III; zcIs4 [hsp-4::gfp] V.
RNA interference
RNA was isolated from a mixed-age population of wild-type C. elegans strain (N2), using RNAzol® RT (RN 190, Molecular Research Center). Using isolated RNA as template, cDNA was synthesized by the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific).
To generate the tag-335 RNAi clone a 1.1 kb cDNA fragment was amplified using Fw: 5′-TATAGATCTTTCCATCAATGAAGGCGCTG-3′ and Rv: 5′-TATGGTACCTTCGACGGAACATTCACAGC-3′ primers and cloned into the vector L4440 (Addgene; plasmid #1654) using BglII and KpnI restriction enzymes.
To silence hsf-1, a published RNAi clone (hsf-1 RNAi A) was used20.
Success of hsf-1 silencing was tested in each experiment by analyzing the induction of hsp-16.2p::gfp (gpIs1) 3 h following heat shock (35 °C for 30 min) in control and hsf-1(RNAi) animals. To knock down hsp-90 we used a published construct derived from Eileen Devaney49. Worms were fed from hatch with E. coli HT115 strain containing an empty vector (control) or expressing double-stranded RNA.
Tunicamycin treatment in C. elegans
Tunicamycin treatment was performed according to Bar-Ziv and colleagues24. Plates were prepared by making NGM containing 50 µg/ml of tunicamycin (Sigma-Aldrich®, T7765-5MG) dissolved in dimethyl sulfoxide (DMSO). Control plates were prepared using DMSO alone. Plates that were seeded with E. coli OP50, or HT115 strain containing control plasmid (L4440) or the given RNAi construct. 1-day‐old adult animals were transferred to tunicamycin plates or to control DMSO plates.
Auxin treatment
The natural auxin indole-3-acetic acid (IAA) was purchased from Merck (Sigma-Aldrich). A 400 mM stock solution in ethanol was prepared and was stored at 4 °C for up to 2–3 months. Auxin was diluted into the NGM agar, cooled to about 50 °C, before pouring plates. Plates were left at room temperature for 24 h to allow bacterial lawn growth. For all auxin treatments, 99.9% ethanol was used as a control. Animals were grown on normal OP50/RNAi plates and transferred to the auxin or control plates at the L4 stage of development and after 24 h they were subjected to microscopy.
Survival assays
Survival assays were performed at 20 °C. Animals were synchronized by timed egg lay. Adult worms were sterilized by 5-fluoro-2′-deoxyuridine (FUdR) in 10 µg/ml concentration. Survival of age-synchronized nematodes was scored every day from the first day of adulthood until all animals were dead (absence of touch response or pharyngeal pumping).Worms that dried out on the edge of the plates or crawled off the plate were censored from the analysis. Statistical analysis was performed using GraphPad Prism and OASIS 250. Each survival experiment was performed at least three times.
Fluorescence microscopy and quantification of GFP expression intensity
For fluorescence microscopy, the worms were synchronized by timed egg laying. Late one-day-old adults (around 80 h after the eggs were laid) were used. The worms were then heat shocked at 35 °C for one hour, then allowed to recover for three hours or left untreated at 20 °C until microscopy. Alternatively, the worms were placed onto plates containing TM or DMSO. Note that in experiments using hsf-1::degron::gfp (TTV859), we found that the GFP intensity of JTL611 was about 20% of the intensity of TTV859 containing also the zcIs4[hsp-4p::gfp] transgene. Expression of hsf-1::degron::gfp (JTL611) was not induced by heat shock, and neither auxin nor hsf-1(RNAi) significantly lowered the intensity of the GFP signal in this genetic background. Before analysis, the worms were immobilized using 100 mM sodium azide, and images were captured using a Zeiss AXIO Imager.M2 epifluorescence microscope with a given exposure time.
Cell culture and drug treatment
HT-1080 and HEK-293T human cell lines (ATCC, Manassas, VA, USA) were cultured according to ATCC guidelines. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Gibco™) supplemented with 10% FBS (Sigma-Aldrich®, St. Louis, MO, USA) in a moisturized incubator with constant supply of 5% CO2 at 37 °C. To inhibit HSF-1, KRIBB11 (Sigma-Aldrich®, 385570-10MG) dissolved in dimethyl sulfoxide (DMSO) was added to cell cultures one hour before stress treatments (heat or tunicamycin) at a final concentration of 10 µM. Control cultures received an equal volume (0.1%) of DMSO. To induce heat stress, cells were transferred to a similar incubator with a constant temperature of 42 °C for 3 h and RNA was isolated immediately without recovery time. To induce ER stress, tunicamycin (Sigma-Aldrich®, T7765-5MG) dissolved in DMSO was added to cell culture medium at the final concentration of 5 µg/mL for 6 h. Tunicamycin treated and control cells (treated with DMSO only), were then harvested for RNA isolation.
RNA isolation and quantitative real-time PCR
Quick-RNA™ Miniprep Kit (Zymo Research) was used to extract total RNA. The integrity of the RNA samples was assessed by agarose gel electrophoresis. RNA was then reverse transcribed to cDNA by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Real-time PCR was performed using Meridian Bioscience™ SensiFAST™ SYBR® No-ROX Mix 2x (Bioline Reagents Ltd). Briefly, RT-qPCR was done with 500 nM of each primer, 100 ng of cDNA, using PikoReal96 real-time PCR device (Thermo Fisher Scientific). Primers are as follows: GAPDH: 5′-TCGGAGTCAACGGATTTGGT-3′ and 5′-CGCGGAGGGAGAGAACAGTGA-3′38; HSPA1A (HSP70): 5′-AGCTGGAGCAGGTGTGTAAC-3′ and 5′-CAGCAATCTTGGAAAGGCCC-3′; XBP1 spliced (XBP1s): 5′-GCTGAGTCCGCAGCAGGT-3′ and 5′-CTGGGTCCAAGTTGTCCAGAAT-3’ and HSPA5 (BIP): 5′-TGTTCAACCAATTATCAGCAAACTC-3′ and 5′-TTCTGCTGTATCCTCTTCACCAGT-3′35.
Bioinformatic analysis
Venny 2.1 was used to construct Venn diagrams (https://bioinfogp.cnb.csic.es/tools/venny/index.html↗). Representation factors of overlapping genes and hypergeometric probability were determined using nemates.org: for ‘total number of genes’ we used 20000. Statistical over-representation tests of gene sets were performed using the PANTHER database (http://PANTHERdb.org↗, Accessed on 20 August 2025)51.
Putative HSEs were identified using FIMO (Find Individual Motif Occurrences) from the MEME Suite23. Promoter sequences (defined as − 2000 bp upstream to the annotated translation start) were extracted from the reference genome. FIMO was run with default background nucleotide frequencies. Statistical significance of motif occurrences was evaluated using the p-value calculated by FIMO based on a log-likelihood ratio scoring scheme.
Statistical analysis
Statistical significance for all assays was determined using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA), and IBM SPSS Statistics (International Business Machines Corporation, Armonk, NY, USA) statistical softwares. Statistics for survival analyses were performed using the Online Application for Survival Analysis (OASIS 2) platform50. Median, mean, and maximum lifespans were calculated by the software. Statistical significance is indicated in the Figures as *p < 0.05, **p < 0.01, and ***p < 0.001.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Supplementary Material 4 Supplementary Material 5 Supplementary Material 6