Stress-dependent activation of PQM-1 orchestrates a second-wave proteostasis response for organismal survival

Abstract

Stress responses are controlled by specialized stress-responsive proteostasis transcription factors that rapidly upregulate protein quality components to re-establish protein homeostasis and safeguard survival. Here we show that the zinc finger transcription factor PQM-1 is crucial for stress survival in response to thermal and oxidative challenges. We provide mechanistic insight into the regulation of PQM-1 during stress that depends on ILS-DAF-16 signaling, as well as phosphorylation on threonine residue 268 that is located within a conserved AKT motif. Our data show that in reproductively mature adults and during well-fed conditions, PQM-1 induction requires DAF-16 and occurs during the recovery period post heat shock. Moreover, PQM-1 co-localizes with DAF-16 in the nucleus during the stress recovery phase. This regulatory dependency on DAF-16 is bypassed under dietary restriction, allowing PQM-1 to promote stress resilience independent of the ILS pathway. During both conditions, PQM-1 is crucial for the upregulation of cytosolic and endoplasmic reticulum stress response genes required for organismal recovery and stress resilience. Our transcriptional and bioinformatic analysis reveals that PQM-1 regulates a distinct set of target genes during the stress recovery phase, suggesting that PQM-1 may be involved in vital secondary wave stress response. Thus, our findings uncover a previously unrecognized mechanism of stress-dependent PQM-1 activation that integrates multiple environmental cues to ensure proteostasis and organismal survival.

Figure 1.. PQM-1 is a stress-responsive transcription factor in a ILS- and DAF-16 dependent manner.

(A) Confocal images of PQM-1::GFP expression in Day 1 adult nematodes grown at 20°C before HS (− 1hr), immediately after a 1-hour 35°C HS (0 hr) and 2 hours post HS (+ 2 hr). PQM-1::GFP is not visible in intestinal nuclei in adults during normal growth conditions or HS, but appears in the nucleus during recovery. Scale bar = 50 μm. (B) Quantification of PQM-1 nuclear and cytosolic localization before (−1hr), immediately after HS (0 hr) and recovery post HS (1 hr – 6 hr post HS). n > 20 per condition; *P<0.05. Error bars represent ± SEM. (C) Western blot analysis of PQM-1::GFP::FLAG expression before (−1hr), immediately after HS (0 hr) and recovery post HS (1 – 12 hrs), using an anti-FLAG antibody. Tubulin was used as a loading control. (D) Heat stress survival of Day 1 N2 Bristol (Wt) and pqm-1(ko) mutant nematodes during control RNAi or hsf-1 RNAi after a 6-hour HS at 35°C. Survival was scored after a 16 hr recovery period at 20°C. n = 20; three biological replicates. Bar graph represent SEM, **P < 0.01; *P<0.05 (E) PQM-1::GFP localization to intestinal nuclei in Day 1 adult control animals (strain OP201) after a 2 hour recovery post HS; and (F) PQM-1::GFP nuclear localization in daf-2 mutants after a 2-hr recovery post HS, either grown on control RNAi (EV) or daf-16 RNAi. (F &H) Quantification of GFP fluorescence intensity of PQM-1 localized to intestinal nuclei immediately after HS (0 hr) and 2 hours post HS, relative to fluorescence intensity levels before HS (−1hr) in (F) control animals and (H) daf-2 mutants. Nematodes were grown on control (EV) or daf-16 RNAi before HS treatment. (I) pqm-1 transcript expression levels of control (wt), daf-2 mutants grown on control (EV) or daf-16 RNAi before (−1hr), after HS (0hr) and 1-hour post HS (+1hr). (F, H, I). Student’s t-test was performed to calculate statistical significance of GFP fluorescence intensity or transcript expression after HS relative to levels before HS (20°C). *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not significant. (J) Heat stress survival rates of age-synchronized Day 1 adult daf-16(mu86) and pqm-1(ko) mutants in a control- or daf-2 mutant background, after a 6-hour HS at 35°C. Bargraph represents S.E.M. *P < 0.05; ***P < 0.001, ****P < 0.0001.

Figure 2.. PQM-1 and DAF-16 co-localize in the nucleus after HS

Confocal images of nuclear localization of DAF-16 (mKate2) and PQM-1 (GFP) in (A) wild type PQM-1 (Biotag::GFP::3xFLAG::PQM-1; strain DLS608) before (−1hr), immediately after HS (35°C; 0hr) or 2 hours post HS. White arrows indicate co-localization of GFP::PQM-1 and DAF-16::mKate2 at the +2-hr timepoint. Scale bar = 25 μm. (B) Schematic of the PQM-1 domain structure, highlighting the three zinc finger domains and the C-terminal phosphorylatable RERTST motif. (C) Confocal images of nuclear localization of DAF-16 (mKate2) and PQM-1 (GFP) in the pqm-1(phospho-null) mutant (Biotag::GFP::FLAG::PQM-1(T266A/S267A/T268A); strain DLS610) before (−1hr), immediately after HS (35°C; 0hr) or 2 hours post HS. White arrows indicate co-localization of GFP::PQM-1 and DAF-16::mKate2 at the +2-hr timepoint. Scale bar = 25 μm. (D) Heat stress survival rates of age-synchronized Day 1 adult pqm-1(ko) mutant and PQM-1 phospho-null mutants T268A and T266A/S267A/T268A, after a 6-hour HS at 35°C. n=3; Bargraphs represent S.E.M. *P < 0.05; ****P < 0.0001. (E) Western blot analysis PQM-1 and DAF-16 expression at 20°C and 2 hrs post HS in Day adult wildtype PQM-1 (strain DLS608) and pqm-1(phospho-null) mutant (strain DLS610) during control (EV) and daf-16 RNAi.

Figure 3.. DR promotes stress resistance via PQM-1, independent of DAF-16

A) PQM-1::GFP::FLAG in Day 1 adults is localized to intestinal nuclei after a 2-, 4-, or 24 hr period of starvation. PQM-1 is not detectable in nuclei of well-fed Day 1 adult nematodes (0 hr). (B) Western blot analysis and quantification of PQM-1::GFP::FLAG expression levels in Day 1 adults before (0 hrs) and during 2, 4, 12 and 24 hours of starvation, using an anti-FLAG antibody. Tubulin was used as a loading control. (C) PQM-1::GFP::FLAG nuclear localization in eat-2 mutants after a 2-hr recovery post HS, and grown on control (EV) or daf-16 RNAi. (D) Quantification of GFP fluorescence intensity of PQM-1 localized to intestinal nuclei immediately after HS (0 hr) and 2 hours post HS, relative to fluorescence intensity levels before HS (−1hr) in eat-2 mutants either grown on control (EV) or daf-16 RNAi. (E) pqm-1 transcript expression levels of eat-2 mutants grown on control (EV) or daf-16 RNAi before (−1hr), immediately after HS (0hr) and 1-hour post HS (+1hr). (F) Western blot analysis of PQM-1::GFP::FLAG expression levels in control (strain OP201), daf-2 and eat-2 mutants before (no HS) and 2 hours post HS and treated with control (EV) and daf-16 RNAi..

Figure 4.. PQM-1 regulates ER resident and cytosolic chaperones in response to heat stress

(A) Venn-diagram analysis of upregulated genes 2 hours post HS to identify genes dependent on the presence of PQM-1, but independent of DAF-16. The red-shaded area represents 203 genes dependent on pqm-1. (B) KEGG pathway enrichment categories of the 203 genes are shown as the −log10 transformation of the Benjamini-Hochberg corrected p-value. (C) Venn-diagram analysis of HS-upregulated genes that are directly bound by PQM-1 in L3 larvae according to previous PQM-1 ChIP-Seq analysis (Niu et al., 2011; Dowen 2019). (D) Heat stress survival rates of wild-type (N2) animals after RNAi-mediated knockdown of 13 PQM-1-dependent HS-induced genes. Statistical analysis one-way ANOVA. n=3; Related to Suppl. Figure 3F. (E-H) Transcript expression of pqm-1-dependent chaperone and stress genes hsp-1 (E), dnj-7 (F), dpy-18 (G), pek-1 (H), in control (N2), pqm-1(ko) mutant and pqm-1 (phospho-null) mutant before and after HS (1h, 35°C). n=3. Error bars represent S. E. M. Statistical analysis one-way ANOVA. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. (I) Heat stress survival rates of age-synchronized Day 1 adult daf-16(mu86) and pqm-1(ko) mutants in a control, or eat-2(ad453) mutant background, after a 6-hour HS at 35°C. (J) Heat stress survival rates of age- synchronized Day 1 adult eat-2 mutants after RNAi-mediated knockdown of hsp-4, hsp-1, dnj-7, and pek-1. n³3; one-way ANOVA. **P<0.01, ****P < 0.0001; n.s. = not significant. Error bars represent S.E.M. (K) Working model of PQM-1 regulation after heat shock in adults. Following heat stress, DAF-16 localizes to the nucleus (1) to regulate the expression of immediate stress response genes. (2) During stress recovery, 2-hours after HS, PQM-1 then accumulates into the nucleus and co-localizes with DAF-16. During dietary restriction (DR), regulation of PQM-1 expression becomes independent of DAF-16 to regulate stress survival. PQM-1 transcriptional activity depends on phosphorylation on its RERTST motif, which is (3) required for transcription of ER resident Hsp70 family chaperones (dnj-7, dpy-18, hsp-4) and cytosolic Hsp70 (hsp-1), to promote stress survival (4).