Research ArticleGENETICS

N6-methyldeoxyadenine and histone methylation mediate transgenerational survival advantages induced by hormetic heat stress

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabc3026
DOI: 10.1126/sciadv.abc3026
  • Fig. 1 Longevity induced by heat stress can be inherited.

    (A to E) Transgenerational inheritance of heat shock induces a survival advantage in C. elegans. Experimental scheme (A) and life-span analyses of with or without treatment P0 (B), F1 (C), F2 (D), and F3 (E). Life span was analyzed using the Kaplan-Meier test; P values were calculated using the log-rank test, and the life-span values of the replicated tests are listed in table S1. (F) Mean life span of generations F1 to F7. Naive progeny from generation F1 to F5 of parents exposed to high temperature had extended life spans; the sixth generation returned to normal life span (mean ± SD, n ≥ 3; *P < 0.05 and ***P < 0.001; NS, not significant; Student’s t test). (G to L) Longevity induced by heat shock is transmitted through both male and female germ lines. Experimental scheme (G and J) and life-span analyses of the progeny of fathers exposed to high temperature (H and I) and the progeny of mothers exposed to high temperature (K and L) (P value by log-rank test). The experiments were repeated at least three times. Detailed life-span values are listed in table S1. WT, wild type.

  • Fig. 2 Effect of heat stress on C. elegans fitness.

    (A and B) Representative images (A) and quantification of muscle α-synuclein aggregates of α-synuclein::YFP worms with or without treatment. (C and D) GFP::LGG-1/Atg8 puncta in the hypodermal seam cells with or without treatment. Means ± SD, n ≥ 30 per condition; P value was calculated by Student’s t test; **P < 0.01 and ***P < 0.001. (E) Representative images of the nuclear localization of HLH-30::GFP in worms with or without treatment. Each experiment was repeated at least three times.

  • Fig. 3 DAF-16, DAF-12, and HSF-1 are required for transgenerational inheritance.

    (A to C) Life-span analyses of (A) hsf-1 mutants, (B) daf-12 mutants, and (C) daf-16 mutants exposed or not exposed to high temperature. (D) Experimental scheme. (E to H) P0 worms were heat shocked without RNAi exposure, and then F1 progeny were exposed to (E) control (HT115), (F) hsf-1, (G) daf-12, and (H) daf-16 RNAi to test the requirement for daf-16, daf-12, and hsf-1 in the F1 generation (P value by log-rank test). (I) Experimental scheme. (J to M) P0 animals were heat shocked, and then F1 progeny were subjected to (J) control (HT115), (K) hsf-1, (L) daf-12, and (M) daf-16 RNAi, and life span of F2 progeny were analyzed. The experiments were repeated at least three times. Detailed life-span values are listed in table S1.

  • Fig. 4 Histone H3K9me3 modifiers are required for transgenerational inheritance induced by heat stress.

    Experimental scheme (A) and transgenerational inheritance tested in (C) set-25 mutants (P0), (D) set-32 mutants (P0), (E) set-25 mutants (recovered F1 and F2), and (G) set-32 mutants (recovered F1 and F2) (P value by log-rank test). The experiment was repeated at least three times. Detailed life-span values were listed in table S1. (B) Heatmap of H3K9me3 ChIP signals approximately 5 kb upstream and downstream of regions that were differentially enriched between the heat shock and control groups. The peaks were ranked in descending order of H3K9me3 intensity within each cluster. (F and H) ChIP-qPCR of H3K9me3 and qPCR of daf-16; hsf-1 and the corresponding target genes of daf-16, hsf-1, and daf-12 in the WT animals with or without treatment (n = 3). The graph data are presented as means ± SD. Statistical analyses were conducted using two-tailed Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 DNA N6-mA mediates the transgenerational inheritance induced by heat stress.

    (A) Experimental scheme. (C) Representative dot blot analysis from three biological replicates used to analyze the global N6-mA level in genomic DNA (gDNA) in control and P0-F6 worms. Methylene blue detected DNA loading. (B and E) Transgenerational inheritance tested in (B) the damt-1 (tm6887) mutants (recovered F1 and F2) and (E) the damt-1 (gk961032) mutants (recovered F1 and F2) (P value by log-rank test). Detailed life-span values are listed in table S1. (D, F, H, and I) Dot blots analyses from heat-shocked and non–heat-shocked WT animals and (D) daf-12 mutants, (F) hsf-1 mutants, and (H and I) daf-16 mutants (means ± SD; two-tailed Student’s t test, ***P < 0.001). Experiment was repeated at least three times. Methylene blue was used as a DNA loading control. (G) Heatmap of N6-mA MeDIP signals in the heat-shocked and non–heat-shocked groups. The peaks were ranked in descending order of N6-mA intensity within each cluster. (J) Profile plot demonstrating the overlapping N6-mA peaks and H3K9me3-associated domains in the exposed and nonexposed WT animals. (K) MeDIP-qPCR analyses of N6-mA occupancy of daf-16, hsf-1, and corresponding target genes of daf-16, hsf-1, and daf-12 in the WT animals (n = 3). Statistical analyses were conducted using two-tailed Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Working model of the transgenerational inheritance of survival advantages induced by hormetic heat stress.

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