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.