Figure 1. Heritable RNAi responses against the oma-1 and gfp genes have different requirements for H3K9me3 methyltransferases.

(A) Scheme depicting the different requirements for H3K9 methyltransferases in RNAi inheritance responses aimed at different genes. In the parental generation, worms are exposed to RNAi by growing on plates seeded with dsRNA-producing bacteria. Next, worms are transferred to plates seeded with control bacteria (that do not express dsRNA) to lay the eggs the next generation. (left) Only worms that inherit small RNAs that silence the temperature-sensitive dominant allele of oma-1 can hatch. Heritable RNAi responses aimed against the endogenous oma-1 gene do not require H3K9me3 methyltransferases. (right) Inheritance of anti-gfp small RNAs lead to heritable silencing of the gfp transgene (Pmex-5::gfp::h2b transgene). Heritable RNAi responses aimed against the foreign gfp gene strongly depends on H3K9me3 methyltransferases. (B) Inheritance of anti-oma-1 RNAi response in H3K9me3 methyltransferase mutants. The percentage of fertile worms per replicate and generation is presented (N = 12, three biological replicates). (upper panel) RNAi inheritance dynamics in met-2;set-25;set-32;oma-1 mutants compared to oma-1 mutants. (lower panel) RNAi inheritance dynamics in set-32;oma-1 mutants compared to oma-1 mutants. (C) Inheritance of anti-gfp RNAi response in H3K9me3 methyltransferase mutants. In each generation the percentage of worms silencing a germline expressed GFP transgene is presented (N > 60, five replicates). (upper panel) RNAi inheritance dynamics in met-2;set-25;set-32 triple mutants. (lower panel) RNAi inheritance dynamics in set-32 single mutants. Error bars represent standard error of mean. *p-value<0.05, **p-value<0.005, ***p-value<0.001, ****p-value<0.0001, Two-way ANOVA, Sidak's multiple comparisons test.

Figure 1—figure supplement 1. Weak anti-gfp heritable RNAi silencing of gfp in methyltransferases mutants.

Scatter column representation of the normalized GFP germline expression levels of RNAi treated animals across generations. The GFP expression levels were normalized to the expression levels of the corresponding (same generation and repeat) untreated control animals (three independent biological repeats). Each dot represent the normalized germline GFP expression level of a specific animal (dots representing different animals with similar expression levels may be visually indistinguishable). (A) Heritable anti-gfp silencing dynamics in met-2;set-25;set-32 triple mutants (Red) and wild type animals (Blue, in total, the germlines of 1350 worms were quantified). (B) Heritable anti-gfp silencing dynamics in set-32 mutants (Red) and wild type animals (Blue, in total, the germlines of 1480 worms were quantified). Error bars represent standard deviations.

Figure 1—figure supplement 2. SET-32 is required for the strong heritable RNAi-induced silencing of gfp in met-2 mutants.

(A) Inheritance of anti-gfp RNAi response in set-32;met-2 double mutants and wild type is shown. In each generation, the percentage of worms silencing a germline expressed GFP transgene is presented (N > 60, three biological replicates). *p-value<0.05, **p-value<0.005, ***p-value<0.001, Two-way ANOVA, Sidak's multiple comparisons test. Error bars represent standard error of mean. (B) Table summarizing the genetic interaction between the H3K9 methyltransferases MET-2, SET-25 and SET-32 and their requirement for RNAi-induced H3K9me3 and heritable silencing of gfp.

Figure 1—figure supplement 3. RNAi-induced silencing of fog-2 or sup-35 is not inherited transgenerationally.

(A) Inheritance of RNAi-induced silencing of sup-35. Silencing of the sup-35 gene by anti-sup-35 RNAi allows the temperature-sensitive pha-1 mutants to develop in restrictive temperatures. The number of developed progeny per worm is indicated (three biological repeats, N = 12). Error bars represent standard error of mean. (B) Inheritance of RNAi-induced silencing of fog-2. Silencing of the fog-2 gene depletes the worm of sperm, and causes stacking of unfertilized oocytes. The average percentage of worms lacking sperm is shown (three biological repeat, N > 30 per repeat). *p-value<0.05, Two-way ANOVA, Sidak's multiple comparisons test.