Figure 6. Stress-induced small RNA resetting depends on the H3K9 methyltransferase MET-2.

(A) Targets of stress-affected small RNAs show significantly increased H3K9me2 marks. An analysis of H3K9me2 signal (based on published data from McMurchy et al., 2017). Presented is the averaged H3K9me2 signal (y-axis) of all protein coding genes (blue) and target genes of stress-affected small RNAs (red). All genes are aligned according to their Transcription Start Sites (TSS), and the regions of 500 base pairs upstream and downstream of the TSS are shown on the x-axis. Each gray line (right panel) represents the average of a random set of genes (500 iterations) equal in size to the set of target genes of stress-affected small RNAs. (B) met-2 mutant worms do not reset heritable small RNAs in response to stress. The graph displays the measured germline GFP fluorescence levels of wild-type and met-2 mutant worms (y-axis) across generations under the indicated condition (x-axis). Each dot represents the value of an individual worm. Shown are the median of each group, with box limits representing the 25th (Q1) and 75th (Q3) percentiles, notch representing a 95% confidence interval, and whiskers indicating Q1-1.5*IQR and Q3+1.5*IQR. FDR-corrected values were obtained using Dunn’s test. Not significant (ns) indicates q ≥ 0.05, (*) indicates q < 0.05, and (***) indicates q < 0.001 (see Materials and methods). (C) A model summarizing stress-induced resetting of heritable small RNAs. Small RNAs from both endogenous and exogenous sources are reset in response to stress. Resetting is mediated by the MAPK pathway, the SKN-1 transcription factor and the H3K9 methyltransferase MET-2. Endogenous small RNAs which are encoded in the genome are re-synthesized in the next generations, while small RNAs from exogenous sources and transient responses are eliminated.

Figure 6—figure supplement 1. Targets of stress-affected small RNAs show unique H3K9 marks and the H3K9 methyltransferase met-2 is required for the execution of small RNA resetting.

Refers to Figure 6. (A) Targets of stress-affected small RNAs show significantly different H3K9me2 and H3K9me3 marks. An analysis of H3K9me2 and H3K9me3 signals (based on published data from McMurchy et al., 2017). All genes are aligned according to their Transcription Start Sites (TSS), and the regions of 500 base pairs upstream and downstream of the TSS are shown on the x axis. The y axis shows the averaged signal of the H3K9me2 (left panel) and H3K9me3 (right panel) modifications as a function of distance from the TSS. The chromatin modification profile is shown for three genes sets: all protein coding (yellow), set of genes that are up-regulated in stress (blue) and set of genes that are down-regulated in stress (red). Each gray line represents the average of a random set of genes equal in size to the set of up-regulated (upper panels) or down-regulated (lower panels) genes. (B) met-2 mutants do not reset heritable silencing even when weaker RNAi inheritance responses are induced. WT and met-2 worms are exposed at the P0 generation to dsRNA-producing bacteria of varying concentrations (either OD = 2 or OD = 0.5) and examined for stress-induced resetting at the F1 generation. The graphs display the measured germline GFP fluorescence levels of WT and met-2 mutant worms (y-axis) under the indicated condition (x-axis). Each dot represents the value of an individual worm. Shown are the median of each group, with box limits representing the 25th (Q1) and 75th (Q3) percentiles, notch representing a 95% confidence interval, and whiskers indicating Q1-1.5*IQR and Q3+1.5*IQR. FDR-corrected values were obtained using Dunn’s test. Not significant (ns) indicates q ≥ 0.05, (**) indicates q < 0.01, and (***) indicates q < 0.001 (see Materials and methods).