Trans-generational inheritance of a phenotypically neutral epimutation enables descendants to reinstate an ancestral silencing episode

Phenotypic differences caused by epimutations rather than changes in DNA sequence have been described in various organisms. Epimutations are potentially adaptive if inherited across generations and might even respond to environmental challenges. Yet experimental evidence is scarce1. Examples of heritable phenotypic changes are paramutation in plants and RNA-induced epigenetic silencing (RNAe) in nematodes, in which small RNAs trigger the formation of silent epialleles that are stable across generations2-7. Consequently, RNA-directed epimutagenesis has been associated with persistent gene repression. Here, using the fission yeast Schizosaccharomyces pombe, we demonstrate that RNA-induced epimutations are stably transmitted to subsequent generations also after reactivation of the silenced gene. This enables descendants to remember and reinstate preceding silencing episodes that had occurred in their ancestors. We show that temporary expression of hairpin-derived small interfering RNAs (siRNAs) triggers RNAe that is stably propagated for at least 18 generations upon inactivation of the polymerase-associated factor 1 complex (Paf1C). In the presence of Paf1C, the silent state is lost, but resumes if Paf1C is again impaired, even in the absence of the original trigger and in later generations. Thus, in yeast RNAe installs a molecular imprint that can be inherited through meiosis with no discernible phenotypic consequences. We show that trimethylated histone H3 lysine 9 (H3K9me3) is a major constituent of this imprint, and that stable propagation thereof is coupled to RNA interference (RNAi). Our findings reveal a distinct form of epigenetic memory in which fission yeast cells acquire heritable, transcriptionally active epialleles that confer gene silencing and manifest a phenotype only in particular, recurring situations. This demonstrates that epigenetic inheritance is adaptive and could potentially constitute a strategy to cope with adverse environmental conditions. If conserved in other eukaryotes, this phenomenon will impact our comprehension of natural variation and epigenetics in evolution. To characterize the phenotypically neutral mark, F10 h- paf1-Q264Stop ade6si3 OFF cells were crossed with h+ paf1+ ade6+ ON cells, that were otherwise wild-type or mutantX (ago1D, dcr1D, rdp1D, clr4D, clrF499Y). 3 - 4 biological replicates of F11 h- paf1+ wild-type/mutantX colonies were sequenced for small RNAs (28 samples). Furthermore, ChIP-seq of H3K9me2, H3K9me3, H3K36me3, H3 total and PolII was performed in triplicates on strains with and without ade6 si3-mark (35 samples). We also performed small RNA sequencing of 61 samples to examine the de novo formation and inheritance of endogenous siRNA peaks.