H3K9me3 is Required for Transgenerational Inheritance of Small RNAs that Target a Unique Subset of Newly Evolved Genes

In Caenorhabditis elegans nematodes, RNA interference responses can be transmitted across generations via heritable small RNAs. Heritable RNAi memory is associated with Histone-3-Lysine-9 tri-methylation marking of the targeted gene. In other organisms, maintenance of silencing responses requires a feed-forward loop between H3K9me3 and small RNAs. In C. elegans, however, we find that the effect of H3K9 methyltransferase mutants on heritable RNAi is more complex. In this paper, we show that not only that H3K9me3 is unnecessary for inheritance, the effect of this modification depends on the identity of the RNAi-targeted gene. We found an asymmetry in the requirement for H3K9me3 and the two H3K9me3 methyltransferases, SET25 and SET-32, for heritable RNAi responses: Both methyltransferases support heritable silencing of the foreign gene gfp, but are dispensable for silencing of the endogenous gene oma-1. Genome-wide examination of heritable endogenous small interfering RNAs (endo-siRNAs) revealed that the heritable endo-siRNAs which are lost in set-25 mutants target newly acquired C. elegans genes, that exhibit high levels of H3K9me3. Thus, “repressive” chromatin marks could be important specifically for heritable RNAi silencing of genes which are flagged as “foreign”, such as gfp. Introduction RNA interference (RNAi) responses are inherited in Caenorhabditis elegans nematodes across generations via heritable small RNAs (Rechavi & Lev, 2017). In worms, exposure to a number of environmental challenges, such as viral infection (Rechavi et al, 2011; Gammon et al, 2017), starvation (Rechavi et al, 2014), and heat (Klosin et al, 2017) induces heritable physiological responses that persist for multiple generations. Inheritance of such transmitted information was linked to inheritance of small RNAs and chromatin modifications, and hypothesized to protect and prepare the progeny for the environmental challenges that the ancestors met. By base-pairing with complementary mRNA sequences, small RNAs in C. elegans control the expression of thousands of genes, and protect the genome from foreign elements (Malone & Hannon, 2009; Luteijn & Ketting, 2013). Via recruitment of RNA-binding proteins, small interfering RNAs (siRNAs) can induce gene silencing also by inhibiting transcription (Castel & Martienssen, 2013). Small RNA-mediated transcription inhibition involves modification of histones, however the exact role that histone marks play in inheritance of RNAi and small RNA synthesis is under debate (Rechavi & Lev, 2017). Worms small RNAs that enter the nucleus were shown to inhibit the elongation phase of Pol II (Guang et al, 2010); In addition, nuclear small RNAs recruit histone modifiers to the target’s chromatin, resulting in deposition of histone marks such as histone H3K9-tri methylation (H3K9me3) and H3K27me3 (Gu et al, 2012; Mao et al, 2015; Lev et al, 2017). The interaction between small RNAs and repressive chromatin marks are reciprocal: in Arabidopsis thaliana (Molnar et al, 2010; Daniel Holoch et al, 2015) and Schizosaccharomyces pombe (Verdel et al, 2004; Moazed et al, 2006), small RNAs and repressive histone marks form a self-reinforcing feedforward loop, where nuclear small RNAs induce deposition of repressive histone marks, and in turn the repressive chromatin marks recruit the small RNA machinery to synthesize additional small RNAs. Whether a similar feedback operates in worms and other organisms, is unclear. In Neurospora crassa, transgene-induced small RNAs work independently of H3K9me3 (Chicas et al, 2005). In C. elegans, it was previously suggested that H3K9me is required for RNAi inheritance (Ashe et al, 2012a). However, studies from different groups have shown that the situation is more complex, and that H3K9me could be dispensable, and can even suppress heritable silencing of some targets (Kalinava et al, 2017; Lev et al, 2017; Minkina & Hunter, 2017). In C. elegans H3K9me is considered to depend mainly on the methyltransferases MET-2, SET-25, and SET-32 (Towbin et al, 2012; Kalinava et al, 2017; Spracklin et al, 2017). H3K9 methylation by MET-2 and SET-25 occurs in a step-wise fashion – after MET-2 deposits the first two methyl groups (H3K9me1/2), SET-25 can add the third methyl group (me3) (Towbin et al, 2012). In the germline, however, SET-25 is capable of tri-methylating H3K9 in a MET-2-independent manner (Bessler et al, 2010; Towbin et al, 2012). SET32-dependent H3K9me3 is at least in part independent of the activity of SET25 or MET-2 (Kalinava et al, 2017). To study the roles of H3K9me3 in the maintenance of heritable small RNAs we examined the inheritance of small RNAs in mutants defective in these histone methyltransferases. Although H3K9me3 was thought to be required for heritable RNAi (Ashe et al, 2012a; Gu et al, 2012), we found the heritable RNAiresponses are greatly potentiated in met-2 mutant background (Lev et al, 2017). Our data indicated that the enhanced strength of the RNAi responses in met-2 mutants stems from a genome-wide massive loss of different endogenous small RNA (endo-siRNAs) species. In normal circumstances, these endo-siRNAs compete with exogenously derived siRNAs over shared biosynthesis components required for small RNA production or inheritance (Lev et al, 2017). In addition, we found that the accumulated sterility (or “Mortal Germline”, Mrt phenotype) of met-2 mutants results from dysfunctional small RNA inheritance (Lev et al, 2017). However, our previous results regarding the role of H3K9me1/2 (deposited by MET-2) did not rule out the possibility that H3K9me3 is yet required for efficient heritable silencing of gfp transgenes: We found that RNAi responses in met-2 mutants nevertheless lead to marking of the target gene’s histones with a heritable H3K9me3 modification. Further, a comparison of the H3K9me3 signal on the gfp locus in different mutants has shown that anti-gfp RNAi responses were strongly inherited only in genetic backgrounds where some H3K9me3 trace could be detected (i.e, in wild type, met-2, and met-2;set25 mutants). In set-25 single mutants, where no statistically significant H3K9me3 footprint could be detected, anti-gfp RNAi was only weakly inherited. Previously, set-25 mutants were reported to be deficient in heritable RNAi responses targeting different fluorescent transgenes (Ashe et al, 2012b; Lev et al, 2017; Spracklin et al, 2017). In contrast to anti-gfp heritable RNAi responses, for which H3K9me3 is important, we detected an enhancement in the inheritance potency of anti-oma1 RNAi in set-25 mutants (Lev et al, 2017). However, in that study we did not examine whether an H3K9me3 footprint was deposited on the endogenous gene oma-1 in the set-25 background (Lev et al, 2017). The publication of a recent paper (Kalinava et al, 2017) which described strong anti-oma-1 RNAi inheritance in met-2;set-25;set-32 triple mutants, despite the absence of a detectable H3K9me3 footprint, prompted us to re-examine the inheritance of anti-gfp RNAi in this triple mutants. We hypothesized that gene-specific characteristics lead to contrasting requirements for H3K9me3 and specific methyltransferases. In this manuscript we describe an asymmetry in the requirement for H3K9me3 and specific methyltransferases for heritable RNAi responses aimed against the endogenous gene oma-1 and the foreign gene gfp. These differences led us to perform a genome-wide analysis of SET-25dependent small RNAs, which revealed that the endo-siRNAs which depend on H3K9me3 target newly acquired C. elegans genes that might be considered “foreign”, similarly to gfp.