RNA interference (RNAi) responses are inherited in Caenorhabditis elegans nematodes across generations via heritable small RNAs (Alcazar et al., 2008; Buckley et al., 2012; Vastenhouw et al., 2006). In worms, exposure to a number of environmental challenges, such as viral infection (Gammon et al., 2017; Rechavi et al., 2011), starvation (Rechavi et al., 2014), heat (Klosin et al., 2017), and growth in liquid (Lev et al., 2018) 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 (Luteijn and Ketting, 2013; Malone and Hannon, 2009). Via recruitment of RNA-binding proteins, small interfering RNAs (siRNAs) can induce gene silencing also by inhibiting transcription (Castel and 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 still not entirely clear (Rechavi and Lev, 2017). In C. elegans 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 are thought to 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; Lev et al., 2017; Mao et al., 2015).

The interactions between small RNAs and repressive chromatin marks are reciprocal: in Arabidopsis thaliana (Holoch and Moazed, 2015; Molnar et al., 2010) and Schizosaccharomyces pombe (Moazed et al., 2006; Verdel et al., 2004; Hall et al., 2002) small RNAs and repressive histone marks form a self-reinforcing feed-forward 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 still under investigation. 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 (Shirayama et al., 2012). 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 and Hunter, 2017).

In C. elegans H3K9me is considered to depend mainly on the methyltransferases MET-2, SET-25, and SET-32 (Kalinava et al., 2017; Spracklin et al., 2017; Towbin et al., 2012). 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). SET-32-dependent H3K9me3 is at least in part independent of the activity of SET-25 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., 2012; Gu et al., 2012), we found the heritable RNAi-responses 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;set-25 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., 2012; Lev et al., 2017).

RNAi silencing of the endogenous oma-1 gene is also inherited transgenerationally. In contrast to anti-gfp heritable RNAi responses, for which H3K9me3 is important, we detected an enhancement in the inheritance potency of anti-oma-1 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 H3K9 methyltransferase-dependent 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.

Recently Kalinava et al. examined the heritable RNAi responses against oma-1 also in a triple mutant, lacking the three main C. elegans H3K9 methyltransferases, SET-25, SET-32 and MET-2 (Kalinava et al., 2017). The authors reported that silencing of oma-1 was independent of H3K9me3, as in these mutants RNAi responses raised against the oma-1 gene were heritable despite the lack of an H3K9me3 trace (Kalinava et al., 2017).

We successfully replicated the results of Kalinava et al., and came to the same conclusion, that the met-2;set-25;set-32 triple mutant worms inherit RNAi responses against the oma-1 gene, also when we used a different assay for inheritance (Figure 1A and Figure 1B, upper panel). Unlike Kalinava et al., which used qPCR to score for downregulation of oma-1 expression, we targeted a redundant, temperature-sensitive and dominant oma-1 allele, that in the restrictive temperatures does not allow the development of embryos unless silenced (as previously described [Alcazar et al., 2008]). Upon shifting to 20 degrees, only worms that silence the oma-1 gene in a heritable manner survive.

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In parallel we discovered, surprisingly, that in contrast to anti-oma-1 inheritance, heritable silencing of a gfp transgene was defective in the same triple mutants (Figure 1C, upper panel, p=0.0014, 2-way ANOVA). In addition, we also confirmed (Spracklin et al., 2017) that while set-32 single mutants are deficient in inheriting RNAi responses raised against the gfp transgene (Figure 1C, lower panel, p=0.0026, 2-way ANOVA), they are capable (Kalinava et al., 2017) of inheriting responses raised against oma-1 (Figure 1B, lower panel, p=0.8487, 2-way ANOVA). Previously we have shown that while set-25 mutants are defective in inheritance of anti-gfp RNAi, weak inheritance responses can still be observed (Lev et al., 2017). Similarly, we were able to detect weak inheritance responses that last at least until the F3 generation also in met-2;set-25;set-32 and set-32 mutants (Figure 1—figure supplement 1, p-value < 0.0001 for met-2;set-25;set-32 and set-32 in the F3 generation, Two-way ANOVA). Together with our previous data, which showed that set-25 is required for inheriting anti-gfp RNAi, but not anti-oma-1 RNAi (Lev et al., 2017), these results suggested that heritable RNAi requires H3K9 methyltransferases in a gene-specific manner.

The levels of RNAi-induced H3K9me3 do not explain the gene-specific requirements of methyltransferases for heritable RNAi

Histone methyltransferase mutants may affect RNAi-induced H3K9me3 levels in a gene-specific manner, thus leading to different inheritance dynamics for each gene. To test this possibility, we performed anti-H3K9me3 Chromatin Immunoprecipitation (ChIP) on F1 met-2;set-25;set-32 triple mutant progeny, that were derived from parents exposed to anti-oma-1 RNAi, anti-gfp RNAi, or untreated controls. Using qPCR we found, as was discovered before (Kalinava et al., 2017) that in met-2;set-25;set-32 triple mutants the RNAi-induced H3K9me3 signal was significantly reduced (p-value=0.0007 and 0.0009, Two-way ANOVA, for gfp and oma-1, respectively). Importantly, this was true for both the oma-1 and gfp loci (Figure 2A). Interestingly, in naive wild-type animals, that were not treated with RNAi, the levels of H3K9me3 on gfp were significantly higher than on oma-1 (Figure 2B, p-value = 0.0039, Two-Way ANOVA) and an additional germline-expressed gene dpy-28 (Figure 2B, p-value = 0.0176, student's t-test). We discuss the possible contribution of this RNAi-independent H3K9me3 signal below. Regardless, as no differences can be found in the RNAi-induced fold changes in H3K9me3 levels between gfp and oma-1 (Figure 2A), the levels of RNAi-induced H3K9me3 cannot explain the gene-specific requirements of methyltransferases for heritable RNAi.

Figure 2

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H3K9me3 methyltransferases are required for the biogenesis of a specific class of endo-siRNAs

Certain germline small RNAs have evolved to confer immunity against foreign genetic elements, while sparing endogenous genes (Malone and Hannon, 2009). The different requirements for particular methyltransferases and H3K9me3 for heritable silencing of gfp and oma-1 may be connected to the fact that gfp is a ‘foreign’ gene, while oma-1 is an endogenous gene. We previously found that exogenous siRNAs that target gfp are lost in set-25 mutants, and hypothesized that endo-siRNAs that target other ‘foreign’ genes would be likewise affected. Therefore, we re-analyzed our previously published small RNA sequencing data, obtained from set-25 mutants (Lev et al., 2017). However, among the targets of these differentially expressed endo-siRNAs, we could not detect striking changes (fold change >1.2) in endo-siRNAs that target transposons and repetitive elements in set-25 mutants (Figure 3A, left panel). In contrast, a subset of endo-siRNAs that target 279 different protein-coding genes was found to exhibit significant changes in set-25 mutants (adj.p <0.1, DESeq2 Figure 3A, right panel). To understand why these small RNAs are uniquely affected by SET-25, we characterized this group and the endo-siRNAs that target them. To compare the endo-siRNA pools that depend on these two H3K9 tri-methyltrasferases, we re-analyzed the recently published small RNA-seq data obtained from set-32 mutants (Kalinava et al., 2018).

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Since in set-25 the loss of exogenous siRNAs coincided with the loss of heritable RNAi-induced H3K9me3 (Lev et al., 2017), we first tested whether genes that were differentially targeted by endo-siRNAs in set-25 mutants were also marked by H3K9me3. By examining publicly available H3K9me3 data (McMurchy et al., 2017), we found that the 151 genes that lost the endo-siRNAs that target them in set-25 mutants were robustly marked by H3K9me3 in wild type animals (Figure 3B). We also found that in contrast, the 128 genes that had increased endo-siRNA levels that target them in set-25 and mutants were not significantly marked by H3K9me3 (Figure 3—figure supplement 1A). By analyzing an available mRNA-seq dataset (Klosin et al., 2017), we also found a significant enrichment for genes that were upregulated (at the mRNA level) in set-25 mutants amongst the list of SET-25-dependent endo-siRNA targets (1.93-fold enrichment, 18/151 genes, p-value=0.006). This suggests that endo-siRNAs that depend on SET-25 silence targeted gene. Recently, Kalinava et al. sequenced endo-siRNAs from set-32 mutants (Kalinava et al., 2018). Our analysis show that the 337 genes that had reduced levels of endo-siRNAs (fold change >2) in set-32 mutants, were also significantly marked by H3K9me3 (Figure 3B). As expected, these genes showed lower levels of H3K9me3 in set-32 mutants (Figure 3—figure supplement 1B), and genes having increased levels of endo-siRNAs were not significantly marked by H3K9me3 (Figure 3—figure supplement 1A). Together, these results support the hypothesis that H3K9me3 methyltransferases directly support the biogenesis of silencing endo-siRNAs by tri-methylating the H3K9 histones of the endo-siRNAs targeted genes.

Next we examined whether genes that display altered endo-siRNAs levels in set-25 and set-32 mutants are expressed in specific tissues. Genes that had significantly reduced levels of endo-siRNAs targeting them in set-25 or in set-32 mutants exhibited significant, but modest, enrichment for expression in the germline (Figure 3C and Figure 3—figure supplement 1C). No significant enrichment was found for other tissues (Figure 3C and Figure 3—figure supplement 1C).

To identify the small RNA pathways which are affected by set-25 and set-32, we tested whether the differentially expressed endo-siRNAs depend on particular argonautes, or associate with specific biosynthesis or functional pathways (Figure 3D). It was previously suggested that the CSR-1 argonaute carries heritable endo-siRNAs that mark endogenous genes (Claycomb et al., 2009), while the HRDE-1 argonaute carries heritable endo-siRNAs that silence foreign or aberrant elements, whose expression could be deleterious, such as transposons (Luteijn et al., 2012; Rechavi, 2014; Shirayama et al., 2012). A strong and significant enrichment (Figure 3D and Figure 3—figure supplement 1C) was found for endo-siRNAs which are carried in the germline by the argonautes WAGO-1 (Gu et al., 2009) and HRDE-1, which is required for inheritance of exogenous siRNAs (Buckley et al., 2012). Both argonautes were found to be involved in gene silencing (Buckley et al., 2012; Gu et al., 2009). Nevertheless, some of the targets of HRDE-1-bound endo-siRNAs are expressed in the germline (Figure 3—figure supplement 2A). This may explain the concurrent enrichment for both germline-expressed genes and targets of HRDE-1-bound endo-siRNAs amongst the gene targets of endo-siRNAs that depend on SET-25 or SET-32. A significant enrichment was also found for Mutator pathway small RNAs (Zhang et al., 2011), ERGO-1-dependent small RNAs, and putative piRNA targeted genes (Bagijn et al., 2012). On the contrary, a significant depletion was found for genes known to be targeted by CSR-1-carried small RNAs, a pathway that was suggested to support the expression of targeted genes (Claycomb et al., 2009; Shen et al., 2018). The helicase EMB-4 (Akay et al., 2017; Tyc et al., 2017) was shown to preferably bind introns of genes targeted by CSR-1; We could not detect a significant enrichment for genes whose introns are bound by EMB-4 (fold change = 1.07 and 0.79, p-value=0.26 and 0.002, for endo-siRNAs dependent on SET-25 or SET-32, respectively). All together, these results suggest that H3K9 methyltransferases are required for the maintenance of a specific sub-class of HRDE-1 and WAGO-1 small RNAs, that are associated with the Mutator and piRNA pathways, and that target protein-coding genes (Figure 3—figure supplement 2B).

Endo-siRNAs that depend on H3K9me3 methyltransferases target a distinctive subset of newly evolved genes

What distinguishes the target genes of endo-siRNAs that depend on SET-25 and SET-32 methyltransferases? It was recently found that periodic A/T (PATC) sequences can shield germline genes from piRNA-induced silencing and allow germline expression of genes in H3K9me3-rich genomic regions (Frøkjær-Jensen et al., 2016; Zhang et al., 2018). Fittingly, we found that genes targeted by SET-25-dependent and SET-32-dependent endo-siRNAs exhibit a moderate (~9–13% in median values) but significant reduction in PATC density compared to all protein coding genes (Figure 4A, p-value = 0.0026 and 0.0011 for SET-25 and SET-32, respectively). This feature is not general for genes targeted by WAGOs (Worm-specific Argonautes, HRDE-1, WAGO-1 and ERGO-1) associated endo-siRNAs, since these targeted genes have a higher PATC density (Figure 4A, 10% increase in average values, p-value=0.034). In addition, genes targeted by endo-siRNAs that are increased in set-25 or set-32 mutants exhibit significantly increased PATC density (Figure 4—figure supplement 1A). However, we posit that this feature is unlikely to be sufficient for distinguishing between oma-1 and gfp, since the oma-1 gene has a very low PATC density (Figure 4—figure supplement 1B).

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The lists of genes which are targeted by SET-25- and SET-32-dependent endo-siRNAs were enriched for genes targeted by ERGO-1-dependent endo-siRNAs (Figure 3D). Many of the genes that are targeted by ERGO-1-bound endo-siRNAs are duplicated genes (Fischer et al., 2011; Vasale et al., 2010). Accordingly, we found an enrichment for duplicated genes amongst the genes that had reduced endo-siRNA levels targeting them in set-25 and set-32 mutants (Figure 4B). An additional characteristic of the set of genes targeted by ERGO-1 endo-siRNAs is an enrichment for poorly conserved genes, that have fewer introns, and possess splicing site sequences that diverge from the consensus sequence (Fischer et al., 2011; Newman et al., 2018). It was recently suggested that these poorly conserved genes are targeted for silencing because their aberrant or”non-self-like’ splicing signals are detected by the splicing machinery (Newman et al., 2018).

Therefore, we examined whether the targets of the endo-siRNAs that depend on SET-25 or SET-32 can be distinguished by their splicing signals. The changes in the endo-siRNA pool in mutants of small nuclear ribonucleoprotein-associated protein RNP-2/U1A (rnp-2) mirrored the endo-siRNA changes found in set-25 mutants (Figure 4C), but not that of set-32 mutants (Figure 4—figure supplement 2A). We also found that genes targeted by SET-25-dependent- but not SET-32-dependent endo-siRNAs bear fewer introns (Figure 4D, median of 3 and 4 compared to 4 of all protein coding genes, p-values=0.0047, and 0.42 for SET-25 and SET-32, respectively). No significant differences in the length of the coding sequences were found, hence, the difference in intron number does not simply derive from differences in gene lengths (Figure 4—figure supplement 2B, p-value = 0.8673). The lists of genes targeted by SET-25-dependent or SET-32-dependent endo-siRNAs were enriched with genes shown to be targeted by intron-targeting small RNAs (Figure 4—figure supplement 2C and D). We could not find, however, small RNAs aligning to the introns of the gfp transgene that we studied (Figure 4—figure supplement 2E, in most cases endo-siRNAs target only exons). We also did not find significant differences in the splicing motif divergence score (obtained from Newman et al., 2018). Since splicing also directly affects the RNAi machinery untangling its role in endogenous RNAi is challenging (Newman et al., 2018). In summary, splicing may be one of the factors that contribute to distinguishing genes targeted by SET-25-dependent endo-siRNAs, but not by SET-32-dependent endo-siRNAs.

In contrast, in the sets of genes targeted by either SET-25-dependent or SET-32-dependent small RNA we found a significant enrichment for newly evolved genes (Figure 4B, fold-change = 2.57 and p-value<0.0001 for both SET-25- and SET-32-dependent endo-siRNAs targets, respectively). We define newly evolved genes here as genes which had no orthologs outside C. elegans (35/151 and 78/337 of genes targeted by SET-25- or SET-32-dependent endo-siRNAs, respectively). Concordantly, in the same gene sets we also found a significant depletion for nematode-conserved genes (Figure 4B). Importantly, the sets of genes targeted by SET-25-dependent endo-siRNAs and SET-32-dependent endo-siRNAs show very small overlap (25 out of 465 genes). Thus, while SET-25 and SET-32 are required for the maintenance of endo-siRNAs that target different genes, the characteristics of these genes are very similar, that is they are distinctively newly evolved genes that have slightly lower levels of PATC sequences. Although the changes in PATC density and intron numbers that distinguish these target genes are moderate, it is possible that the cumulative effect of these small differences may result in the exposure of foreign genes that need to be silenced.

In general, we find that certain sub-classes of endo-siRNA, such as ERGO-1 and HRDE-1 bound small RNAs, target gene sets enriched for newly evolved genes (Figure 4—figure supplement 3). The significant enrichment for newly evolved genes among SET-25- and SET-32-dependent endo-siRNAs is maintained, however, even after excluding genes that are also targeted by HRDE-1, ERGO-1, WAGO-1 or Mutator endo-siRNAs (SET-25: 59/151 genes are not shared, fold enrichment = 2.97, p-value=0.0001, SET-32: 153/337 genes are not shared, fold-enrichment = 1.89, p-value=0.0012). Thus, the enrichment of newly evolved genes amongst the targets of SET-25- and SET-32-dependent endo-siRNAs is not simply due to a general preference for newly evolved genes by endo-siRNA pathways. Further, we find that newly evolved genes are marked by higher levels of H3K9me3 in comparison to the average level of H3K9me3 on protein coding genes (Figure 4—figure supplement 3). Likewise, in the absence of RNAi, in wild-type animals, gfp, the example for a foreign (non-nematode) gene that we investigated, has higher levels of H3K9me3, in comparison to the well-conserved oma-1 gene (Figure 2B). The fact that across the genome SET-25-dependent- and SET-32-dependent endo-siRNAs target newly evolved and H3K9me3 methylated genes (Figure 3B and Figure 4B), may explain why inheritance of RNAi responses raised against gfp, but not oma-1, depends on SET-25 and SET-32 (Figure 1).

In summary, our experiments reveal a specific role for histone modifications in small RNA inheritance. While in S. pombe and A. thaliana a feedback between H3K9me3 and small RNAs was suggested to be required for silencing, the worm’s RNAi inheritance machinery may use H3K9me3 as a mark that distinguishes genes identified as ‘new’. Since newly evolved genes can be disruptive, small RNAs survey these H3K9me3-flagged elements transgenerationally.

Our study began from an investigation of a perplexing asymmetry in the requirement of specific H3K9 methyltransferases for heritable silencing of the endogenous gene oma-1 and the ‘foreign’ gene gfp. Single mutants of set-25 and set-32 and the met-2;set-25;set-32 triple mutant displayed different heritable dynamics when either the gfp or the oma-1 gene were targeted by RNAi. These results are not unique to the specific gfp transgene that was tested, since similar observations have been made with other transgenes (Klosin et al., 2017; Lev et al., 2017; Shirayama et al., 2012; Spracklin et al., 2017).

Unlike mutations in these histone methyltransferases, which negatively affect heritable silencing of gfp, but not oma-1, mutations in genes required for small RNA inheritance negatively affect heritable silencing of both oma-1 and gfp. For example, the argonaute HRDE-1 is required for inheritance of RNAi responses against both genes (Ashe et al., 2012; Buckley et al., 2012; Kalinava et al., 2017; Shirayama et al., 2012; Weiser et al., 2017). The fact that heritable RNAi responses aimed at different genes are affected by different proteins should be taken into account when studying transgenerational inheritance. Specifically, when screening for genes that affect such inheritance, one must acknowledge that heritable silencing of different targets requires different chromatin modifiers.

Future studies will hopefully reveal why some recently evolved genes, but not others, display high levels of H3K9me3 (in the absence of RNAi), and are targeted by endo-siRNAs. Recent studies examined why transgenes are sensitive to silencing by synthetic piRNAs, while endogenous germline expressed genes, including oma-1, are not. This protection was suggested to be conferred at least in part by PATC sequences, and to be independent of the genomic location of the gene (Zhang et al., 2018). PATC sequences were previously shown to allow expression of transgenes in the germline in heterochromatic areas (Frøkjær-Jensen et al., 2016). Similarly, our analysis revealed that the gene targets of SET-25-dependent and SET-32-dependent endo-siRNAs have lower levels of PATC density (Figure 4A). However, the oma-1 gene does not possess many PATC sequences (Figure 4—figure supplement 1B). An additional theory suggested that an intrinsic unknown coding-sequence feature confers resistance to silencing by piRNAs. Seth et al. have studied why a fusion between oma-1 and gfp can trans-activate silenced gfp transgenes (an effect known as ‘RNAa’,(Seth et al., 2013)). While unique ‘protective’ sequence features were not described in that work, the authors showed that an unknown coding-sequence feature, not related to the codon usage or the translation of the protein, grants the oma-1 gene with its ability to activate silenced transgenes (Seth et al., 2018). It is possible that the gene targets of SET-25- dependent and SET-32-dependent small RNAs that we describe here have unique intrinsic sequences that distinguish them as well. The different requirement of methyltransferases for heritable silencing of some genes but not others may be related to such intrinsic sequence features. Alternatively, it is possible, as was suggested in the past, that new genes are silenced because they are not licensed transgenerationally by heritable small RNAs for expression (Claycomb et al., 2009; Shen et al., 2018). If this is the case, future studies will hopefully reveal how such license is granted (See Figure 5 for Scheme).

Figure 5

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All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Itamar Lev

   Department of Neurobiology, Wise Faculty of Life Sciences & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   itamai.et@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9100-5100

2. Hila Gingold

   Department of Neurobiology, Wise Faculty of Life Sciences & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

   Contribution

   Conceptualization, Software, Formal analysis, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

3. Oded Rechavi

   Department of Neurobiology, Wise Faculty of Life Sciences & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   odedrechavi@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6172-3024

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