The nuclear Argonaute HRDE-1 directs target gene re-localization and shuttles to nuage to promote small RNA-mediated inherited silencing

SUMMARY Argonaute/small RNA pathways and heterochromatin work together to propagate transgenerational gene silencing, but the mechanisms behind their interaction are not well understood. Here, we show that induction of heterochromatin silencing in C. elegans by RNAi or by artificially tethering pathway components to target RNA causes co-localization of target alleles in pachytene nuclei. Tethering the nuclear Argonaute WAGO-9/HRDE-1 induces heterochromatin formation and independently induces small RNA amplification. Consistent with this finding, HRDE-1, while predominantly nuclear, also localizes to peri-nuclear nuage domains, where amplification is thought to occur. Tethering a heterochromatin-silencing factor, NRDE-2, induces heterochromatin formation, which subsequently causes de novo synthesis of HRDE-1 guide RNAs. HRDE-1 then acts to further amplify small RNAs that load on downstream Argonautes. These findings suggest that HRDE-1 plays a dual role, acting upstream to initiate heterochromatin silencing and downstream to stimulate a new cycle of small RNA amplification, thus establishing a self-enforcing mechanism that propagates gene silencing to future generations.

by cellular RNA-dependent RNA polymerase. Once established, inherited silencing can be propagated independently of the initiating cues via continuous cycles of WAGO 22G-RNA amplification and transmission of the WAGO Argonautes and their small RNA co-factors to progeny. 8,[12][13][14] The nuclear WAGO Argonaute, HRDE-1/WAGO-9, plays a central role in transgenerational silencing in C. elegans. 15,16 HRDE-1 is thought to engage nascent transcripts at target loci to induce heterochromatin and transcriptional silencing through the nuclear RNAi pathway. 15,17 HRDE-1 promotes the transgenerational silencing of many genes 18 and is thought to do so by recruiting chromatin remodeling factors, including the nucleosome remodeling and deacetylase complex (NuRD) and histone methyltransferases (e.g., MET-2, SET-25, SET-32). 9,18,19 The nuclear RNAi pathway is also required for the spreading of secondary small RNAs from piRNA target sites. 14,20 Transgenerational silencing requires a series of events that are thought to occur in the nuage, nucleus, and cytoplasm. Because all of these events are essential for the cycle of inherited silencing, their order has been difficult to determine. For example, it is not known whether the nuclear Argonaute HRDE-1 directly triggers RdRP recruitment and amplification of small RNAs or whether it must first induce heterochromatin at its targets to elicit small RNA amplification. Here, we use the phage lambda N (λN)-boxB tethering system [21][22][23][24][25] to recruit-i.e., tether-HRDE-1 or the nuclear silencing factor NRDE-2 to a reporter mRNA. In principle, tethering enables initiation of silencing in the absence of upstream initiators such as piRNAs or dsRNA and, with appropriate genetic tests, can be used to order events in the pathway. We show that tethering either HRDE-1 or NRDE-2 can induce a complete silencing response, including small RNA amplification and transgenerational silencing that persists even after the λN-fusion protein is crossed from the strain. Tethering NRDE-2 initiates chromatin silencing through nrde-4 and independently of hrde-1 but requires hrde-1 for small RNA amplification. By contrast, tethering HRDE-1 stimulates chromatin silencing through NRDE-2 and NRDE-4 but can elicit small RNA amplification independently of both these chromatin-silencing factors. Mutations that block HRDE-1 from binding small RNA disarm silencing and cause HRDE-1 to become cytoplasmic, but tethering HRDE-1 in these mutants nevertheless initiates a strong silencing response that requires small RNA amplification proximal to the tether site. The small RNA amplification machinery is recruited to the tether site by sequences in the N-terminal half of HRDE-1 (the N-terminal domain [NTD]). Like full-length HRDE-1 protein, HRDE-1 NTD co-localizes with MUT-16 in Mutator foci, subdomains of cytoplasmic nuage where the small RNA amplification machinery resides. 26 Our findings suggest that HRDE-1 lies at a nexus in the silencing pathway, shuttling from the nucleus to the nuage and back, to coordinate the nuclear and cytoplasmic events of transgenerational silencing. germline ( Figure 1A). We hypothesized that if artificial recruitment of a silencing factor mimics a physiological event, then it should elicit a silencing response that is independent of upstream factors but depends on known downstream factors. For example, directly tethering a chromatin factor should, in principle, induce silencing without requiring machinery necessary to amplify the small RNAs that would normally guide the chromatin silencing machinery to the appropriate targets.
Having established that tethering induces inherited silencing that depends genetically on known components of the RNA silencing pathway, we asked which factors were required for silencing when the tethered protein was continuously present. For example, because the λN-boxB interaction recruits HRDE-1 and NRDE-2 independently of a guide RNA, we reasoned that the small RNA amplification machinery should be unnecessary when nuclear silencing factors are tethered to the reporter. Consistent with this idea, we found that λN::NRDE-2 silenced the reporter in the absence of rde-3, mut-16, and hrde-1 (Figures 2E, S2A-S2C) but failed to silence it in the absence of nrde-4 ( Figures 2E and S2D). These results suggest that NRDE-2 acts downstream of HRDE-1 and upstream of NRDE-4 in nuclear silencing.
In wild-type animals without tethering, inherited silencing requires nuclear chromatin silencing factors (e.g., nrde-2 and nrde-4) and nuage-localized factors (e.g., rde-3 and mut-16; Figure 1G), indicating that these pathways function together, possibly sequentially, to propagate inherited silencing. In contrast, when λN::HRDE-1 was tethered to the reporter, we found that leaving either pathway intact was sufficient to maintain silencing, as monitored by GFP epifluorescence. For example, silencing of the reporter GFP was maintained independently of nrde-2, nrde-4, or rde-3 and only partly required mut-16 activity ( Figures 2F and S2E-S2H). To completely prevent silencing, it was necessary to Ding et al. simultaneously mutate components of both the small RNA amplification machinery (rde-3 or mut-16) and components of the chromatin nuclear silencing machinery (nrde-2 or nrde-4) (Figures 2F and S2E-S2K). HRDE-1 tethering in wild-type worms reduced the unspliced pre-mRNA reporter level by 2-fold and the spliced RNA level by 100-fold, as measured by quantitative PCR (qPCR) ( Figure 2C). For unknown reasons, nrde-2 mutants exhibited a 4-fold increase in reporter pre-mRNA both with and without HRDE-1 tethering ( Figure 2C) but exhibited discordant effects on spliced reporter RNA levels. Removing nrde-2 activity in animals without tethering had little effect on spliced reporter mRNA levels (a slight 1.2-fold increase) compared with wild type, but removing nrde-2 activity in the context of tethering caused spliced RNA levels to increase (compared with levels in wild-type HRDE-1-tethered animals), reaching levels of approximately 40% of wild-type mRNA levels. It is important to note that the qPCR assay cannot distinguish mRNA from template RNA being silenced, as template RNAs derive from spliced RNAs. Moreover, the high levels of spliced RNA in λN::HRDE-1;nrde-2 worms correlate with a marked accumulation of reporter RNA localized in nuage (via RNA fluorescence in situ hybridization [FISH], shown below). Thus, the accumulated spliced RNA likely reflects template RNA engaged in amplifying the small RNA silencing signal, perhaps to compensate for the loss of heterochromatin silencing. Further study is needed to understand the effects of nrde-2 mutants on pre-mRNA levels, such as whether increased pre-mRNA levels in nrde-2 mutants reflect processing defects. 30 Nevertheless, in the nrde-2 background, HRDE-1 tethering reduces mRNA and pre-mRNA levels by 2-to 3-fold, suggesting that tethered HRDE-1 can exert effects on both mRNA and pre-mRNA levels independently of NRDE-2. Taken together, our findings suggest that HRDE-1 functions twice during inherited silencing-upstream of nuclear silencing to recruit NRDE-2 and NRDE-4 and again downstream of these factors to induce small RNA amplification and post-transcriptional clearance of mRNA. While these events likely occur sequentially and thus depend on each other during the normal course of inherited silencing, 31 tethering HRDE-1 initiates both modes of silencing independently, either of which is sufficient to prevent reporter GFP expression.
λN::HRDE-1 tethering induced abundant small RNA accumulation that was independent of nrde-2 and nrde-4 ( Figures 3F, 3G, and S3A). However, interestingly, both the distribution of small RNAs and their levels of accumulation along the target mRNA were dramatically altered in the nrde mutants. Small RNA levels were markedly increased adjacent to the boxB sites and were diminished on the gfp coding sequences ( Figures 3F, 3G, S3A, and S3B). Small RNAs targeting the reporter were greatly reduced by mutations in rde-3 and mut-16, as expected, (Figures 3H and 3I). Interestingly, however, a low level of small RNAs persisted directly adjacent to the boxB sites when λN::HRDE-1 was tethered in the absence of rde-3 but not in the absence of mut-16 ( Figure 3H). This result is consistent with the observation that tethering of λN::HRDE-1 can bypass rde-3 but cannot fully bypass mut-16 ( Figure 2F).
When outcrossed to a hrde-1(+) background to segregate away λN::HRDE-1, the reporter remained silent for at least 13 generations, with no change in penetrance. Moreover, we observed only a slight reduction in small RNA levels primarily in regions juxtaposed to the boxB hairpins ( Figure 3J). In contrast, when outcrossed to a hrde-1 null background, the reporter was fully de-silenced, and small RNAs were absent ( Figure 3K). As expected, the maintenance of silencing, and of small RNA levels, also required rde-3(+) and mut-16(+) ( Figures 3L and 3M). Taken together, these findings suggest that heterochromatin formation at the target locus induces de novo transcription and loading of small RNAs onto the nuclear Argonaute HRDE-1. HRDE-1, in turn, further promotes small RNA amplification and then functions again, perhaps in the next life cycle, to reinitiate heterochromatin silencing (see discussion).

HRDE-1 guide RNA loading is not required for small RNA amplification
The finding that λN::HRDE-1 can direct chromatin silencing in rde-3 and mut-16 mutants, which are defective in small RNA amplification, suggests that the unloaded Argonaute can direct chromatin silencing when tethered. To further test this idea, we monitored silencing (1) by λN::HRDE-1 in an hrde-2 mutant, which is defective in HRDE-1 small RNA loading 13 and (2) by a λN::HRDE-1(Y669E) mutant, predicted by structural work to be defective in guide RNA binding ( Figure S5B). 32 In both cases, tethering completely silenced the boxB reporter as monitored by GFP fluorescence ( Figure 4C and S4D) and by quantitative reverse transcription PCR (qRT-PCR) of the mRNA ( Figure 4D). For unknown reasons, compromising nuclear silencing by hrde1-(Y669E) caused elevated pre-mRNA levels as measured by qRT-PCR ( Figure 4D), similar to nrde-2 mutants. As expected, the hrde-1(Y669E) mutant was defective in silencing a piRNA reporter ( Figure S4A) and showed a collapse of small RNAs resembling that in hrde-1(null) (Figures S4B and  S4C). However, in these mutant contexts, loss of rde-3 alone was sufficient to completely de-silence the reporter ( Figures S4E and 4C), suggesting that in the absence of guide RNA loading, HRDE-1 fails to engage the NRDE heterochromatin machinery. Deep sequencing revealed an abundant accumulation of rde-3-dependent small RNAs targeting the boxB reporter in λN::HRDE-1(Y669E) animals ( Figures 4E and 4F). Notably, the pattern and levels of small RNA accumulation induced by λN::HRDE-1(Y669E) resembled those observed when wild-type λN::HRDE-1 is tethered in a nrde-2 mutant (compare Figures   4E-3G)-i.e., resulting in increased levels of small RNAs targeting sequences adjacent to the boxB sites and reduced levels targeting GFP sequences. Taken together, these results suggest that tethering of unloaded HRDE-1 can induce local small RNA amplification and silencing but that tethered HRDE-1 must be loaded with small RNAs to induce chromatin silencing, which is in turn required for small RNA targeting to spread into the 5′ sequences of the target mRNA.

HRDE-1 promotes small RNA amplification through its NTD
We next attempted to dissect functional domains of HRDE-1 required for small RNA amplification. We used CRISPR to make a series of λN::hrde-1 truncation mutants ( Figure 5A). These studies identified the N-terminal half (herein the NTD) as the minimal fragment of HRDE-1 that could fully silence the reporter. The NTD and the remaining C-terminal domain (CTD) truncations of HRDE-1 are predicted by I-TASSER 33 to fold into self-contained globular structures, with subdomains similar to those identified in atomic resolution studies on humanAgo2 34 (Figures 5B, S5A, and S5B). As expected, in the absence of tethering, hrde-1(NTD) and hrde-1(CTD) alleles failed to silence a piRNA sensor ( Figure S4A).
Silencing by λN::NTD required rde-3 but not nrde-2 ( Figures 5C and S5C), and deep sequencing revealed that λN::NTD induces abundant rde-3-dependent small RNAs targeting the boxB reporter ( Figures 5D and 5E). Truncations that failed to silence the reporter did not trigger small RNA generation ( Figure S5D). The small RNA pattern induced by λN::NTD resembled the patterns caused by λN::HRDE-1 in nrde-2 mutants or by λN::HRDE-1(Y669E)-i.e., dramatically increased levels of small RNAs proximal to the boxB sites and reduced levels of small RNAs targeting GFP sequences. Interestingly, the magnitude of small RNA accumulation induced by λN::NTD at the boxB sites was ~4-fold greater than that induced by either λN::HRDE-1 in nrde-2 mutants or by λN::HRDE-1(Y669E) (compare Figure 5D with Figures 3G and 4E). These results suggest that the NTD of HRDE-1 robustly recruits the small-RNA amplification machinery to the target and promotes silencing that is independent of the NRDE-2 nuclear silencing pathway.

HRDE-1 tethering promotes accumulation of poly-UG-modified target fragments
During RNA silencing in worms, truncated target RNAs are converted into templates for small RNA production via the RDE-3-dependent addition of poly-UG tails. 27 We therefore used a qPCR assay 27 to detect poly-UG additions to reporter RNA in the absence of a λN fusion or in worms expressing λN::HRDE-1, λN::NTD, or λN::HRDE-1(Y669E) ( Figures  5G and 5H). Priming from an endogenous UGUG motif in the reporter 3′ UTR serves as a control for the presence of full-length mRNA. This analysis revealed that faster-migrating, poly-UG-modified RNAs accumulated in strains where silencing was active. In wild-type λN::HRDE-1 worms, poly-UG-modified RNAs were most robustly detected at truncations within the GFP sequences ( Figures 5G and 5H). As expected, only full-length mRNA was detected in rde-3 mutants, confirming that RDE-3 is absolutely required for poly-UG RNA accumulation. Notably, mutation of nrde-2 or tethering the NTD or Y669E mutants shifted poly-UG addition toward the 3′ end of the reporter, close to the boxB elements ( Figures  5G and 5H). These results suggest that HRDE-1 tethering induces RDE-3-dependent poly-UG modification of truncation products that are generated near the tethering sites and that nuclear silencing promotes the induction of additional truncations far away from the tethering sites that likely support the 5′ spread of small RNA amplification.
To further analyze changes in target RNA caused by tethering, we used qRT-PCR. Surprisingly, whereas tethering wild-type λN::HRDE-1 reduced the reporter pre-mRNA by 50% and mRNA by 99% ( Figure 2C), λN::NTD increased the reporter pre-mRNA by ~2.5-fold and reduced the mRNA by ~40% ( Figure 5F). This result was surprising given that GFP fluorescence was undetectable in λN::NTD worms (Figures 5C and S5C) and suggested that the accumulating species in λN::NTD animals might reflect the accumulation of nearly full-length pUG RNA.

Functional HRDE-1 RNA-induced silencing complex (RISC) is not required parentally for transmission of silencing to offspring
We next asked if λN::NTD can initiate inherited silencing. To do this, we first established reporter silencing by tethering λN::NTD in otherwise wild-type worms. We then crossed to a reporter strain homozygous for a hrde-1 null allele to generate animals heterozygous for the tethering construct. Finally, we crossed these λN::NTD/null heterozygotes (either as males or hermaphrodites) to a hrde-1(+) reporter strain, resulting in two types of cross progeny-λN::NTD/+ or null/+ heterozygotes. Remarkably, although the λN::NTD/null parents lacked a functional HRDE-1 RISC, they nevertheless robustly transmitted silencing to the next generation ( Figures S7A and S7B). As expected, HRDE-1(+) was required in the inheriting generation for silencing to occur (Buckley et al. 15 and Figure 1F). Since the NTD fails to establish heterochromatin upon tethering and cannot directly form a RISC complex, these findings suggest that parentally established heterochromatin and HRDE-1 RISC are not required in gametes for inheritance, a finding consistent with previous work in which hrde-1 homozygous mutant hermaphrodites were shown to transmit silencing to their heterozygous progeny. 15 Rather, in the parental generation, the tethered NTD can stimulate amplification of small RNAs that likely engage with other Argonautes to propagate silencing to offspring (see discussion).

Silencing by dsRNA or tethering causes target genes to co-localize
To understand how HRDE-1 and nuclear silencing regulate their target genes and RNAs, we performed RNA and DNA FISH studies to visualize the boxB reporter mRNA and DNA. In the absence of silencing, reporter RNA foci were detected throughout the germline cytoplasm ( Figures 6E and S6C). In addition, we observed prominent RNA signals in the majority (~70%) of pachytene nuclei (most nuclei, 57%, exhibited at least two closely paired nuclear dots, while the remainder exhibited a single dot; Figures 6E and 6I). The positions of these nuclear signals adjacent to DAPI-stained chromosomes suggests that they correspond to sites of transcription on the paired sister chromatids within the axial loops of synapsed meiotic homologs. Silencing, induced either by exposure to dsRNA targeting the reporter or by tethering λN::HRDE-1, eliminated cytoplasmic reporter RNA signal and greatly reduced the nuclear signal ( Figures 6F, 6L, and S6C). More than 80% of the pachytene nuclei with visible RNA signal exhibited a single nuclear focus ( Figures 6F, 6L, 6I, and 6O). The changes in nuclear RNA signal induced by silencing correlated with changes in the reporter DNA FISH signal. In the absence of silencing, we observed a pair of nuclear DNA FISH signals in approximately 50% of pachytene nuclei that have visible DNA signal ( Figures 6P  and 6T), while in the presence of silencing, we observed a single focus of DNA FISH signal in approximately 90% of pachytene nuclei with visible DNA signal ( Figures 6Q, 6J, 6T, and S6E). These results suggest that nuclear silencing mediated by HRDE-1 causes the target alleles to become merged from predominantly paired DNA FISH signals into a single focus containing all 4 silenced alleles.

Mutations that disarm nuclear silencing cause target RNA to accumulate in nuage subdomains that resemble Mutator foci
We next examined how mutations that disarm only the nuclear silencing pathway impact RNA and DNA localization after RNAi or tethering. To do this, we performed RNA and DNA FISH on λN::NTD worms and on nrde-2 mutants. In these mutants, where nuclear silencing is disarmed, we found that nuclear RNA and DNA FISH signals resembled the nuclear signals observed in wild-type animals in the absence of silencing: predominantly two foci of RNA and DNA FISH signals detected in each background ( Figures 6M, 6N, 6I, 6J, 6O, 6T, and S6D). In contrast, however, the cytoplasmic RNA FISH signals were dramatically altered. While RNA signal was absent from the bulk cytoplasm throughout the gonad, consistent with cytoplasmic post-transcriptional silencing, we noticed pronounced accumulation of reporter RNA signals in multiple peri-nuclear foci surrounding pachytene nuclei. Co-staining experiments with GFP::GLH-1 or MUT-16::GFP revealed that these RNA foci coincide with most of the nuage subdomains that express MUT-16::GFP ( Figures  6G, 6H, 6M, and 6N). The accumulation of target RNA in the MUT-16 foci required RDE-3(+) activity ( Figure S6F), suggesting that these RNA signals may correspond to RdRP templates engaged in small RNA amplification.
MUT-16 promotes the nuclear localization of GFP::HRDE-1 but not its nuage localization MUT-16 is required for the co-localization of small RNA amplification factors within Mutator foci. 26,28,35 We therefore wondered if MUT-16 is also required for the colocalization of HRDE-1 in Mutator foci. To answer this question, we introduced a null allele of mut-16 into worms expressing both GFP::HRDE-1 and mCherry::GLH-1. As shown previously, 24 we found that MUT-16 activity is required for the nuclear localization of HRDE-1 (Figures 7A and 7B). MUT-16 was not, however, required for the localization of GFP::HRDE-1 to nuage (Figures 7A and 7B). The localization of GFP::HRDE-1 in nuage appeared more obvious in mut-16 mutants, but the levels of GFP::HRDE-1 within nuage and the approximate numbers of foci appeared similar with or without mut-16 activity ( Figures  7A and 7B). Finally, the localization of MUT-16 itself to nuage was not disrupted in hrde-1 mutants (data not shown), thus HRDE-1 and MUT-16 localize within a nuage subdomain (or domains) independently of each other.

DISCUSSION
In many eukaryotes, the installation and maintenance of chromatin silencing is coupled to Argonaute small RNA pathways that promote transmission to offspring. Here, we have explored the role of a nuclear Argonaute HRDE-1 in coordinating transgenerational silencing in the C. elegans germline. In addition to its known role in directing heterochromatin silencing downstream of RNAi 13,15 and Piwi Argonaute silencing, 8,9,14 our tethering studies have shown that HRDE-1 is also de novo loaded with small RNA, downstream of heterochromatin silencing, enabling it to prime a new round of small RNA amplification within nuage (Figure 7C, model).
The nuclear silencing events that depend on HRDE-1 cause the target alleles to co-localize into a single focus of DNA FISH signal ( Figures 6P-6S and S6E). Presumably, the heterochromatinized alleles within this focus are transcribed at low levels to produce template RNA that feeds transgenerational silencing; indeed, the continued expression of the target locus after heterochromatin induction is a conserved feature of co-transcriptional small RNA silencing. 36 Consistent with this idea, the inactivation of heterochromatin silencing caused target alleles to remain separated and increased the levels of the nuclearand nuage-localized RNA signals as measured by RNA FISH. The failure to engage nuclear silencing did not de-silence protein expression in the context of our tethering studies nor indeed in previously published studies on nuclear-silencing mutants when an RNAi trigger is present. 13,15 Instead, our RNA FISH studies suggest that unabated transcription of the target gene feeds increased levels of target RNA localization in nuage (also noted in a recent study by Ouyang et al. 37 ) and that small RNA levels also increase dramatically to compensate and silence mRNA expression. Taken together, our findings suggest that when the nuclear heterochromatin pathways are inactive, the target mRNA is silenced by a combination of cytoplasmic clearance or trapping in the P granule.
In the yeast S. pombe, the RNAi-induced transcriptional silencing complex (RITS), which includes an RdRP and a nuclear Argonaute AGO1p, resides in heterochromatin. A previous study showed that tethering of AGO1p to RNA via a boxB reporter system, similar to the one used here, was sufficient to recruit the RITS complex, induce small RNA amplification, and drive reporter silencing 25 .
HRDE-1 associates with NRDE-2 and components of the nucleosome re-modeling and deacetylase NuRD complex to establish heterochromatin silencing. 15,18,38 How heterochromatin leads to de novo programming of HRDE-1 is nevertheless unknown. In C. elegans, the RdRP EGO-1 has been shown to associate with germline chromatin, 39,40 and several of our findings would be consistent with a cycle of nuclear small RNA transcription and de novo HRDE-1 loading within heterochromatin. Such a mechanism could explain why tethering NRDE-2 in the absence of HRDE-1 initiates heterochromatin silencing but not small RNA amplification (Figures 2E and 3E). Perhaps after a nuclear cycle of HRDE-1 loading, the protein exits the nucleus along with nascent target/template RNA to further amplify small RNA production. Consistent with this idea, we have shown that the N-terminal half of HRDE-1 is sufficient to stimulate small RNA amplification and loading and that both the NTD and full-length HRDE-1 (as well as target RNA) localize within a specialized nuage domain known as Mutator foci.
Mutator foci accumulate poly-UG-modified templates derived from target RNA 27 and are thought to serve in the amplification of small RNA signals that are propagated to offspring. Thus, our findings suggest that HRDE-1 shuttles out of the nucleus to nuage to promote small RNA amplification. A mutant HRDE-1 protein incapable of binding guide RNA was sufficient (when tethered) to induce silencing that transmits to offspring via either the sperm or the egg (Figures S7A and S7B). Thus, as previously reported, 15 a functional HRDE-1 RISC is not required in gametes for transgenerational silencing but is required in offspring to renew silencing for another generation (Buckley et al. 15 and Figure 1F). In the parental germline, Mutator foci likely serve as locations where HRDE-1 and other upstream Argonautes trigger the expansion of small RNAs that are loaded onto downstream WAGO Argonautes, including the two prominent nuage-localized Argonautes WAGO-1 8 and WAGO-4. 41 Consistent with this idea, silencing induced by λN::HRDE-1(Y669E) was partially dependent on wago-1 (75% de-silenced, N = 32, and Figure S4G).
Taken together, our findings suggest that heterochromatin renews small RNA silencing (and vice versa) during each germline life cycle. For example, small RNAs guide heterochromatin formation in the zygote, and heterochromatin then propagates silencing before feeding back into the de novo synthesis of guide RNAs that load onto HRDE-1. HRDE-1 promotes expansion of small RNAs that are then transmitted to offspring through HRDE-1 and other WAGOs to re-establish heterochromatin. Heterochromatin then, in turn, transcribes RNA that forms templates for RdRP-dependent amplification, renewing the cycle. Consistent with these ideas, neither pathway, small RNA or heterochromatin alone, is sufficient to stably transmit silencing signals for multiple generations 8,9,13,15 (Figures S7C-S7F). Given the similarities between the worm and yeast mechanisms-and by extension, the intriguing relationships between long non-coding RNAs and chromatin modifiers in flies and mammals 7 -feedforward RNA-chromatin circuits that amplify and maintain silencing across cell divisions or generations will likely be a common feature of gene regulation in eukaryotes.

Limitations of the study
In this study, we use an artificial mechanism to recruit RNA silencing factors to their targets. Recruiting, factors via the λN/boxB system may elicit non-physiological mechanisms that block gene expression. For example, tethering factors to the reporter UTR could prevent proper recruitment of translation-initiation machinery or 3′ end processing factors. Transcripts that are not processed properly (for example, unspliced mRNA 11 ) could trigger default recruitment of the same RNA silencing factors that mediate physiological silencing in response to bona fide Argonaute-guided silencing. To control for such possibilities, we used genetics to dissect the nature of the silencing pathways induced by tethering and found that tethering different factors elicited different genetic dependencies for silencing. For example, λN::NRDE-2 required nrde-4(+) activity for silencing but λN::HRDE-1 tethering did not. We have controlled for possible artifacts by initiating parallel studies on untethered factors and by using a combination of genetics, microscopy, and RNA-expression profiling. Together, these studies give us high confidence that tethering, in these instances, has faithfully replicated actual physiological steps in silencing.

RESOURCE AVAILABILITY
Lead contact-Further information and requests for resources and materials should be directed to and will be fulfilled by the lead contact, Craig Mello (Craig.Mello@umassmed.edu).
Materials availability-All materials generated in this study are available from the lead contact without restrictions. This study did not generate any new code, but the scripts used in the study are available from the lead contact upon request.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
All the strains used in this study were derived from C. elegans Bristol N2 (CGC) and cultured on nematode growth media (NGM) plates with E. coli OP50 43 or E. coli HT115 for RNAi experiments. Strains used in this study were generated by CRISPR-cas9 method or Cross (see Table S1 for details).

METHOD DETAILS
CRISPR-Cas9 genome editing-The Cas9 ribonucleoprotein (RNP) CRISPR strategy 44 were used to edit the genome. Plasmid pRF4 containing rol-6 (su-1006) was used as co-injection marker. For short insertions like λN and deletion mutations, synthesized single-strand DNAs were used as the donor; for long insertions like GFP, mCherry, and 5xBoxB, the annealed PCR products were used instead. The gRNA and donor sequences were listed in Table S2. The BoxB reporter strain was constructed based on a single copy insertion of Ppie-1:GFP::his-58:unc-54UTR (WM701). The 5xBoxB sequence amplified from a previously published strain JMC002 22 was inserted before the unc-54 UTR.
Live worm fluorescent image-Young adult worms were transferred to glass slide in M9 buffer with 0.4mM Tetramisole. Epifluorescence and differential interference contrast (DIC) microscopy were performed on a Zeiss Axio Imager M2 Microscope and images were processed with ZEN Microscopy Software (Zeiss). Confocal images were taken by a Andor Dragonfly Spinning Disk confocal microscope. Confocal images were processed with Imaris Microscopy Image Analysis Software.
Quantifying reporter RNA using qPCR-Young adult worms were collected and washed with M9 for three times and ddH 2 O once. Total RNA was extracted with TRIZOL and treated with DNase I to remove DNA contamination. First strand cDNA was synthesized by Superscript IV with random hexamers. Quantitative PCR was performed on a Quant studio 5 Real-time PCR machine together with Fast SYBR Green Master Mix. Actin was used as internal reference (primer set S5265 and S527). Primer set of oYD826 and oYD827 were used for reporter. All primers used were listed in Table S2. 45 was applied to the young adult worm samples. Anti H3K9me3 antibody (Upstate 07523) and CHIP grade IgA/G magnetic beads were used for the immunoprecipitation. During elution, RNase A and Protease K were used to remove RNA and proteins. For qPCR, actin was used as internal reference. All primers used were listed in Table S2.

CHIP-qPCR-A traditional worm CHIP method
Small RNA cloning and data analysis-Small RNA cloning was conducted as previously reported. 6 Synchronized young adult worms were collected and total RNA were purified with Trizol. Two biological repeats were included for each strain. Small RNAs were enriched using a mirVana miRNA isolation kit. Homemade PIR-1 was used to remove the di or triphosphate at the 5′ to generate 5′ monophosphorylated small RNA. Adaptors of 3' (DA35) and 5' (DA4) were ligated to the small RNA by T4 RNA ligase 2 (NEB) and T4 ligase 1 (NEB) sequentially. Reverse transcription was performed with SuperScript III and RT primer (DA5). After PCR amplification, productions around 150 bp were separated by 12% SDS-PAGE and equally mixed. Libraries were sequenced on a NextSeq 550 sequencer with the illumina NextSeq 500/550 high output kit in 75bp single-end sequencing mod. Reads were trimmed by cutadapt and mapped using Bowtie2. 42 For small RNAs mapped to the reporter, total reads with length longer than 16 nt were used to normalized between samples. Plots were generated by R and R studio.

QUANTIFICATION AND STATISTICAL ANALYSIS
To determine the genes with increased or decreased antisense small RNAs ( Figures S4B  and S4C), small RNAs were cloned and sequenced as described above with two biological repeats for each strain. DEseq2 package in R was used to find out genes with 2-fold decrease of antisense small RNA (p value ≤ 0.05) in hrde-1(null) or hrde-1(Y669E) compared to WT.
Structure prediction-The 3D structure of HRDE-1 was predicted by I-TASSER online server 33 with default setting. HRDE-1 structure was aligned with hAgo2 by PyMOL 46 and its domains were annotated based on the alignment. pUG RNA analysis-As previously reported, 27 total RNAs were extracted with Trizol. SuperScript IV was used to generate the first strand DNA with reverse transcription primer oYD1001. A pair of outer primers (oYD998 and oYD1002) were used for the first round PCR amplification with Taq DNA polymerase. After 100-fold dilution, another round of PCR was performed with a pair of inner primers (oYD256 and oYD1003). PCR products were analyzed by 1.5% agarose gels. DNA bands were purified, cloned with TOPO TA Cloning Kit and sent for sanger sequencing. gsa-1 served as a control for pUG PCR analysis.
RNA FISH-Worms at young adult stage were dissected in Happy Buffer (81mM HEPES pH 6.9, 42mM NaCl, 5mM KCl, 2mM MgCl2, 1mM EGTA) (From personal correspondence with James Priess). Dissected gonads were transferred to poly-lysine treated dish with 80 μl of Happy Buffer and fixed by adding equal volume of 5% formaldehyde in PBST (PBS+0.1% Tween 20) for 30 min. After one wash with PBST, gonads were treated with PBST-Triton (PBST+0.1% Triton) for 10 min, washed with PBST again and emerged in 70% ethanol for 30 min to overnight. Before hybridization, samples were washed with fresh wash buffer (2xSSC +10% formamide) for 5 min hybridization was performed at 37°C for 18 h to overnight in hybridization buffer (900 μl Stellaris RNA FISH Hybridization Buffer+ 100ul formamide) with 10 pmol RNA FISH probes. Samples were washed with wash buffer, once quick wash, one wash for 30 min at 37°C and two quick washes. Mounting medium with DAPI was added to preserve the signal. Confocal images were taken with an Andor Dragonfly Spinning Disk confocal microscope and processed with Fusion and Imaris.
DNA FISH-Same to RNA FISH, gonads were dissected, fixed and washed with PBST and treated with 70% ethanol. Then, samples were washed with wash buffer three times, one at room temperature for 5 min, one at 95°C for 3 min, and one at 60°C for 20 min. Hybridization was performed in hybridization buffer (700 μl Stellaris RNA FISH Hybridization Buffer +300 μl formamide + primary probes (final 10 pmol) + detection probe (final 10 pmol)) at 95°C for 5 min and then transferred to 37°C for 3 h to overnight. After hybridization, samples were wash with 2xSSC for 20 min at 60°C, and then 2xSSCT (2xSSC +0.3% Triton X-100) for 5 min at 60°C and another 20 min at 60°C.
After another wash with 2xSSCT for 5 min at room temperature, samples were preserved in the mounting medium with DAPI. Confocal images were taken with an Andor Dragonfly Spinning Disk confocal microscope and processed with Fusion and Imaris. Primary probes of DNA FISH were picked from the oligo lists generated by OligoMiner. 47 RNAi experiments-Synchronous L1 worms of the reporter strain were plated on NGM plates for 48 h. Then the worms were collected and washed with M9. About 100 worms were plated on every IPTG plate with the gfp RNAi food. After 24 h, worms were dissected for the FISH experiment. RNA FISH and DNA FISH were performed as described above.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

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Nuclear Argonaute HRDE-1 separately induces heterochromatin and small RNA production   were scored for GFP expression: ON or OFF, as indicated. N > 30 worms scored for each genotype. *GFP is ON, but signal is weak (see Figure S2H).  (G and H) Analysis of poly-UG modification of reporter RNA in response to tethering in the indicated mutants. Poly-UG PCR products in (G) were cloned and sequenced to identify the precise positions of poly-UG addition (H), indicated by arrowheads. A gsa-1-specific PCR was used as loading control.