Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Dec 22.
Published in final edited form as: Cell. 2013 Aug 29;154(5):1010–1022. doi: 10.1016/j.cell.2013.08.006

Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor

Bi-Tzen Juang 1, Chen Gu 1,2, Linda Starnes 1,3, Francesca Palladino 4, Andrei Goga 1, Scott Kennedy 5, Noelle D L'Etoile 1,6
PMCID: PMC4274153  NIHMSID: NIHMS514739  PMID: 23993094

Summary

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs) remains unclear. Here we show the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene who's down regulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation.

Introduction

RNA interference (RNAi) has been exploited as a powerful experimental tool in both somatic and germ cells for over a decade (Fire, et al., 1998), and organisms ranging in complexity from yeast to humans produce a range of endogenous small RNAs of 20-30 nucleotides in length. Although it is apparent that almost all cells of an organism are actively engaged in some form of endogenous RNAi, its role, particularly in somatic cells, remains unclear (reviewed in Ketting, 2011; Ghildiyal and Zamore, 2009).

Endogenous small RNAs are grouped into three classes according to their biosynthetic origin and the proteins they bind: piwi-RNAs (piRNAs), micro RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). piRNAs and miRNAs are encoded by genes while in C. elegans, endo- siRNAs are produced by RNA dependent RNA polymerases that use thousands of cellular mRNAs as templates to produce anti-sense small RNAs ((Ghildiyal and Zamore, 2009; Ketting, 2011; Gent et al., 2009; Gu et al., 2009). Small RNAs have been linked to synaptic function and memory formation in mammals (McNeill and Van Vactor 2012). For instance, the microRNA miR134 was shown to repress context dependent fear learning and Long Term Potentiation in mice (Gao et al., 2010), and a piRNA has been shown to promote long-term synaptic facilitation of cultured Aplysia sensory neurons (Rajasethupathy et al., 2012). However, the extent to which small RNAs couple environmental stimuli to synaptic plasticity and the mechanism by which small RNAs regulate experience-induced behavioral changes remain a mystery.

Prolonged odor exposure induces a form of behavioral plasticity termed adaptation. C. elegans are innately attracted to food-related odors, but the attraction is diminished if starvation accompanies the odor. The resulting odor-adapted state lasts until the animal is fed (Colbert and Bargmann 1997; Lee et al., 2010). Odor sensation (Bargmann et al.1993) and adaptation occur within the sensory AWC neuron. While odor sensation requires the guanylyl cyclase (GC), ODR-1, odor adaptation requires down regulation of ODR-1 (L'Etoile and Bargmann, 2000). Decreased intracellular cGMP, in part, drives the cGMP-dependent protein kinase, EGL-4, into the AWC nucleus (O'Halloran et al., 2012). Once in the nucleus, EGL-4 is both necessary and sufficient to induce long-lasting odor adaptation (Lee et al., 2010). The mechanism by which nuclear EGL-4 triggers long-lasting odor adaptation is not known.

Small RNAs can regulate gene expression in both the cytoplasm and nucleus. For instance, miRNAs and siRNAs act as guides to target mRNAs for repression in the cytoplasm (reviewed in Ketting, 2011; Ghildiyal and Zamore, 2009). piRNAs and siRNAs can enter nuclei to trigger co-transcriptional gene silencing (nuclear RNAi) (Guang et al., 2008; LeThomas et al., 2013). During nuclear RNAi in C. elegans, the Argonaute (Ago) NRDE-3 shuttles siRNAs into the nucleus and binds nascent transcripts that exhibit sequence complementarity to NRDE-3 associated siRNAs (Guang et al., 2008; Guang et al., 2010). NRDE-3 recruits the conserved nuclear protein NRDE-2, and two nematode specific proteins NRDE-1 and perhaps NRDE-4, to RNAi-targeted nascent transcripts to inhibit RNA Polymerase II (RNAP II) elongation (Guang et al., 2010; Burkhart et al., 2011). In addition, genes targeted for silencing by the nuclear RNAi pathway accumulate the repressive chromatin mark, H3K9me3 (Guang et al., 2010; Burton et al., 2011). In the C. elegans germline, piRNAs and siRNAs trigger nuclear RNAi at thousands of genomic loci (Claycomb et al., 2009; Gu et al., 2009; Ashe et al., 2012; Lee et al., 2012; Shirayama et al., 2012) and the silencing effects can endure for more than five generations (Vastenhouw et al., 2006; Buckley et al., 2012). When nuclear RNAi is disabled, C. elegans germlines lose their immortal character (Buckley et al., 2012).

In this paper we examine the role of RNAi in neurons. Four lines of evidence indicate that in the AWC olfactory sensory neurons of adult behaving C. elegans, endogenous RNAi promotes odor adaptation by repressing the odr-1 gene. First, we show that the nuclear RNAi Ago, NRDE-3, is required in the AWC neuron to promote adaptation. Second, NRDE-3 co-immunoprecipitates (coIPs) odr-1 directed endo-siRNAs, and in adapted animals we find the levels of NRDE-3 bound odr-1 siRNA increase. Third, odor exposure diminishes the levels of odr-1 mRNA. Fourth, in odor adaptation, HPL-2, a heterochromatin binding protein, is loaded onto the odr-1 locus. Additionally we find that phosphorylation of HPL-2 at sites that are in vitro targets of the odor-responsive kinase, EGL-4, is both necessary and sufficient to promote odor adaptation in the AWC neurons of an intact animal. Our work indicates a mechanism by which environmentally relevant experiences may regulate gene expression, thereby shaping behavior in a specific and dynamic fashion.

Results

The Nuclear RNAi Argonaute NRDE-3 is Required in the AWC Sensory Neuron for Odor Adaptation

C. elegans are innately attracted to the odor, butanone. Attraction is assessed by the chemotaxis assay shown in Figure 1A, which allows quantification of the behavior in the form of a chemotaxis index (CI) (Bargmann et al., 1993). Naïve wild type animals exhibit a high CI to butanone, which decreases after eighty minutes of butanone-exposure in the absence of food (Colbert and Bargmann 1995). This experience-dependent decrease in CI is termed long-term olfactory adaptation. If the adapted CI is greater than one half of the naive CI, a strain is considered adaptation defective.

Figure 1. HPL-2 and MUT-7 act at the time of odor exposure in the AWC neurons to promote adaptation to butanone.

Figure 1

Open in a new tab

(A) Olfactory adaptation paradigm. Animals exposed to buffer alone (naïve), or butanone (adapted) for 80 minutes are placed at the “origin” of an agar-lined 10cm Petri dish. Butanone is at red and ethanol at the black “X” the anesthetic, sodium azide was added to each spot. Animals roam plates 2 hours before counting. The chemotaxis index (CI) is calculated by subtracting the number of animals at the diluent from the number at the odor and dividing this by the number of animals that left the origin.

(B) Initial screen of mutant strains defective for small interfering RNA pathways. Bars represent mean CIs of strains of the indicated genotype that had either been incubated with buffer (-) or buffer-diluted butanone (+) for 80′. Bars for wildtype represent the mean CI of pooled controls for all the strains. All error bars are SEM. The side-by-side comparisons of each strain with wild type controls are shown in Figure S2. **=p< 0.005, *=p<0.05, “ n.s.” =p >0.05 and unless otherwise noted, all tests were two-tailed Student's t and all assays were performed on separate days with > 100 animals per assay. drsh-1, alg-2, dcr-1; n=4; rde-1, mut-7 n=6; hpl-2 n=5.

(C) HPL-2 and MUT-7 are expressed in AWCs. Fluorescent confocal images of wildtype animals expressing the putative hpl-2 (top) or mut-7 (bottom) promoters driving GFP tagged versions of each protein. AWC is marked with ceh-36prom3 promoter driving mCherry. Anterior is at the left for both images. Figure S1B is associated with this panel.

(D) Expression of HPL-2 or MUT-7 in AWC rescued the adaptation defects of each mutant. HPL-2 (left graph, 3rd pair of bars) or GFP-tagged MUT-7 (right graph, 3rd pair of bars) was expressed in AWC from pceh-36prom3 in hpl-2(tm1489) or mut-7(pk204), respectively. **p=0.002, *p=0.0035, n>5 for each.

(E) Expression of HPL-2 or MUT-7 at the time of odor exposure rescued adaptation defects. hpl-2(tm1489) (left) or mut-7(pk204) (right) transgenic for the respective cDNA under the control of the heatshock promoter (phsp16-2) were heated (+) one hour before odor exposure. Heat-treated animals' exposed CI's were significantly different from either before heating (p=0.02, hpl-2; p<0.005, mut-7) or from non-transgenic animals that had been heated (P=0.005, hpl-2; p<0.005, mut-7) n>5 for each.

(F) HPL-2 and MUT-7 act in the same genetic pathway for adaptation. Mean naïve (-) and exposed (+) CIs of animals of the indicted genotype. The adaptation defects of the fbf-1(ok91) strain are due to loss of the translational control pathway that acts in parallel with hpl-2 (Kaye et al., 2009).

(G) Expression of NRDE-3 in AWC rescued the adaptation defects of the nrde-3 mutant strain. Mean CI of naive (-) and exposed (+) wildtype, ndre-3(gg66) and NRDE- 3 expressed in AWC (pceh-36prom3) of the nrde-3(gg66) mutant strain. Figure S1D is associated with this figure.

To investigate the role of small RNAs in long-term olfactory adaptation, we examined butanone adaptation in strains defective for major pathways producing RNAi in the soma including the microRNA, exogenous RNA (exo-RNAi) and endogenous RNAi pathways. Animals lacking Dicer (DCR-1) were defective for adaptation (Figure 1B). Dicer, an RNAase III, processes double stranded (ds) RNA into small non-coding RNAs (Grishok et al., 2001, Knight and Bass, 2001, Duchaine et al., 2006) that feed into the microRNA, exo- and endo-siRNA interference pathways (Grishok et al., 2001; Knight and Bass 2001; Grishok et al., 2005). These data suggest that Dicer-mediated processing of dsRNA is required for adaptation. By contrast, the adapted CI of strains bearing mutations in the pri-miRNA processing RNase III enzyme Drosha, DRSH-1 (Denli et al., 2004), the miRNA-binding Ago, ALG-2 (Vasquez-Rifo et al., 2012), or the exo-RNAi pathway Ago, RDE-1 (Tabara et al., 1999), were not significantly different from the CI of wild type controls (Figure 1B, supplementary material). These data suggest that if Dicer-mediated double stranded RNA processing is required for butanone adaptation, microRNAs or the exoRNAi pathway are unlikely to mediate this process.

MUT-7, a putative 3′-5′ exonuclease, is required for accumulation of endogenous 22 nucleotide siRNAs that bind the WAGO clade of Agos (Yigit et al., 2006; Lee et al., 2006; Gu et al., 2009) and accumulation of 26 nucleotide siRNAs (Zhang et al., 2011) as well as transposon and transgene silencing, exogenous RNAi and proper chromosome segregation (Ketting et al., 1999; Tabara et al., 1999; Dernburg et al., 2000 Tops et al., 2005). MUT-7 is also required for nuclear accumulation of NRDE-3 (Guang et al., 2008). HPL-2 is one of two C. elegans homologs of Heterochromatin Protein 1 (HP1) (Couteau et al., 2002). HPL-2 is involved in multiple cellular events, including gene regulation, DNA replication and repair (Couteau et al., 2002; Coustham et al., 2006; Black and Whetstine 2011) as well as transgene silencing and piRNA mediated gene silencing in the gonad (Grishok et al., 2005; Burkhart et al., 2011; Ashe et al., 2012; Buckley et al., 2012; Shirayama et al., 2012). Strains that lacked MUT-7 or HPL-2 were defective for butanone adaptation (Figure 1B). These results suggest heterochromatin and possibly small RNAs promote odor adaptation downstream of Dicer.

Using mut-7 and hpl-2 promoter fusions to drive expression of GFP-tagged MUT-7 or HPL-2 respectively, we observed GFP expression in many cells including both AWCs (Figure 1C). To determine if MUT-7 and HPL-2 act in the AWC neurons, the site of odor sensation and adaptation, we asked if cell-specific expression of MUT-7 and HPL-2 could rescue the odor adaptation defect of each corresponding mutant strain. Expressing MUT-7 or HPL-2 solely within the AWC neurons (from the AWC-specific ceh-36prom3 promoter (Etchberger et al., 2007)) of the respective mutant strain rescued its adaptation defects (Figure 1D). These data indicate that MUT-7 and HPL-2 act within AWC neurons to promote odor adaptation.

These factors could be required at the time of odor exposure or developmentally. To distinguish between these possibilities, we used the heatshock promoter phsp-16.2 (Stringham et al., 1992) to express each factor in the adult immediately prior to odor exposure. Heat shock driven expression restored adaptation to the mut-7 and hpl-2 strains (Figure 1E). Consistent with a requirement in the adult, neither morphology nor cell fate of the AWC was altered by loss of HPL-2 or MUT-7 (supplementary material S1B,C). Together, these results indicate that the adaptation defects of mut-7 and hpl-2 deficient animals are not due to developmental defects.

To address whether MUT-7 and HPL-2 act in the same molecular pathway, we created mut-7; hpl-2 and control fbf-1;hpl-2 double mutant animals. We found that the ability of hpl-2 or mut-7 single mutant animals to adapt to odors was similar to the ability of mut-7; hpl-2 double mutant animals (Figure 1F) but the adaptation defects of hpl-2 were enhanced in the fbf-1;hpl-2 double mutant strain. These data indicate that MUT-7 and HPL-2 likely act in the same pathway within AWC to promote odor adaptation at the time of odor exposure.

To probe the involvement of nuclear RNAi in adaptation, we examined the nuclear Ago, NRDE-3. NRDE-3 interacts with a subset of endo-22GRNAs and shuttles them into the nucleus where they direct cotranscriptional gene silencing (Guang et al., 2008). NRDE-3 is expressed in the AWC neurons (data not shown) and the NRDE-3 null (nrde-3(gg66)) was unable to adapt to butanone (Figure 1G). These adaptation defects were rescued by expressing NRDE-3 solely in the AWC neuron (Figure 1G, supplementary material S3), demonstrating that the nuclear RNAi Argonaute NRDE-3 acts in AWC to promote odor adaptation.

To better characterize the nuclear RNAi pathway, we surveyed adaptation in small interfering RNA-defective strains that were deemed chemotaxis proficient (Table 1 and supplementary material S2). In C. elegans, RNAi can be broken down into three steps: trigger processing, amplification, and silencing (reviewed in Pak et al., 2012). We found that trigger processing factors, Dicer and its partner, RDE-4 (Tabara et al., 2002; Duchaine et al., 2006) are required for adaptation. The siRNA amplifying RNA dependent RNA polymerase (RdRP), RRF-3 (Simmer et al., 2002) was partially required as rrf-3(pk1426) animals failed to adapt in five out of eight trials. The silencing factor NRDE-3 along with its nuclear complex of NRDE-2 (Guang et al., 2010) and NRDE-1 (Burkhart et al., 2011) were each required. These results suggest that adaptation requires trigger processing, possibly RdRP amplification, and nuclear Ago-mediated silencing.

Table 1. Olfactory adaptation Requires a Nuclear RNAi Pathway.

Gene (Allele) Gene Function Butanone Adaptationa
dcr-1(ok247)b RNase III nuclease defective
rde-4(ne337) Double-stranded RNA binding defective
rrf-1(pk1417) RNA-dependent RNA polymerase wild-type
rrf-2(ok210) RNA-dependent RNA polymerase wild-type
rrf-3(pk1426) RNA-dependent RNA polymerase partial defective
drh-1(tm1329) RNA helicase (RIG-I) wild-type
drh-2(ok951) RNA helicase (RIG-I) defective
drh-3(ne4253) RNA helicase wild-type
rde-3(ne3364) beta-nucleotidyl transferase chemotaxis defective
rde-1(ne300) exo-RNAi Argonaute wild-type
ergo-1(gg98) Argonaute wild-type
quintuple 5 Argonautes wild-type
MAGO12 12 Argonautes defective
nrde-3(gg66) Nuclear RNAi Argonaute defective
nrde-2(gg91) NRDE-3 binding nuclear factor defective
nrde-1(gg88) NRDE-2/3-chromatin associated defective
hpl-2(tm1489) Histone H3 lysine 9 trimethyl binding (HP1) defective
Open in a new tab
a

Behavioral assays are shown in Supplemental Figure 1A

b

Heterozygous animals are marked with hT2∷GFP(I,III)

Biochemical and genetic analyses have implicated additional factors in RNAi. Of the many factors shown to associate with Dicer, DRH-2 (a Dicer related DExH-box helicase (Tabara et al., 2002)) and RDE-3 (a beta nucleotidyl transferase) (Duchaine et al., 2006)) were required for adaptation. Taken as a whole, our genetic analysis indicates that the nuclear RNAi pathway is likely to act in the AWC neuron to promote odor adaptation downstream of DCR-1/RDE-4 mediated small RNA production.

odr-1 mRNA Decreases in Odor Adapted Animals

To identify a target for siRNA in adaptation, we used qRTPCR to probe endo-22GRNAs that map to AWC expressed genes (see Supplemental Material). We found the odr-1 derived 22GRNAs, odr-1.6 and odr-1.7, as well as the unc-40 derived 22GRNA, unc-40.2, gave the most robust signals. odr-1 encodes a GC whose down regulation is required for odor adaptation (L'Etoile and Bargmann, 2000) and unc-40 encodes an axon guidance and synaptogenesis factor (Hedgecock et al., 1990). The gene structure, along with the amplicons corresponding to mRNA, 22GRNA and genomic DNA are indicated in Figure 2A.

Figure 2. Prolonged odor stimulation dynamically regulates odr-1 derived 22G RNAs, association of HPL-2 with the odr-1 locus and levels of odr-1 mRNA.

Figure 2

Open in a new tab

(A) Diagram of the odr-1 and unc-40 genes. The odr-1 and unc-40 22GRNAs examined are indicated below the gene. In green, the PCR amplicons for ChIP-qPCR. PCR amplicons for mRNA analysis are in red. (B) Prolonged odor exposure decreases odr-1 mRNA levels. Bars represent the mean fold change in unc-40 (grey) or odr-1 (black) mRNA level as a function of odor-exposure (adapted/naive). RNA from animals of the indicated genotype was normalized to act-3 mRNA, all error is SEM. The red line indicates “ no change” and the significance of the difference between a sample and “ no change” was assessed using a two-tailed Wilcox signed rank test. ** indicates median of sample and “ no change” are different, p< 0.005. # indicates a difference of p=0.034 (non-parametric, pair-wise comparison) in medians between the naïve and adapted values of the mRNA. P values displayed are from two-tailed Mann-Whitney test of medians. Chemotaxis behavior for each population and the individual data points for each pair are shown in Figure S2A. (C) Chemotaxis behavior correlates with the level of odr-1 mRNA in butanone adapted animals. The butanone CI of odor-exposed animals was compared with their odr-1 mRNA level (mRNA levels normalized to act-3 mRNA). Red circles indicate wildtypes and blue triangles mut-7(pk204) animals expressing MUT-7 solely within the AWC neurons. r is Pearson's correlation coefficient, and p is from a two tailed Student t-test (wt n=8, and AWC MUT-7 rescue n=5). (D) Prolonged odor exposure increases NRDE-3 bound odr-1 22GRNA levels. First 5 bars represent fold change in total 22GRNAs normalized to odor-insensitive sn2343 RNA in adapted versus naive animals of the indicated genotype. Red line indicates no change. *=p<0.03, Wilcox sign Rank test for median values vs. “ no change” . P values displayed are the comparison of medians using an unpaired two sample Mann Whitney non-parametric t-test, n >3. Figure S2B is associated with this panel. Last 3 bars represent pnrde-3∷NRDE-3 coIPed 22GRNA (n=6) normalized to the odor-insensitive X-cluster. *=p<0.04, Wilcox sign Rank test for median values vs. “ no change” . Displayed p values are from a pairwise, 1-tailed t-test p=0.0469 of medians. Figure S2C shows the behavior. (E) Prolonged odor exposure specifically increases HPL-2 binding to the odr-1 locus in a MUT-7 dependent fashion. The ratio of 3XFLAG-HPL-2 expressed in AWC (podr-3) coIPed odr-1 (dark bars) or unc-40 (light bars) in adapted versus naive animals is shown above the genotype of each population. Also indicated is the PCR amplified, coIPed region of each locus corresponding to “ A” “ B” and “ C” in panel A. CoIPed DNA from each locus was normalized to input. This was then normalized to the ratio of IPed act-3 to input. act-3 levels were odor-insensitive. *=p=0.031, one tailed Wilcox Signed rank test comparing median values to “ no change” (the red line). The median value of odr-1 B was compared to unc-40 B, p=0.0079 using a two tailed, Mann Whitney test. n=5, error is SEM. The final set of bars represents background from non-transgenic animals. Figure Figure S2D shows the behavior.

Nuclear RNAi silences gene expression leading to lower levels of target mRNA. To determine if odr-1 message levels are decreased in odor-adapted populations, we performed qRTPCR on RNA collected from the same samples that showed behavioral adaptation to butanone (Figure S2A). We found that odr-1 mRNA decreased by approximately one half in odor-adapted as compared to naïve populations (Figure 2B, 2nd bar). By comparison, unc-40 mRNA levels were unchanged (Figure 2B, first bar). In mut-7(pk204) animals, odr-1 mRNA levels were not odor-responsive (Figure 2B, third bar, Figure S2A for behavior) but expression of MUT-7 solely within AWC partially restored odor-responsiveness (Figure 2B, fourth bar). Thus, in odor-adapted populations, the odr-1 mRNA decreases, and these changes depend on odor exposure as well as functional MUT-7.

To understand whether the modest decrease in odr-1 mRNA (Figure 2B) has a behavioral consequence, we asked whether the level of odr-1 mRNA correlates with the chemotaxis index (CI) in odor adapted animals. We found that the levels of odr-1 mRNA correlated strongly with odor attractiveness (CI). The correlation between CI and odr-1 mRNA was even stronger in the mut-7(pk204) strains that expressed MUT-7 solely in the AWC neuron (Figure 2C). This indicates that the decreases we observe in odr-1 mRNA in AWC could be responsible for the stably diminished odor-attractiveness that is the hallmark of long-term adaptation.

In analysis described above, we examined mRNA from whole worms, but two lines of evidence indicate that this drop in mRNA occurs within the AWC neurons: loss of odr-1 leads to the adapted phenotype and this is rescued by expression of ODR-1 in the AWC neurons (L'Etoile and Bargmann, 2000) and overexpression of ODR-1 in AWC alone blocks adaptation (L'Etoile and Bargmann, 2000). Taken together, the data implicate down-regulation of the odr-1 gene in AWC in butanone adaptation.

odr-1 Directed 22GRNA Increases in the AWC Sensory Neuron of Adapted Animals

To determine if there is evidence for the endo-RNAi pathway acting in adaptation, we used qRTPCR to compare the levels of odr-1 and unc-40 22GRNA species in naïve and butanone adapted populations. We found expression of the odr-1 22GRNA odr-1.7 increased by more than two-fold in adapted animals compared to naive controls (Figure 2D second bar, Figure S2B). The levels of a less abundant 22GRNA, odr-1.6, and unc-40.2, however, did not change significantly (red line indicates a ratio of 1:1 for adapted to naïve levels) (Figure 2D, first and third bars, Figure S2B). Thus, a 22GRNA (odr-1.7) complementary to the odr-1 gene increases in animals adapted to odor.

These measurements of 22GRNAs were obtained via analysis of the whole animal, including the germline (Gu et al., 2009). To determine if odr-1.7 22GRNA is regulated by odor specifically in AWC, we expressed MUT-7 only in AWC (Figure 1D). Though total odr-1.7 22GRNA levels were insensitive to odor exposure in mut-7 defective animals, expression of MUT-7 in AWC restored odor responsiveness to this species of 22GRNA (Figure 2D, 4th and 5th bars, Figure S2B). Thus, the levels of odr-1.7 22GRNA are increased by odor-exposure when a factor required for 22GRNA accumulation (Gu et al., 2009) is expressed solely within the AWC neuron.

odr-1 siRNAs are Loaded onto NRDE-3 in adaptation

To better understand how the nuclear RNAi pathway might function in odor adaptation, we asked whether odr-1.7 or unc-40.2 22GRNAs associate with NRDE-3. We probed this association by IPing 3XFLAG tagged NRDE-3. We found odr-1.6, 1.7 and unc-40.2 coIPed with NRDE-3. The level of odr-1.7 22GRNA in association with NRDE-3 was increased significantly in adapted animals ((Figure 2D), 7th bar). An increase in coIPed odr-1.6 or unc-40.2 22GRNA was not seen in the same animals, . indicating that NRDE-3 specifically binds more odr-1.7 22GRNA in adapted animals. This finding supports a model in which ODR-1 mRNA is reduced by NRDE-3/odr-1.7 22GRNA mediating down-regulation of the odr-1 gene.

HPL-2 associates with the odr-1 locus in odor adapted AWC neurons

One biochemical readout of siRNA/NRDE-3 directed silencing is increased heterochromatin deposition at the targeted locus (Burkhart et al., 2011; Guang et al., 2010; Gu et al., 2012). To understand if odr-1.7/NRDE-3 might target the odr-1 locus in the odor adapted AWC neuron, we expressed 3XFLAG tagged heterochromatin associated factor, HPL-2 from the odr-3 promoter (which drives expression in AWCs and four other neurons). When we performed chromatin immunoprecipitation (ChIP) of HPL-2 followed by qPCR on naive and behaviorally adapted populations, we found that ChIP of the odr-1 locus was increased in adapted AWC neurons (Figure 2E). The greatest increase in HPL-2 associated ChIP (eight fold higher in adapted than in naïve) was located just downstream of the region encoding odr-1.7. Further, the odor-dependent increase was not seen at the unc-40 locus. As a specificity control for the 22GRNA pathway, we performed ChIP from mut-7 loss of function animals, which show no increase in odr-1.7 22GRNA levels in response to odor and likewise show no increase in odr-1 ChIP (Figure 2E). These results show that odr-1 is a target for increased HPL-2 association in the odor adapted AWC. Though this is not the only interpretation, these results are most consistent with nuclear RNAi targeting this locus.

HPL-2 is a Direct Phosphorylation Target of the Odor-Responsive Kinase, EGL-4

How might an environmental signal such as odor intersect with the endogenous nuclear RNAi pathway to mediate adaptation? Prolonged odor stimulation causes nuclear accumulation of the cGMP-dependent protein kinase EGL-4 (Figure 3A, (O'Halloran et al., 2009; Lee et al., 2010) and nuclear EGL-4 is both necessary and sufficient to induce long-term odor adaptation. Indeed, expression of constitutively nuclear EGL-4 (NLS-EGL-4) in AWC decreased chemotaxis towards inherently attractive odors even in naïve animals (Figure 3B, Lee et al., 2010; O'Halloran et al., 2009). MUT-7 or HPL-2 could thus act by promoting nuclear accumulation of EGL-4. However, we found that nuclear accumulation of EGL-4 was not altered in in mut-7 or hpl-2 mutant strains (Figure 3C). Three lines of evidence led us to hypothesize instead, that EGL-4 promotes adaptation by phosphorylating and activating MUT-7 and HPL-2. First, we found that that constitutively nuclear EGL-4 required both HPL-2 and MUT-7 to induce adaptation in naive animals (Figure 3B). Second, predicted EGL-4 phosphorylation sites within MUT-7 and HPL-2 (Figure 4A) are required for adaptation (Figure 4B,C, Figure S3). Third, expression of phospho-defective MUT-7 in wild-type animals caused adaptation defects, suggesting that of MUT-7 phosphorylation is required for this behavioral change (Figure S3D).

Figure 3. HPL-2 and MUT-7 act downstream of nuclear EGL-4.

Figure 3

Open in a new tab

(A) Current model for long-term olfactory adaptation of the AWC neuron Acute stimulation of AWC localized G-protein coupled receptors (GPCR) by odor (left) causes animals to chemotax towards the odor. After prolonged odor-exposure (right), the cGMP-dependent protein kinase (PKG) EGL-4 translocates to the nucleus to cause animals to ignore the odor for prolonged periods of time. (B) Once in the nucleus, EGL-4 requires HPL-2 and MUT-7 to promote adaptation. The chemotaxis index of the indicated strains that express constitutively nuclear EGL-4 from a transgene (+) were compared to their siblings that did not carry this transgene (-). rde-2 is a control, adaptation-proficient strain (Figure S1A). Importantly, all animals were naïve to butanone. n>3 with >100 animals analyzed per assay. **=p<0.0001, two-tailed Student t-test. Error is SEM. (C) HPL-2 and MUT-7 are not required for odor-induced nuclear entry of EGL-4. GFP tagged EGL-4 was expressed in either wild type, hpl-2(tm1489) or mut-7(pk204) strains. Animals were exposed to buffer alone (naïve) or butanone for 80 minutes before imaging. The percentage of the population that showed nuclear EGL-4 in one AWC neuron was determined.

Figure 4. Phosphorylation of HPL-2 and MUT-7 at predicted PKG sites is required for adaptation.

Figure 4

Open in a new tab

(A). Schematic of HPL-2 and MUT-7 (Top) HPL-2 contains an N-terminal chromo domain, a C-terminal chromo shadow domain and four predicted PKG phosphorylation sites. (Bottom) MUT-7 contains two predicted functional domains: cytosolic fatty acid binding domain, 3′ to 5′ exonuclease and seven predicted PKG phosphorylation sites.

(B). Phosphorylation of predicted PKG target sites in MUT-7 is required for adaptation. Mean CIs of wild type or mut-7(pk204) strains expressing the indicated form of MUT-7 in AWC. Figure S3D shows individual lines. n=3, p value is from a two-tailed Student's t-test. The lines rescued the sterility defects of mut-7(pk204) (Figure S3B).

(C). Phosphorylation of HPL-2 at predicted PKG sites is required for adaptation. CIs of animals of the indicated genotype that expressed the indicated form of HPL-2 cDNA in AWC are shown. n > 3 with >100 individuals per assay. P value is from an unpaired Student t-test. (D) Nuclear EGL-4 phosphorylates HPL-2 in vitro. (Left) 3XFLAG-Nuclear EGL-4 kinase was immunopurified from worms (behavior in Figure S3F) and incubated with purified HPL-2 and 32P ATP. The reactions were resolved on a gel and stained with Coomassie blue as loading control (lower) followed by autoradiography (upper). (Right) Quantification of five independent kinase assays. 32P phosphorylated HPL-2 was normalized to Coomassie stained band. Values shown for mutant HPL-2 substrate are shown as fold reduction of phosphorylation relative to HPL2-WT, which was set to 1. Error bars represent mean ± SEM (p < 0.0001; two-tailed Student t test, n=5). (E) Phosphorylation of HPL-2 at a predicted PKG phosphorylation site in the CSD is sufficient to decrease butanone chemotaxis in naïve animals. CIs of wildtype animals expressing the indicated form of HPL-2 in AWC, n>3. Figure S3E shows CIs of individual lines. All strains expressed similar levels of the indicated transgenes as assessed by GFP intensity. P values are from a two tailed Student- t-test. (F) Phosphorylation of HPL-2 at the EGL-4 phosphorylated sites is sufficient to promote adaptation in naïve animals. CIs of wildtype, osm-9 or mut-7 naive animals either expressing the phosphomemetic HPL-2(S/Tto E) (+) or not (-). Error is SEM and. n >5, p value is from an unpaired two tailed Student t-test.

MUT-7 and HPL-2 might be direct targets of the EGL-4 kinase, thus, we asked whether NLS-EGL-4 phosphorylates these factors in vitro. We were unable to purify full length MUT-7 so we focused on HPL-2. We found that C. elegans expressed, immunopurified NLS-EGL-4 phosphorylated recombinant HPL-2 and that the level of 32P incorporation diminished when the predicted PKG phosphorylation sites within HPL-2 were mutated (Figure 4D and Figure 3G). We therefore conclude that these sites are direct targets of EGL-4 in vitro. Thus it is likely that HPL-2, a nuclear protein, is directly phosphorylated by EGL-4 once it enters the AWC nucleus.

Phosphorylation of HPL-2 at EGL-4 Target Sites is Both Necessary and Sufficient to Promote Odor Adaptation

If nuclear EGL-4 promotes odor adaptation by phosphorylating HPL-2 or MUT-7, then mimicking phosphorylation at consensus sites is predicted to promote adaptation in naïve animals. To test this, we replaced the serines and threonines at each predicted EGL-4 phosphorylation site in MUT-7 and each in vitro verified site in HPL-2 with the phosphomimetic, glutamic acid (Mansour et al., 1994). Expression of the phosphomimetic form of MUT-7 in wild type worms had no effect on chemotaxis. Since only ∼50% of known functions of phosphorylated residues can be mimicked by glutamic acid substitutions (Maciejewski et al., 1995) we can make no conclusions about MUT-7 phosphorylation. However, expressing the phosphomimetic form of HPL-2 in wild type animals substantially reduced naïve attraction to butanone while expression of the wild type HPL-2 had no effect (Figure 4E). Thus, mimicking phosphorylation of HPL-2 at EGL-4 target residues is sufficient to promote behavior that resembles the adapted state. When each site was analyzed individually, we found that HPL-2(S155E), which lies in the chromo-shadow domain (CSD), had the greatest effect (Figure S3E).

HPL-2 (all S/T to E) could act as a gain of function mutation that engages the adaptation machinery in the absence of odor, or it could non-specifically diminish AWC function. To distinguish between these possibilities, we expressed HPL-2 (all S/T to E) in mutants that lack the downstream adaptation-promoting factor, OSM-9 (Colbert and Bargmann, 1995). These animals were able to chemotax significantly better to butanone than the parental strain (Figure 4F). Thus HPL-2(all S/T to E) promotes adaptation upstream of OSM-9. We conclude that phosphorylation of HPL-2 at EGL-4 target sites is sufficient to promote adaptation even in the absence of odor-exposure. Importantly, EGL-4 is the only PKG in C. elegans that is required for odor adaptation (Figure S3H). Thus, it is likely that odor acts via EGL-4 to activate HPL-2.

To understand if the siRNA pathway was required for HPL-2(all S/T to E) to induce adaptation, we asked whether mut-7 was required for this gain-of-function phenotype. Loss of function MUT-7 (mut-7(pk204)) suppressed the ectopic adaptation seen in naïve animals expressing HPL-2(all S/T to E) (Figure 4F). Thus, phosphorylation of HPL-2 is both necessary and sufficient for adaptation but it requires fully functional MUT-7. This is consistent with the ChIP studies in Figure 3C that show accumulation of HPL-2 at the odr-1 locus of adapted worms requires functional MUT-7. The observation that HPL-2(allS/T to E) promotes adaptation in the naïve animal, and yet loss of MUT-7 blocks this adaptation, indicates that in the naïve animal there is sufficient MUT-7 dependent RNAi to engage the adaptation process.

Discussion

An emerging paradigm is that small non-coding RNAs provide memory of nonself gene expression (Shirayama et al., 2012); this work extends the role of siRNAs to encoding memory of the environment and experience. We provided evidence that in the olfactory sensory neurons (AWCs) of adult behaving C. elegans, endogenous RNAi promotes odor adaptation by repressing the odr-1 gene (Figure 5). Our data show that in response to prolonged odor exposure, odr-1 directed 22GRNAs increase and this increase is most likely restricted to the AWC neuron (Figure 2B). We demonstrated that these 22GRNAs are loaded onto the nuclear Ago, NRDE-3 (Figure 2D) that acts in AWC (Figure 1G). NRDE-3 may shuttle the odr-1 22GRNA into the AWC nucleus and we have direct evidence that the HP1 homolog, HPL-2, is loaded on to the odr-1 gene in response to odor (Figure 2E). We provide in vitro evidence that HPL-2 can be phosphorylated by nuclear EGL-4 (Figure 4). Mimicking phosphorylation of HPL-2 is sufficient to evoke adaptation behavior. Phosphorylation of HPL-2 would repress the odr-1 gene and ultimately lead to the reduced levels of odr-1 mRNA seen in adapted animals (Figure 2B and 2C). This reduction in odr-1 mRNA correlates strongly with behavior (Figure 2C). One gap in this model is that we do not know if NRDE-3 or odr-1 22GRNA bind the odr-1 locus. An alternate explanation is that odr-1 is repressed by a factor that is itself negatively regulated by a second factor that is repressed by NRDE-3 and the RNAi pathway. In this scheme, the repressive factor that binds to the odr-1 regulatory regions would set up repressive chromatin marks that center at the same part of the odr-1 gene that encodes the odr-1 22GRNA bound by NRDE-3. However, the proposed model is more parsimonious and consistent with the data, than the alternative model, and leads to the exciting hypothesis that RNAi may act broadly as a biological rheostat to allow stimulation to dampen gene expression, and promote cells to alter their responses as a function of previous stimulation.

Figure 5. Prolonged stimulation induces long-term olfactory adaptation in the AWC neurons via an siRNA and chromatin remodeling dependent process.

Figure 5

Open in a new tab

Model for how prolonged butanone stimulation may lead to long-lasting olfactory adaptation in the AWC neuron. Asterisks indicate processes and factors shown to act in AWC. Odor exposure stimulates a seven transmembrane G-protein coupled receptor at the cell membrane and causes EGL-4 to enter the nucleus where it phosphorylates HPL-2 (solid arrow) and may also phosphorylate MUT-7 (dashed arrow). Phosphorylated HPL-2 promotes adaptation, in a 22GRNA dependent process, by binding to H3K9me3 (yellow flags). Phosphorylated MUT-7 boosts levels of odr-1 22G RNAs. These siRNAs act as guides within the NRDE-3 Ago complex to direct H3K9me3 to odr-1 gene. Phosphorylated HPL-2 would repress transcription of the odr-1 gene (red inhibitory bar). Lower levels of odr-1 mRNA decreases the animal's attraction to butanone.

Specificity of Odor Adaptation within AWC neurons

Butanone adaptation does not affect attraction to benzaldehyde and isoamyl alchohol (Colbert and Bargmann 1995) so, how would down regulation of ODR-1, a GC required for all AWC responses (L'Etoile and Bargmann, 2000), specifically adapt the butanone response? The other odors are sensed by both AWCs and butanone is sensed by only one AWC (Wes and Bargmann, 2001). Thus, reducing the levels of ODR-1 in the butanone responsive neuron should not affect chemotaxis mediated by the other AWC. Indeed, prolonged butanone exposure results in nuclear EGL-4 in only one AWC (Lee et al., 2010). Furthermore, each odor requires different factors for adaptation (Colbert and Bargmann, 1995) and, thus, each response may have unique sensitivity to the level of ODR-1.

The Nuclear RNAi Pathway Acts with HP1 in Odor Adaptation

We found that the nuclear Argonaute, NRDE-3 is required in AWC for odor adaptation and it binds odr-1 siRNA in an odor-dependent fashion. Prior work showed that NRDE-3 acts in the nucleus along with NRDE-2, NRDE-1 and NRDE-4 to establish H3K9me3 marks on the target locus, thereby silencing transcription (Burkhart et al., 2011; Burton et al., 2011; Gu et al., 2012; Guang et al., 2010). This connection between endo-siRNA, H3K9me3 marks and gene silencing was originally reported in S. pombe where silencing involves deposition of H3K9me3 marks directed by siRNAs produced from peri-centromeric repeat regions and the mating type locus (Aygun and Grewal, 2010). In pombe, these siRNAs induce a transcriptional silencing complex (RITS) that localizes chromatin to specific nascent transcripts. A feed-forward silencing loop is established as chromodomain proteins, including the HP1 homolog, and methyl-transferases are nucleated by RITS complexes, and in turn recruit more methyl-transferases. Concurrently, RNA-directed RNA polymerase complexes (RDRCs) are recruited, thus increasing siRNA production (Hayashi et al., 2012; Rougemaille et al., 2012; Yamanaka et al., 2013). A direct link between chromatin, RNAi and RITS was demonstrated when the CSD of pombe HP1 was shown to interact with several members of the RNAi and RITS machinery via the HP1 binding protein, Ers1 (Rougemaille et al., 2012). Because Ers1 interacted specifically with the CSD of yeast HP1, and we show that in C. elegans, phosphorylation of this domain is sufficient to induce adaptation, we speculate that the C. elegans HPL-2 CSD likewise nucleates RNAi factors on genes such as odr-1 whose silencing promotes adaptation. Indeed, since loss of mut-7 suppressed the gain of function HPL-2(S155E), MUT-7 may either act along with or downstream of activated HPL-2. Thus, our data is consistent with HPL-2 being recruited to siRNA targeted loci by H3K9me3 marks and perhaps also nucleating an RNAi based feed forward loop in an analogous fashion to its role in S. pombe.

Chromatin Marks in Behavior

HPL-2 loads onto the odr-1 locus in odor adapted AWCs. This may reflect deposition of a heterochromatic mark. Such marks have been implicated in both neuronal development as well as in stimulus induced changes in behavior. H3K9me3 mediated silencing of all but the active olfactory receptor allows for mono-allelic expression of odor receptors in the mammalian nasal epithelium (Magklara et al., 2011). In rodents, behavioral addiction to cocaine has been shown to increase H3K9me2 marks within a key brain reward region (Renthal et al., 2009) and regulation of H3K9 methylation is important for addiction-induced neuroplasticity (Maze et al., 2010). These studies highlight the importance of histone methylation marks in regulating long-term behavioral states and may indicate that recruitment of these marks to specific locations could be a key regulated process. It remains to be seen whether such marks can be directed to genes via the action of endo-siRNAs.

Evidence that mammals have a dicer-dependent class of 22GRNAs is currently lacking (Babiarz et al., 2011). In S. pombe, however, siRNA species derived from protein coding genes were not identified until nuclear exosome deficient cells were used (Yamanaka et al., 2013). Such degradation processes might also conceal endo-siRNAs in higher eukaryotes. Though no RNA-dependent RNA polymerase has yet been identified in mammals, it is possible that other classes of small RNAs such as mitrons (miRNAs processed from introns) play an analogous function in the mammalian brain or that RNA polymerase I, II or III might be recruited to produce small antisense RNAs (Filipovska and Konarska 2000; Lehmann et al., 2007; Greco-Stewart et al., 2009). These RNAs could similarly direct deposition of chromatin marks and affect behavior.

Odor Regulates Chromatin Changes

Our work indicates that an environmental signal is likely to act via a kinase to amplify the small RNA-directed process. Kinases have been widely appreciated to effect behavioral responses: mitogen-activated protein kinases, calcium calmodulin-dependent protein kinase II, protein kinase C and protein kinase A can each contribute to the formation of long-term memory subsequent to repeated training (Dash et al., 2007; Gerstner et al., 2009). Indeed, EGL-4 acts via a histone deacetylase (HDA class I) in the nucleus of uterine epithelium cells to promote egg laying (Hao et al., 2011). Here we demonstrate that the HP1 homolog, HPL-2, is a direct target of this odor-dependent kinase.

In both yeast and mammals, HP1 phosphorylation has been shown to provide a dynamic pathway for the differential regulation of repressive domains in response to inter- and intracellular signals. While many studies highlighted the important role played by modifications of the CD (Shimada and Murakami 2010), our observations suggest that modifications of the CSD may be equally important. The CSD serves as a platform for the assembly of other chromatin (Couteau et al., 2002) and RNAi (Rougemaille et al., 2012) associated proteins, and may therefore represent an attractive target for dynamic regulation of transcriptional states. CSD is required for HP1 homodimerization and formation of an interaction platform with proteins containing the PxVxL interaction motif (Cowieson et al., 2000; Thiru et al., 2004). Though basal silencing requires phosphorylation of the CSD (Zhao and Eissenberg, 2001), our data indicate that CSD phosphorylation may also be used for signal responsive silencing in neurons.

Other kinases may act in a similar fashion to EGL-4 in other cells and organisms to allow developmental or environmental signals to enhance small RNA dependent gene silencing. By regulating RITS, all siRNA-producing loci could be coordinately silenced at a point in time and the ensuing chromatin changes would ensure stable silencing. Such widespread silencing by siRNAs may allow experiences to alter expression of whole cohorts of genes in the context of both development and dynamic behavior.

Experimental Procedures

Worm strains

For a complete list of strains used please consult Supplemental Material. Bristol N2 was the wild-type strain.

Plasmid construction and transgenic strains

Details of plasmid construction can be found in the Supplemental Material.

Behavior

Behavioral assays were conducted on day one adults as described (Colbert and Bargmann, 1995). More details are presented in Supplemental Material. For heat-shock experiments, worms on their original growth plates were exposed to 30°C for 1 hour and then recovered at 20°C for 2 hours prior to behavioral assays.

Kinase assay with nuclear EGL-4

To evaluate nuclear NLS FLAG-EGL-4 kinase activity, 100 μg of worm lysate was immunoprecipiated using anti-FLAG M2 magnetic beads (Sigma-Aldrich). Immunoprecipitates were washed before performing kinase assays were performed directly on the beads with 1.5 μg of substrate (HPL-2 WT, HPL-2(all S/T-A), or Histone H1), 2 μCi 32P ATP (PerkinElmer) and of 25 μM cGMP (Sigma-Aldrich).

Details are in Supplemental Materials.

Isolation of NRDE-3 associated small RNA

50-60 plates of adult animals expressing 3XFLAG∷GFP∷NRDE-3 were collected and half the population was incubated either SBasal alone and the other half with SBasal plus butanone for 80 minutes. Behavior of ∼100 animals from each was assessed. Extracts were made from the remaining animals as described (Guang et al., 2008 and Supplemental Material).

Isolation of HPL-2 associated DNA

podr-3∷3XFLAG∷GFP∷HPL-2 was integrated into the genome and outcrossed five times. 100 plates of adult animals were harvested, half were exposed to buffer and half to butanone and buffer. ∼100 animals from each were assayed. The remaining animals were processed for ChIP (Gerstein et al., 2011). Only populations that showed an adapted CI of 0.05-0.3 were used. Details of the ChIP are in Supplemental Material. Quantitation of coIPed DNA is described in the Supplemental Material.

Quantitative Real-Time PCR

For RNA analysis, 5 plates of day one adult animals were collected, treated to the adaptation protocol and their behavior assessed. Total RNA was isolated as described in Supplemental Material. Total RNA from entire worms was used in 22GRNA and mRNA quantitation as described in Supplemental Materials.

To quantify HPL-2 associated DNA, ChIP results were analyzed by qPCR using Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent Technologies).

The primers were specific to the odr-1, unc-40 or act-3 loci. The levels of the housekeeping gene, act-3 did not change with odor. Please see Supplemental Material for details and primers.

Supplementary Material

01
02

Highlights.

  • Behavioral adaptation requires nuclear RNAi in adult olfactory sensory neurons

  • Odor adaptation increases nuclear Argonaute, NRDE-3-bound small interfering RNAs

  • Odor adaptation increases HPL-2′s association with an endo-siRNA targeted locus

  • HP1 odor responsive kinase target sites are necessary and sufficient for adaptation

Acknowledgments

We thank Maria Gallegos, Mehrdad Matloubian, Bassem Al-Sady, Jonathan Isaiah Gent, Georgia Woods and Robert Blelloch, members of the L'Etoile lab: Damien O'Halloran, Scott Hamilton, Sarah Gerhart, Mary Bethke, Chantal Brueggemann; members of the Kennedy lab: Bethany Buckley and Kirk Burkhart, for critical reading and helpful discussions. We thank Shi-Yu Chen for purified MUT-7 and HPL-2. We thank Ahmed Elewa and Craig Mello for good discussions and worm strains and odr-1 22G RNA sequences; Ebenezer Yamoah for use of his qPCR system; Kohta Ikegami and Susan Strome for ChIP protocols and advice. We also thank Nadia Gronachon, Kim Collins, Christopher Morales, Anu Gupta and Aarati Asundi for help with worm culture; Bethany Buckley, the Caenorhabditis Genetics Center and the National Bioresource Project for numerous strains, and Yuji Kohara for yk cDNA clones. We thank Leah Frater (Anderson lab), Derek Pavelec (Kennedy lab), Jay Maniar and Jonathan Gent (Fire lab), for producing and sharing unpublished sequencing reads. NDL acknowledges support from NSF 0954258 and NIH R01DC005991

Footnotes

Author Contributions: BJ conceived of and carried out experiments and co-wrote the manuscript. LS developed, optimized and performed kinase assays. CG performed RNA and DNA analysis. AG, FP and SK provided unpublished protocols, reagents, support and insight. NL analyzed data and wrote the manuscript.

References

  1. Ashe A, Sapetschnig A, Weick EM, Mitchell J, Bagijn MP, Cording AC, Doebley AL, Goldstein LD, Lehrbach NJ, Le Pen J, Pintacuda G, Sakaguchi A, Sarkies P, Ahmed S, Miska EA. piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans. Cell. 2012;150:88–99. doi: 10.1016/j.cell.2012.06.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aygün O, Grewal SI. Assembly and functions of heterochromatin in the fission yeast genome. Cold Spring Harb Symp Quant Biol. 2010;75:259–267. doi: 10.1101/sqb.2010.75.055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Babiarz JE, Hsu R, Melton C, Thomas M, Ullian EM, Blelloch R. A role for noncanonical microRNAs in the mammalian brain revealed by phenotypic differences in Dgcr8 versus Dicer1 knockouts and small RNA sequencing. RNA. 2011;17:1489–1501. doi: 10.1261/rna.2442211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bargmann CI, Hartwieg E, Horvitz HR. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 1993;74:515–527. doi: 10.1016/0092-8674(93)80053-h. [DOI] [PubMed] [Google Scholar]
  5. Black JC, Allen A, Van Rechem C, Forbes E, Longworth M, Tschop K, Rinehart C, Quiton J, Walsh R, Smallwood A, Dyson NJ, Whetstine JR. Conserved antagonism between JMJD2A/KDM4A and HP1gamma during cell cycle progression. Mol Cell. 2010;40:736–748. doi: 10.1016/j.molcel.2010.11.008. [DOI] [PubMed] [Google Scholar]
  6. Black JC, Whetstine JR. Chromatin landscape: methylation beyond transcription. Epigenetics. 2011;6:9–15. doi: 10.4161/epi.6.1.13331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brower-Toland B, Findley SD, Jiang L, Liu L, Yin H, Dus M, Zhou P, Elgin SC, Lin H. Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 2007;21:2300–2311. doi: 10.1101/gad.1564307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buckley BA, Burkhart KB, Gu SG, Spracklin G, Kershner A, Fritz H, Kimble J, Fire A, Kennedy S. A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality. Nature. 2012;489:447–451. doi: 10.1038/nature11352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burkhart KB, Guang S, Buckley BA, Wong L, Bochner AF, Kennedy S. A pre-mRNA-associating factor links endogenous siRNAs to chromatin regulation. PLoS Genet. 2011;7:e1002249. doi: 10.1371/journal.pgen.1002249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burton NO, Burkhart KB, Kennedy S. Nuclear RNAi maintains heritable gene silencing in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2011;49:19683–19688. doi: 10.1073/pnas.1113310108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Claycomb JM, Batista PJ, Pang KM, Gu W, Vasale JJ, van Wolfswinkel JC, Chaves DA, Shirayama M, Mitani S, Ketting RF, Conte D, Jr, Mello CC. The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell. 2009;139:123–134. doi: 10.1016/j.cell.2009.09.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Colbert HA, Bargmann CI. Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron. 1995;14:803–812. doi: 10.1016/0896-6273(95)90224-4. [DOI] [PubMed] [Google Scholar]
  13. Colbert HA, Bargmann CI. Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans. Learn Mem. 1997;4:179–191. doi: 10.1101/lm.4.2.179. [DOI] [PubMed] [Google Scholar]
  14. Coustham V, Bedet C, Monier K, Schott S, Karali M, Palladino F. The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development. Dev Biol. 2006;297:308–322. doi: 10.1016/j.ydbio.2006.04.474. [DOI] [PubMed] [Google Scholar]
  15. Couteau F, Guerry F, Muller F, Palladino F. A heterochromatin protein 1 homologue in Caenorhabditis elegans acts in germline and vulval development. EMBO Rep. 2002;3:235–241. doi: 10.1093/embo-reports/kvf051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cowieson NP, Partridge JF, Allshire RC, McLaughlin PJ. Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis. Curr Biol. 2000;10:517–525. doi: 10.1016/s0960-9822(00)00467-x. [DOI] [PubMed] [Google Scholar]
  17. Dash PK, Moore AN, Kobori N, Runyan JD. Molecular activity underlying working memory. Learn Mem. 2007;14:554–563. doi: 10.1101/lm.558707. [DOI] [PubMed] [Google Scholar]
  18. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. Processing of primary microRNAs by the Microprocessor complex. Nature. 2004;432:231–235. doi: 10.1038/nature03049. [DOI] [PubMed] [Google Scholar]
  19. Dernburg AF, Zalevsky J, Colaiacovo MP, Villeneuve AM. Transgene-mediated cosuppression in the C. elegans germ line. Genes Dev. 2000;14:1578–1583. [PMC free article] [PubMed] [Google Scholar]
  20. Duchaine TF, Wohlschlegel JA, Kennedy S, Bei Y, Conte D, Jr, Pang K, Brownell DR, Harding S, Mitani S, Ruvkun G, Yates JR, 3rd, Mello CC. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell. 2006;124:343–354. doi: 10.1016/j.cell.2005.11.036. [DOI] [PubMed] [Google Scholar]
  21. Etchberger JF, Lorch A, Sleumer MC, Zapf R, Jones SJ, Marra MA, Holt RA, Moerman DG, Hobert O. The molecular signature and cis-regulatory architecture of a C. elegans gustatory neuron. Genes Dev. 2007;21:1653–1674. doi: 10.1101/gad.1560107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Filipovska J, Konarska MM. Specific HDV RNA-templated transcription by pol II in vitro. RNA. 2000;6:41–54. doi: 10.1017/s1355838200991167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. doi: 10.1038/35888. [DOI] [PubMed] [Google Scholar]
  24. Gao J, Wang WY, Mao YW, Graff J, Guan JS, Pan L, Mak G, Kim D, Su SC, Tsai LH. A novel pathway regulates memory and plasticity via SIRT1 and miR-134. Nature. 2010;466:1105–1109. doi: 10.1038/nature09271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gerstner JR, Lyons LC, Wright KP, Jr, Loh DH, Rawashdeh O, Eckel-Mahan KL, Roman GW. Cycling behavior and memory formation. J Neurosci. 2009;29:12824–12830. doi: 10.1523/JNEUROSCI.3353-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gerstein MB, Lu ZJ, Van Nostrand EL, Cheng C, Arshinoff BI, Liu T, Yip KY, Robilotto R, Rechtsteiner A, Ikegami K. Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project. Science. 2011;330:1775–1787. doi: 10.1126/science.1196914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S, Xu J, Kittler EL, Zapp ML, Weng Z, Zamore PD. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science. 2008;320:1077–1081. doi: 10.1126/science.1157396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009;2:94–108. doi: 10.1038/nrg2504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Greco-Stewart VS, Schissel E, Pelchat M. The hepatitis delta virus RNA genome interacts with the human RNA polymerases I and III. Virology. 2009;386:12–15. doi: 10.1016/j.virol.2009.02.007. [DOI] [PubMed] [Google Scholar]
  30. Grewal, Jia S. Heterochromatin revisited. Nature Reviews Genetics. 2007;8:35–46. doi: 10.1038/nrg2008. [DOI] [PubMed] [Google Scholar]
  31. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell. 2001;106:23–34. doi: 10.1016/s0092-8674(01)00431-7. [DOI] [PubMed] [Google Scholar]
  32. Grishok A, Sinskey JL, Sharp PA. Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. Genes Dev. 2005;19:683–696. doi: 10.1101/gad.1247705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Gu SG, Pak J, Guang S, Maniar JM, Kennedy S, Fire A. Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint. Nat Genet. 2012;44:157–164. doi: 10.1038/ng.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Gu W, Shirayama M, Conte D, Jr, Vasale J, Batista PJ, Claycomb JM, Moresco JJ, Youngman EM, Keys J, Stoltz MJ, Chen CC, Chaves DA, Duan S, Kasschau KD, Fahlgren N, Yates JR, 3rd, Mitani S, Carrington JC, Mello CC. Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol Cell. 2009;36:231–244. doi: 10.1016/j.molcel.2009.09.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Guang S, Bochner AF, Burkhart KB, Burton N, Pavelec DM, Kennedy S. Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription. Nature. 2010;465:1097–1101. doi: 10.1038/nature09095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Guang S, Bochner AF, Pavelec DM, Burkhart KB, Harding S, Lachowiec J, Kennedy S. An Argonaute transports siRNAs from the cytoplasm to the nucleus. Science. 2008;321:537–541. doi: 10.1126/science.1157647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Hao Y, Xu N, Box AC, Schaefer L, Kannan K, Zhang Y, Florens L, Seidel C, Washburn MP, Wiegraebe W, Mak HY. Nuclear cGMP-dependent kinase regulates gene expression via activity-dependent recruitment of a conserved histone deacetylase complex. PLoS Genet. 2011;7:e1002065. doi: 10.1371/journal.pgen.1002065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Hayashi A, Ishida M, Kawaguchi R, Urano T, Murakami Y, Nakayama J. Heterochromatin protein 1 homologue Swi6 acts in concert with Ers1 to regulate RNAi-directed heterochromatin assembly. Proc Natl Acad Sci U S A. 2012;109:6159–6164. doi: 10.1073/pnas.1116972109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Hedgecock EM, Culotti JG, Hall DH. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron. 1990;4:61–85. doi: 10.1016/0896-6273(90)90444-k. [DOI] [PubMed] [Google Scholar]
  40. Kaye JA, Rose NC, Goldsworthy B, Goga A, L'Etoile ND. A 3′UTR pumiliobinding element directs translational activation in olfactory sensory neurons. Neuron. 2009;61:57–70. doi: 10.1016/j.neuron.2008.11.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ketting R. The Many Faces of RNAi. Developmental Cell. 2011;20:148–161. doi: 10.1016/j.devcel.2011.01.012. [DOI] [PubMed] [Google Scholar]
  42. Ketting RF, Haverkamp TH, van Luenen HG, Plasterk RH. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell. 1999;99:133–141. doi: 10.1016/s0092-8674(00)81645-1. [DOI] [PubMed] [Google Scholar]
  43. Knight SW, Bass BL. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science. 2001;293:2269–2271. doi: 10.1126/science.1062039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. L'Etoile ND, Bargmann CI. Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1. Neuron. 2000;25:575–586. doi: 10.1016/s0896-6273(00)81061-2. [DOI] [PubMed] [Google Scholar]
  45. L'Etoile ND, Coburn CM, Eastham J, Kistler A, Gallegos G, Bargmann CI. The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans. Neuron. 2002;36:1079–1089. doi: 10.1016/s0896-6273(02)01066-8. [DOI] [PubMed] [Google Scholar]
  46. Le Thomas A, Rogers AK, Webster A, Marinov GK, Liao SE, Perkins EM, Hur JK, Aravin AA, Toth KF. Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state. Genes Dev. 2013;27:390–399. doi: 10.1101/gad.209841.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Lee HC, Gu W, Shirayama M, Youngman E, Conte D, Jr, Mello CC. C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts. Cell. 2012;150:78–87. doi: 10.1016/j.cell.2012.06.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Lee RC, Hammell CM, Ambros V. Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans. RNA. 2006;12:589–597. doi: 10.1261/rna.2231506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Lee JI, O'Halloran DM, Eastham-Anderson J, Juang BT, Kaye JA, Scott Hamilton O, Lesch B, Goga A, L'Etoile ND. Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation. Proc Natl Acad Sci U S A. 2010;107:6016–6021. doi: 10.1073/pnas.1000866107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Lehmann E, Brueckner F, Cramer P. Molecular basis of RNA-dependent RNA polymerase II activity. Nature. 2007;450:445–449. doi: 10.1038/nature06290. [DOI] [PubMed] [Google Scholar]
  51. Maciejewski PM, Peterson FC, Anderson PJ, Brooks CL. Mutation of serine 90 to glutamic acid mimics phosphorylation of bovine prolactin. J Biol Chem. 1995;270:27661–27665. doi: 10.1074/jbc.270.46.27661. [DOI] [PubMed] [Google Scholar]
  52. Magklara A, Yen A, Colquitt BM, Clowney EJ, Allen W, Markenscoff-Papadimitriou E, Evans ZA, Kheradpour P, Mountoufaris G, Carey C, Barnea G, Kellis M, Lomvardas S. An epigenetic signature for monoallelic olfactory receptor expression. Cell. 2011;145:555–570. doi: 10.1016/j.cell.2011.03.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ahn NG. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994;265:966–970. doi: 10.1126/science.8052857. [DOI] [PubMed] [Google Scholar]
  54. Maze I, Covington HE, 3rd, Dietz DM, LaPlant Q, Renthal W, Russo SJ, Mechanic M, Mouzon E, Neve RL, Haggarty SJ, Ren Y, Sampath SC, Hurd YL, Greengard P, Tarakhovsky A, Schaefer A, Nestler EJ. Essential role of the histone methyltransferase G9a in cocaine-induced plasticity. Science. 2010;327:213–216. doi: 10.1126/science.1179438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. McNeill E, Van Vactor D. MicroRNAs shape the neuronal landscape. Neuron. 2012;75:363–379. doi: 10.1016/j.neuron.2012.07.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. O'Halloran DM, Altshuler-Keylin S, Lee JI, L'Etoile ND. Regulators of AWC-mediated olfactory plasticity in Caenorhabditis elegans. PLoS Genet. 2009;5:e1000761. doi: 10.1371/journal.pgen.1000761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. O'Halloran DM, Hamilton OS, Lee JI, Gallegos M, L'Etoile ND. Changes in cGMP levels affect the localization of EGL-4 in AWC in Caenorhabditis elegans. PLoS One. 2012;7:e31614. doi: 10.1371/journal.pone.0031614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Pak J, Maniar JM, Mello CC, Fire A. Protection from feed-forward amplification in an amplified RNAi mechanism. Cell. 2012;151:885–899. doi: 10.1016/j.cell.2012.10.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Rajasethupathy P, Antonov I, Sheridan R, Frey S, Sander C, Tuschl T, Kandel ER. A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity. Cell. 2012;149:693–707. doi: 10.1016/j.cell.2012.02.057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Renthal W, Kumar A, Xiao G, Wilkinson M, Covington HE, 3rd, Maze I, Sikder D, Robison AJ, LaPlant Q, Dietz DM, Russo SJ, Vialou V, Chakravarty S, Kodadek TJ, Stack A, Kabbaj M, Nestler EJ. Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins. Neuron. 2009;62:335–348. doi: 10.1016/j.neuron.2009.03.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Rougemaille M, Braun S, Coyle S, Dumesic PA, Garcia JF, Isaac RS, Libri D, Narlikar GJ, Madhani HD. Ers1 links HP1 to RNAi. Proc Natl Acad Sci U S A. 2012;109:11258–11263. doi: 10.1073/pnas.1204947109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Shimada A, Murakami Y. Dynamic regulation of heterochromatin function via phosphorylation of HP1-family proteins. Epigenetics. 2010;5:30–33. doi: 10.4161/epi.5.1.10605. [DOI] [PubMed] [Google Scholar]
  63. Shirayama M, Seth M, Lee HC, Gu W, Ishidate T, Conte D, Jr, Mello CC. piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell. 2012;150:65–77. doi: 10.1016/j.cell.2012.06.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Simmer F, Tijsterman M, Parrish S, Koushika SP, Nonet ML, Fire A, Ahringer J, Plasterk RH. Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr Biol. 2002;12:1317–1319. doi: 10.1016/s0960-9822(02)01041-2. [DOI] [PubMed] [Google Scholar]
  65. Stringham EG, Dixon DK, Jones D, Candido EP. Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans. Mol Biol Cell. 1992;3:221–233. doi: 10.1091/mbc.3.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell. 1999;99:123–132. doi: 10.1016/s0092-8674(00)81644-x. [DOI] [PubMed] [Google Scholar]
  67. Tabara H, Yigit E, Siomi H, Mello CC. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DexH-Box helicase to direct RNAi in C. elegans. Cell. 2002;109:861–871. doi: 10.1016/s0092-8674(02)00793-6. [DOI] [PubMed] [Google Scholar]
  68. Thiru A, Nietlispach D, Mott HR, Okuwaki M, Lyon D, Nielsen PR, Hirshberg M, Verreault A, Murzina NV, Laue ED. Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin. EMBO J. 2004;23:489–499. doi: 10.1038/sj.emboj.7600088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Tops BB, Tabara H, Sijen T, Simmer F, Mello CC, Plasterk RH, Ketting RF. RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans. Nucleic Acids Res. 2005;33:347–355. doi: 10.1093/nar/gki183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Vasquez-Rifo A, Jannot G, Armisen J, Labouesse M, Bukhari SIA, et al. Developmental Characterization of the MicroRNA-Specific C. elegans Argonautes alg-1 and alg-2. PLoS ONE. 2012;7(3):e33750. doi: 10.1371/journal.pone.0033750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Vastenhouw NL, Brunschwig K, Okihara KL, Muller F, Tijsterman M, Plasterk RH. Gene expression: long-term gene silencing by RNAi. Nature. 2006;442:882. doi: 10.1038/442882a. [DOI] [PubMed] [Google Scholar]
  72. Wes PD, Bargmann CI. C. elegans odour discrimination requires asymmetric diversity in olfactory neurons. Nature. 2001;410:698–701. doi: 10.1038/35070581. [DOI] [PubMed] [Google Scholar]
  73. Yamanaka S, Mehta S, Reyes-Turcu FE, Zhuang F, Fuchs RT, Rong Y, Robb GB, Grewal SI. RNAi triggered by specialized machinery silences developmental genes and retrotransposons. Nature. 2013;493:557–560. doi: 10.1038/nature11716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Yigit E, Batista PJ, Bei Y, Pang KM, Chen CC, Tolia NH, Joshua-Tor L, Mitani S, Simard MJ, Mello CC. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell. 2006;127:747–757. doi: 10.1016/j.cell.2006.09.033. [DOI] [PubMed] [Google Scholar]
  75. Zhang C, Montgomery TA, Gabel HW, Fischer SE, Phillips CM, Fahlgren N, Sullivan CM, Carrington JC, Ruvkun G. mut-16 and other mutator class genes modulate 22G and 26 G siRNA pathways in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2011;108:1201–8. doi: 10.1073/pnas.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Zhao T, Heyduk T, Eissenberg JC. Phosphorylation site mutations in heterochromatin protein 1 (HP1) reduce or eliminate silencing activity. J Biol Chem. 2001;276:9512–9518. doi: 10.1074/jbc.M010098200. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS514739-supplement-01.pdf (2.9MB, pdf)
02
NIHMS514739-supplement-02.pdf (206KB, pdf)

RESOURCES