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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nature. Author manuscript; available in PMC 2013 March 20.
Published in final edited form as:
PMCID: PMC3509936
NIHMSID: NIHMS412246

A nuclear Argonaute promotes multi-generational epigenetic inheritance and germline immortality

Abstract

Epigenetic information is frequently erased near the start of each new generation (1). In some cases, however, epigenetic information can be transmitted from parent to progeny (epigenetic inheritance) (2). A particularly striking example of epigenetic inheritance is dsRNA-mediated gene silencing (RNAi) in C. elegans, which can be inherited for more than five generations (38). To understand this process we conducted a genetic screen for animals defective for transmitting RNAi silencing signals to future generations. This screen identified the gene heritable RNAi defective (hrde)-1. hrde-1 encodes an Argonaute (Ago) that associates with small interfering (si)RNAs in germ cells of the progeny of animals exposed to dsRNA. In nuclei of these germ cells, HRDE-1 engages the Nrde nuclear RNAi pathway to direct H3K9me3 at RNAi targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed siRNAs, which direct nuclear gene silencing in germ cells. In hrde-1 or nuclear RNAi deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Ago HRDE-1 directs gene-silencing events in germ cell nuclei, which drive multi-generational RNAi inheritance and promote immortality of the germ cell lineage. We propose that C. elegans uses the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

We conducted a genetic screen to identify factors required for multi-generational RNAi inheritance. We mutagenized animals carrying a germline gfp reporter gene and screened for mutant animals that retained the ability to silence gfp when exposed directly to gfp dsRNA, but failed to silence gfp in subsequent generations. Among fourteen mutant alleles fulfilling these criteria, four of these alleles defined a gene we term here heritable RNAi defective (hrde)-1 (Fig. S2). hrde-1 mutant animals silenced GFP when exposed to gfp dsRNA, but failed to transmit this silencing to subsequent generations (Fig. 1a, and Fig. S3). Similarly, hrde-1 mutants silenced the germline expressed oma-1 gene when treated directly with oma-1 dsRNA, but were defective for silencing inheritance (Fig. S4). We conclude that hrde-1 promotes multi-generational RNAi silencing in the germline.

Figure 1
hrde-1 encodes a nuclear Ago that acts in inheriting generations to promote multi-generational germline RNAi inheritance

We mapped hrde-1 to a genomic region containing the gene c16c10.3. c16c10.3 is predicted to encode an Ago not known to contribute to gene-silencing (9,10). c16c10.3 encodes a predicted bipartite nuclear localization signal (NLS), a PAZ, and a PIWI domain (Fig. S5). We found that hrde-1 is c16c10.3 (Fig. S5). c16c10.3 was referred to once previously in the literature as worm Ago (wago)-9 (10). Henceforth, we refer to this Ago as hrde-1/wago-9. hrde-1 is a member of a worm-specific clade of Agos (wagos) (9). HRDE-1 appeared to be relatively unique amongst the WAGOs in its contribution to germline RNAi inheritance (Fig. S6). We constructed a fusion gene between gfp and a full-length genomic copy of hrde-1 (gfp::hrde-1). gfp::hrde-1 rescued RNAi inheritance in hrde-1(−) animals, indicating that GFP::HRDE-1 is functional (Fig. S5). GFP::HRDE-1 was expressed in nuclei of male and female germ cells (Fig. 1b, and Fig. S7). These data indicate that hrde-1 encodes a germline Ago that localizes to the nucleus.

HRDE-1 could conceivably promote multi-generational RNAi inheritance by acting in animals exposed directly to dsRNA (RNAi generation) or by acting in the progeny of these animals (inheriting generation). In C. elegans, dsRNA exposure induces the expression of siRNAs in inheriting generations ((7,8) and Fig S8). HRDE-1 co-precipitated with siRNAs for multiple generations after RNAi, consistent with the idea that HRDE-1 acts in inheriting generations to promote RNAi inheritance (Fig. 1c). Note: maintenance of HRDE-1 siRNA populations across generations is likely mediated by RdRPs (see supplemental discussion). The following genetic analyses confirmed that HRDE-1 acts in inheriting generations. Animals that were hrde-1(+/−) in the RNAi generation, but were hrde-1(−/−) in the F1 inheriting generation, failed to inherit RNAi silencing (Fig. 1d, and Table S1). Similarly, HRDE-1 activity was required in the F2 generation for F1 to F2 RNAi inheritance, and in the F3 generation for F2 to F3 RNAi inheritance (Fig. 1d). Conversely, animals that lacked HRDE-1 in the RNAi generation, but expressed HRDE-1 in the inheriting generation, were able to inherit RNAi silencing (Table S1). Thus, HRDE-1 acts in inheriting progeny to facilitate the memory of RNAi silencing events that occurred in previous generations. Altogether, these data establish that C. elegans possess machinery dedicated to propagating epigenetic information across generational boundaries.

The nuclear RNAi factors NRDE-1/2/3/4 comprise a sub-branch of the C. elegans RNAi silencing machinery that is required for dsRNA-based silencing of nuclear-localized RNAs (1113). According to our current model, siRNAs bound to the somatically expressed Ago NRDE-3 recognize and bind nascent RNA transcripts and recruit NRDE-1/2/4 (termed downstream Nrde factors) to genomic sites of RNAi in somatic cells. Together, the Nrde factors direct nuclear gene silencing events, which include the deposition of the repressive chromatin mark histone H3 lysine-9 me3 (H3K9me3), and the inhibition of RNA Polymerase II elongation (1113). The Nrde factors contribute to heritable gene silencing events that are manifest in somatic cells (7). Five lines of evidence indicate that HRDE-1 engages the downstream Nrde factors to direct nuclear RNAi, and, consequently, RNAi inheritance in germ cells. First, the downstream Nrde factors were required for gfp or pos-1 germline RNAi inheritance (Fig. 2a, and Fig. S9). Second, like HRDE-1, the downstream Nrde factor NRDE-2 acted in inheriting generations to promote memory of RNAi in germ cells (Fig. 2b). Third, HRDE-1 was required for RNAi-mediated recruitment of NRDE-2 to a germline pre-mRNA, indicating that HRDE-1 acts as a specificity factor in germ cells to recruit a downstream Nrde factor to genomic sites of RNAi (Fig. 2c). Fourth, the ability of dsRNA to induce H3K9me3 was lost in mutant strains that eliminate hrde-1 or the downstream Nrde factors (Fig. 2d, Fig. S10, and see supplemental discussion). Fifth, consistent with the idea that hrde-1 and the downstream Nrde factors act together in the germline, hrde-1(−) animals share a germline mortality phenotype with nrde-1/2/4(−) animals (see below). These data indicate that NRDE-1/2/4 are required for multi-generational RNAi inheritance and support a model whereby HRDE-1 and NRDE-1/2/4 comprise a germline RNAi pathway that drives RNAi inheritance by inducing gene silencing in the nuclei of inheriting progeny. Henceforth, we refer to HRDE-1 and NRDE-1/2/4 as the germline RNAi inheritance machinery.

Figure 2
HRDE-1 engages the Nrde nuclear RNAi pathway to direct multi-generational RNAi inheritance

We asked if, under normal reproductive conditions, the germline RNAi inheritance machinery transmits endogenous RNAi silencing signals across generations. To test this idea, we first used H3K9me3 as a read-out for endogenous nuclear RNAi in germ cells. We isolated wild-type or nrde-2/3/4(−) animals, conducted H3K9me3 ChIP, and subjected H3K9me3 co-precipitating nucleosome core DNA to high-throughput sequencing (8). We identified 320 predicted genes that were depleted for H3K9me3 >2 fold in both nrde-2(−) and nrde-4(−) animals relative to wild-type (Fig. 3a, and Table S2). H3K9me3 ChIP, followed by directed qRT-PCR analysis, confirmed the nrde-2/4 dependence of H3K9me3 at four out of four of these loci (data not shown). Nrde-dependent H3K9me3 was present in germ cells; in glp-4(ts) mutants (14), which lack most germ cells, H3K9me3 was significantly reduced at most nrde-2/4-dependent sites (Fig. 3a, p-value 2×10−13). Together, these data show that nrde-1/2/4 contribute to H3K9me3 at multiple loci in germ cells. Henceforth, we refer to these loci as the endogenous Nrde “germline target genes”.

Figure 3
The RNAi inheritance machinery transmits endogenous epigenetic information across generations

HRDE-1 co-precipitated with endogenous small RNAs (Fig. 3b). We sequenced these small RNAs and found that HRDE-1 bound 22G endogenous (endo) siRNAs, which were expressed in germ cells, and were antisense to ~1500 predicted coding genes (Table S2, and Fig. S11). HRDE-1 22G siRNAs also targeted pseudogenes and cryptic loci (Table S2). 22G siRNAs are synthesized by RNA dependent RNA Polymerases (RdRPs) acting on cellular RNAs templates (10,15), suggesting that the HRDE-1 22G siRNAs are synthesized via RdRP activity in germ cells (see supplemental discussion). Three lines of evidence link HRDE-1 to the regulation of gene expression at Nrde germline target genes. First, we observed a correlation between genomic sites homologous to the most abundant (top 200) HRDE-1 bound siRNAs and genomic sites depleted for H3K9me3 in nrde-2/4(−) animals (p-value 2×10−16) (Fig. 3c, Fig. S12, Table S2). Second, we conducted H3K9me3 ChIP on hrde-1(−) animals and quantified H3K9me3 at fourteen Nrde germline target genes. At thirteen of these loci, H3K9me3 was depleted in hrde-1(−) animals (Fig. 3d, and Fig. S13). Third, we observed increased pre-mRNA expression from many germline target genes in hrde-1(−) animals, indicating that the RNAi inheritance machinery silences germline target genes co-transcriptionally during the normal course of reproduction (Fig. 3e). These data indicate that HRDE-1 contributes to H3K9me3 in the germline and support a model whereby HRDE-1 uses 22G endo siRNAs as specificity factors to direct nuclear RNAi in germ cells.

We asked if HRDE-1-mediated nuclear RNAi at germline target genes was heritable. We out-crossed hrde-1(−) animals with wild-type animals, isolated hrde-1(−) progeny, and conducted H3K9me3 ChIP on these hrde-1(−) animals and their progeny. H3K9me3 at germline target genes was progressively lost over generations in hrde-1(−) animals (Fig. 3f, Fig. S14). Similar results were seen with nrde-1(−) animals (Fig. S14). Coincident with loss of H3K9me3, germline target gene over-expression became more pronounced in late generations hrde-1(−) animals (Fig. S15). These data show that the RNAi inheritance machinery transmits endogenous gene regulatory information across generational boundaries.

Why might an organism transmit gene regulatory information across generations? During the course of our studies, we noticed that our RNAi inheritance defective strains would periodically become sterile; stock plates would contain hundreds of adults, but no progeny. We hypothesized that the RNAi inheritance machinery might be required to maintain the integrity of the germ cell lineage. To test this idea, we out-crossed two independently isolated alleles each of hrde-1(−) and nrde-1/2/4(−) to wild-type and then monitored fertility across generations. After out-crossing hrde-1(−) and nrde-1/2/4(−) animals exhibited near wild-type fertility (early generations), but became sterile in subsequent generations (late generations) (Fig. 4a, and Fig. S16). These data show that RNAi inheritance defective animals exhibit a mortal germline (Mrt) phenotype (16). Animals lacking the somatic Ago NRDE-3 were not Mrt (Fig. S17). The Mrt phenotype of hrde-1(−) animals was temperature sensitive: hrde-1(−) animals were Mrt at 25°C, but not 20°C, indicating that growth at higher temperatures is required to reveal defects associated with loss of HRDE-1 (Fig. S18). Most late generation hrde-1(−) mutants (grown at 25°C) failed to produce mature oocytes or sperm, showing that one reason hrde-1(−) animals do not produce progeny is due to defects in gametogenesis (Fig. 4b, and Fig. S19). 26% of late generation hrde-1(−) animals were able to produce sperm and oocytes (Fig. 4b). Most of these gametes, however, are unlikely to be functional as fecundity of hrde-1(−) animals in this late generation was only 1% that of wild-type animals (Fig. 4a). Finally, late generation hrde-1(−) animals exhibited a high incidence of male (Him) phenotype, suggesting that loss of RNAi inheritance may cause defects in chromosome pairing and/or segregation (Fig. S19). We conclude that the RNAi inheritance machinery is required to maintain the immortality of the germline and that, over generations, disabling the RNAi inheritance machinery causes progressive and diverse defects in germ cell formation and function.

Figure 4
The RNAi inheritance machinery promotes germline immortality

Here we show that C. elegans possess dedicated regulatory machinery that promotes an epigenetic memory of RNAi-silencing events that occurred in distant ancestors (Fig. S1). The Ago HRDE-1 is at the heart of this process, binding heritable specificity determinants (siRNAs) to direct nuclear RNAi and promote RNAi inheritance in germ cells. Nuclear RNAi also promotes RNA inheritance in somatic cells (7), indicating that nuclear gene silencing events promote RNAi inheritance in both somatic and germ cells. Finally, we show that the germline RNAi inheritance machinery transmits endogenous epigenetic information across generational boundaries while promoting germline immortality (Fig. S1). Our data suggest a model in which endogenous heritable RNAs that engage HRDE-1 act as specificity factors to direct epigenomic maintenance and immortality of the germ cell lineage. Additional work is needed to determine how defects in epigenomic maintenance relate to germline mortality (see supplemental discussion). We note, however, that both processes depend upon the same complement of factors (hrde-1 and nrde-1/2/4), and in animals lacking these factors, defects in epigenome maintenance and defects in germ cell viability are coincident over generational time. Therefore, we propose that one biological function of the RNAi inheritance machinery is to transmit “germline immortality” small RNAs, selected during species evolution for their ability to promote fertility, across generational boundaries to promote fertility in future generations.

Methods Summary

RNAi. RNAi experiments were conducted as described previously (11). The oma-1 and pos-1 constructs were taken from the Ahringer library.

RNA IP (RIP). RIPs were performed as described previously (11), with the exception that adult animals were used for all RIPs. Adult animals were frozen and dounced 10x prior to RIP. FLAG::NRDE-2 protein was immunoprecipitated with anti-FLAG M2 antibody (Sigma, A2220).

Chromatin IP (ChIP). ChIP experiments were performed as described previously (12), except that gravid adult animals were used. Worms were frozen prior to cross linking and were dounced 10x prior to sonicating. H3K9me3 antibody was from Upstate (07–523).

oma-1 siRNA TaqMan assay. TaqMan assay was performed as described previously (7). TaqMan probe set #1 was used in Fig. 2a (see Supplemental Methods).

Unless indicated otherwise, the following mutant alleles were used in this study: hrde-1(tm1200), nrde-1(gg088), nrde-2(gg091), nrde-3(gg066), nrde-4(gg129).

Supplementary Material

2

Acknowledgements

We thank Dr. Phil Anderson, Dr. Hopa Licious, and Dr. David Wassarman for thoughtful discussions. We thank Dr. Shawn Ahmed and member of the Ahmed lab for sharing unpublished data concerning the role of nrde-1 in germline immortality. This work was supported by grants from the Pew and Shaw scholar’s programs, and the National Institutes of Health GM88289 (S.K), GM37706 (A.F.), and (JK).

Footnotes

Full Methods and any associated references are available in the online version of this paper at www.nature.com/nature.

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Author contributions: B.B. contributed to Fig. 1abc, 2bd, S3, S4, S5b, S6, S8, S10, S13, K.B. contributed to Fig. 2c, 3def, 4ab, S13, S14, S15, S16abc, S17, S18, S19c, S.G.G. and A.F. contributed to Fig. 3ac, Table S2, Figs. S11, S12, G.S. contributed to Fig. S2, S5A, S16d, A.K. and J.K contributed to Fig. 4b, S7, S19a, H.F. contributed to Fig. 4a, S16abc, S17, S18, S19c, S.K. to contributed to Fig. 1acd, 2a, 3b, Table S1, Figs. S2, S6b, S9, S10b, S19b. S.K, B.B, and K.B. wrote the manuscript.

Accession numbers: nrde-2 ChIP-seq data (published previously): GSE32631. Other ChIP-seq and HRDE-1 siRNA data: GSE38041

Competing financial interests: The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature.

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