A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality

Abstract

Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, 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 Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

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

(a) pie-1::gfp::h2b expressing animals were exposed to gfp dsRNA. F1 progeny were grown in the absence of dsRNA, and gfp expression in oocytes was visualized with fluorescence microscopy. WT, wild-type. % fluorescent animals is indicated, n>100. rde-1 is required for RNAi (17). (b) GFP::HRDE-1 was visualized by fluorescent microscopy. Small arrow, L1 animal; inset, magnification showing GFP::HRDE-1 in primordial germ cells. Large arrow, oocyte nucleus. (c) 3xFLAG::HRDE-1 was immunoprecipitated with a-FLAG antibody and HRDE-1 co-precipitating RNA was isolated and oma-1 siRNAs were quantified with oma-1 TaqMan probe set. Data is expressed as a ratio (+/− oma-1 RNAi) (n=3, +/− s.d.). Note: RNA dependent RNA Polymerases likely maintain HRDE-1 siRNA populations during RNAi inheritance (see supplemental discussion) (d) pie-1::gfp::h2b fluorescence was scored in hrde-1(+/−)/(+/+) or hrde-1(−/−) inheriting progeny. The hrde-1(−) chromosome was marked with unc-93 (unc-93 is ~1.3 cM from hrde-1), and hrde-1 genotypes were inferred by Unc phenotypes. % fluorescent animals is indicated.

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

(a) pie-1::gfp::h2b fluorescence is shown. >98% of animals of each genotype exhibited phenotypes similar to that of image. (b) P0 animals expressing pie-1::gfp::h2b were exposed to gfp dsRNA. F1 progeny were exposed to nrde-2 or oma-1 dsRNA (n=3). (c) FLAG::NRDE-2 was precipitated with a-FLAG antibody and NRDE-2 co-precipitating oma-1 pre-mRNA was quantified with qRT-PCR using exon/intron primer sets. Data are expressed as ratio (+/−) oma-1 RNAi. (n=3, +/− s.e.m.). (d) The F1 progeny of oma-1 dsRNA-treated animals were subjected to H3K9me3 Chromatin Immunoprecipiation (ChIP). Co-precipitating oma-1 DNA was quantified with qRT-PCR. Data were normalized to co-precipitating eft-3 DNA and expressed as a ratio +/− oma-1 RNAi (1=no change). X-axis, 0 denotes predicted start codon of oma-1. (bp), base pair. rde-4 is required for RNAi (18) (n=3–4, +/− s.e.m.).

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

(a) H3K9me3 in mutant (y-axis) vs. wild type (x-axis, N2). Levels of H3K9me3 at C. elegans genes are ratio of IP nucleosome/ input nucleosome counts. smg-1 (positive control) (8) (blue dot) and nrde-2/4-dependent genes (red) are highlighted. N2 and nrde-2 ChIP-seq data were published previously (accession number: GSE32631) (8). Other ChIP-seq data, and HRDE-1 siRNA data (accession number: GSE38041) (b) FLAG::HRDE-1 co-precipitating RNA was ³²P-radiolabeled and analyzed by PAGE. (nt) nucleotide. (c) Example of HRDE-1 target gene. (d) qRT-PCR quantification of H3K9me3 ChIP (animals grown at 25°C) (+/− HRDE-1) at eleven genes targeted by HRDE-1 siRNAs (HRDE-1 targets). Four genes, which are not targeted by HRDE-1 siRNAs and exhibit Nrde-independent H3K9me3 in the germline (non-HRDE-1 targets), do not exhibit H3K9me3 loss, showing that H3K9me3 loss in hrde-1 mutants is not simply due to loss of germ cells. Data is expressed as % of input DNA recovered by ChIP (n=3, +/− s.d.). (e) Total RNA was isolated from +/− HRDE-1 animals (25°C) and qRT-PCR was used to quantify mRNA or pre-mRNA levels from eleven HRDE-1 targeted genes. Three genes not targeted by HRDE-1 siRNAs, but expressed in germ cells, are also shown. Data is normalized to nos-3 mRNA (germline only) and expressed as a ratio (hrde-1(−)/wild-type) (n=3, +/− s.d.). (f) dpy-17 or hrde-1; dpy-17 animals were out-crossed 5x, and Dpy adult animals (P0) and adult progeny (F1, F2, F6) were isolated (20°C) and H3K9me3 was quantified with qRT-PCR. Data are expressed as a ratio (wild-type/hrde-1(−)) (n=1 for P0-F1, and n=3 for F2 and F6, +/− s.e.m.). Note: in this panel increased H3K9me3 signal means loss of H3K9me3 in hrde-1(−) animals.

Figure 4. The RNAi inheritance machinery promotes germline immortality

(a) Animals of indicated genotypes were out-crossed to wild-type 2–4x and brood sizes scored across generations at 25°C. n=5, +/− s.e.m. Note: for unknown reasons, nrde-1 and nrde-4 mutants are Mrt at both 20°C and 25°C, but hrde-1 and nrde-2 mutants are only Mrt ~25°C (n=4). (b) hrde-1(−) animals were out-crossed 3x and (+/+) or (−/−) siblings were isolated. Gonads were isolated and stained with DAPI (DNA), and immuno-fluorescence was used to detect sperm (green) and oocytes (red) (see Methods) in F1 and F5 generations. Scale bar = 100µm.