piRNAs Can Trigger a Multigenerational Epigenetic Memory in the Germline of C. elegans

SummaryTransgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C.elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.Graphical AbstractHighlights► Multigenerational inheritance and piRNAs converge on same nuclear silencing pathway ► HRDE1/WAGO-9 and chromatin factors required for inheritance of piRNA silencing ► piRNAs can induce multigenerational silencing for more than 20 generations. ► Long-term memory independent of piRNA triggers but remains dependent on nuclear pathwayMultigenerational inheritance and piRNAs converge on same silencing pathway, in which both nuclear WAGOs and chromatin factors are required. The piRNA trigger can be lost, but the nuclear silencing pathway maintains the silencing for more than 20 generations

Piwi and piRNAs Act Upstream of an Endogenous siRNA Pathway to Suppress Tc3 Transposon Mobility in the Caenorhabditis elegans Germline

The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function

<i>henn-1</i> affects 21UR1 sensor activity.

<p>(A) Activity of a transgene expressing GFP (21UR1 sensor), silenced by 21U species 21UR1 in wild-type, <i>prg-1(pk2298)</i> and <i>henn-1(pk2295)</i> mutant backgrounds. The gonads are outlined with a white dashed line. (B) Northern blot analysis of 21UR1 in young adult animals of the indicated genotypes. “Sensor” refers to the 21UR1 sensor also shown in panel A. Signal intensities are related to <i>let-7</i>, and the 21UR1:<i>let-7</i> ratio in N2 is set at one. The 21-mer and 20-mer signals of the 21UR1 probe have also been quantified separately. The signal intensity of the 20-mer relative to the total 21UR1 signal is presented. Repetition of this blot with two independent biological samples has shown that the apparent differences are not reproducible. The 22G-sensor blot shows the signals obtained after hybridizing probes homologous to the 21UR1 sensor.</p

HENN-1 is the <i>C. elegans</i> homolog of Hen1.

<p>(A) Left panel: protein gel stained with PageBlue shows purified GST, GST-HENN-1 and GST-HENN-1(D151N) proteins used to perform methyltransferase assays as shown in the right panel and B. Right panel: <i>in vitro</i> methyltransferase activity assay. RNA oligos were incubated with indicated proteins and 14C-labelled SAM. Reaction products were run on a 12% acryl-amide gel. (B) <i>In vitro</i> methyltransferase assay using different RNA substrates each differing in the identity of the most 3′ nucleotide. (C) Northern blot analysis using RNA from wild-type, <i>henn-1(pk2452)</i>, <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> animals. Blots were probed for 21UR1 and 26G species siR26-263. Probing for <i>let-7</i> serves as loading and as oxidation-β-elimination control. (D) Response of wild type (N2), <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:henn-1:GFP</i> to <i>dpy-13</i> RNAi. <i>Henn-1(pk2295)</i> sensitivity is significantly higher than both controls (two-tailed t-test, n = 5: p<0.05 for both). (E) Response of wild type (N2), <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> to <i>pos-1</i> RNAi, delivered at three different dosages: undiluted (100%), diluted one to one (50%) and diluted one to four (25%). At 50% <i>pos-1</i> RNAi, <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> animals display significant rescue (p = 0.01) of the <i>henn-1(pk2295)</i> RNAi defect (p<0.0005). The p-values at 25% <i>pos-1</i> RNAi are p = 0.04 for both the <i>henn-1(pk2295)</i> RNAi defect and the rescue. P-values were calculated with a two-tailed t-test, n = 10.</p

Global effects of <i>henn-1</i> on 21U RNAs.

<p>(A) Bar diagram displaying the different annotated small RNA reads obtained after deep-sequencing. Reads from structural RNAs were removed before analysis. (B) The expression level of 21U RNAs in wild-type and <i>henn-1</i> mutant backgrounds. Total 21U reads are normalized to total miRNA reads. The differences between wild-type and <i>henn-1</i> mutant samples are significant (Chi-squared test; p<10⁻¹⁰). (C) Length distribution plot of 21U RNAs. (D) Scatter plot displaying the fraction of 20-mer species of individual 21U RNA loci that were represented by at least 250 raw reads (20+21-mers) in each of the libraries used for this analysis (<i>henn-1(pk2452)</i> and <i>henn-1(pk2295)</i>). (E) Scatter plot displaying individual 21U loci represented by at least 250 raw reads (20+21-mers) in each of the libraries used for this analysis (wild-type and <i>henn-1(pk2295)</i>). X-axis: fold loss of reads in the <i>henn-1(pk2295)</i> background relative to wild-type. Y-axis: fold increase of 20-mer species in the <i>henn-1(pk2295)</i> background relative to wild-type. (F) Bar diagram displaying the frequencies of non-templated base additions found on 21U reads, as a percentage of the total 21U read count.</p

Effects of <i>henn-1</i> 22G and 26G RNAs.

<p>(A) Length distribution plots of ‘siRNA’ category, containing both 22G and 26G RNAs, in diverse libraries. (B) Bar diagram displaying the frequencies of non-templated base additions found on 26G reads, as a percentage of the total 26G read count. P<0.0001 for <i>henn-1(pk2452)</i> and <i>henn-1(pk2295)</i> relative to wild-type (Chi-squared test). (C) Ratio of ALG-3/4 and ERGO-1 bound 26G RNAs as derived by previously described annotation (see main text), in diverse libraries. P-values of all differences <0.001 (Chi-squared test). (D) The 22G counts, in rpm, for all genes in total, ERGO-1, ALG-3/4 and CSR-1 target genes. 22G count for ‘Total’ was divided by 100 for better visualization.</p

HENN-1 expression analysis.

<p>(A) Western blot analysis for HENN-1 expression in different mutant backgrounds. <i>Glp-4(bn2)</i> animals contain almost no germ cells. Tubulin is shown as loading control. (B) Western blot analysis for HENN-1 expression at different time points during development of <i>C. elegans</i>. <i>Glp-4(bn2)</i> animals contain almost no germ cells. Tubulin is shown as loading control. (C) Confocal images (single z-plane) of HENN-1::GFP expressing animals at different developmental stages. Nuclei of embryonic germ cells are outlined in white boxes. Blastomere identities in the four-cell stage embryos are indicated. Scale bars are 10 µm. (D) Immuno-fluorescence with anti-PGL-1 and anti-GFP antibodies. The white arrowhead indicates a site of co-localization. Scale bars are 10 µm. (E) Western blots for HENN-1 (top) and PRG-1 (bottom) on fractions obtained after gel filtration.</p

Differential Impact of the HEN1 Homolog HENN-1 on 21U and 26G RNAs in the Germline of Caenorhabditis elegans

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