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Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants

Abstract

PIWI-interacting RNAs (piRNAs) promote fertility in many animals. However, whether this is due to their conserved role in repressing repetitive elements (REs) remains unclear. Here, we show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is not caused by derepression of REs or other piRNA targets but, rather, is mediated by epigenetic silencing of all of the replicative histone genes. In the absence of piRNAs, downstream components of the piRNA pathway relocalize from germ granules and piRNA targets to histone mRNAs to synthesize antisense small RNAs (sRNAs) and induce transgenerational silencing. Removal of the downstream components of the piRNA pathway restores histone mRNA expression and fertility in piRNA mutants, and the inheritance of histone sRNAs in wild-type worms adversely affects their fertility for multiple generations. We conclude that sRNA-mediated silencing of histone genes impairs the fertility of piRNA mutants and may serve to maintain piRNAs across evolution.

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Fig. 1: Silencing of histone mRNA correlates with progressive sterility in piwi-mutant worms.
Fig. 2: Loading histone 22G-RNAs into WAGO-1 after disruption of the piRNA-induced silencing complex.
Fig. 3: WAGO-1 loses interactions with germ-granule components across generations of piwi-mutant worms and remains cytoplasmic.
Fig. 4: The CSR-1 pathway triggers the biogenesis of histone 22G-RNAs in piwi mutant worms.
Fig. 5: Removal of histone 22G-RNAs rescues piwi mutant transgenerational sterility.
Fig. 6: Histone 22G-RNAs facilitate the epigenetic inheritance of a piwi-like phenotype in wild-type worms.

Data availability

All sequencing data (GRO-seq, RNA-seq and sRNA-seq from total lysate or IP experiments) are available at the Gene Expression Omnibus (GEO) under accession code GSE125601. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012557. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are available online for Figs. 16 and Extended Data Figs. 16.

Code availability

The custom scripts generate for this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the members of the Cecere laboratory, D. Canzio, N. Iovino, R. Sawarkar and P. Andersen for discussions of the manuscript; the Miska, the Mello, the Kennedy, the Seydoux, the Claycomb, the Strome and the Dumont laboratories for sharing strains and/or antibodies. Some strains were provided by the CGC, funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This project has received funding from the Institut Pasteur, the CNRS and the European Research Council (ERC) under the EU Horizon 2020 research and innovation programme under grant agreement no. ERC-StG- 679243. G.B. is part of the Pasteur–Paris University (PPU) International PhD Program and has received funding from the EU Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 665807. E.C. was supported by a Pasteur-Cantarini Fellowship program. F.D. and D.L. have received funding from Région Ile-de-France and Fondation pour la Recherche Médicale grants to support this study.

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Authors and Affiliations

Authors

Contributions

G.C. identified and developed the core questions addressed in the project and analysed the results of all of the experiments. G.B. performed most of the experiments and helped to analyse the results. E.C. conceived and generated all of the CRISPR–Cas9 lines used in this study, designed and performed the experiment using a catalytic mutant of CSR-1 and performed all of the confocal live-imaging experiments. M.S. performed all of the co-IP and IP experiments for MS and co-IPs. F.D. and D.L. performed the MS and analysed the data together with M.S. and G.C. B.L. performed the bioinformatics analysis of all sequencing data. M.U. performed some RNA extractions and the RT–qPCR experiment. A.S. performed the brood-size analysis of the RNAi experiments under the supervision of G.B. C.D. performed the brood-size analysis of some of the RNAi and crossing experiments together with G.C. P.Q. performed the GRO-seq. E.C. and P.Q. contributed to collecting some of the RNA samples that were used for the initial RNA-seq experiments. G.C. wrote the paper with contributions from G.B., E.C., M.S. and B.L.

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Correspondence to Germano Cecere.

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Extended data

Extended Data Fig. 1 Histone silencing occurs at the post-transcriptional level only in mutants of the piRNA biogenesis pathway.

a, Brood size assay of wild-type, prg-1 (n4357) and hrde-1 (tm1200) mutant worms as in Fig. 1d. The black lines indicate the mean, the error bars the standard deviation, and the n (animals) is indicated above in parenthesis. b, Number of individual misregulated REs by RNA-seq (≥ 2-fold and padj < 0.05, Wald test) in piwi or hrde-1 mutants. Data shown represent average of two biologically independent replicates. c, RT-qPCR log2 fold change of histone mRNAs and piRNA-dependent 22G-RNA targets in prg-1 (n4357), prde-1 (mj207), and hrde-1 (tm1200) mutants compared to wild-type worms. The bars indicate the mean and error bars indicate the standard deviation. n = 3 biologically independent experiments. d, RT-qPCR showing log2 fold change of individual DNA or RNA transposons in mutant vs. wild-type. Up-regulated transposons by RNA-seq are labelled in red. The bars indicate the mean and the black dots individual data from two biologically independent experiments. e, mRNA log2 fold change (y axis) and 22G-RNA log2 fold change (x axis) in hrde-1(tm1200) mutant vs. wild-type worms for protein-coding genes as in Fig. 1a. Wald test was used to calculate the p value. Data shown represent average of two biologically independent replicates. f, nascent RNA (nRNA) log2 fold change (y axis) and 22G-RNA log2 fold change (x axis) in prg-1 (n4357) mutant vs. wild-type worms for protein-coding genes. Red dots indicate the replicative histone genes. Wald test was used to calculate the p value. Data shown represent average of two biologically independent replicates. g, Box plot showing transcriptional (GRO-seq) and post-transcriptional (RNA-seq) histone gene expression changes in prg-1 (n4357) mutant vs. wild-type worms. The median (line), first and third quartiles (box), and whiskers (5th and 95th percentile) are shown. Two-tailed p value calculated with the Mann-Whitney-Wilcoxon tests is shown, using the sample size n (number of genes) = 61. Source data are available in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Transgenerational gene expression changes of protein-coding genes and REs in piwi mutant.

ac, MA-plot showing mRNA log2 fold change between piwi mutant and wild-type lines at different generations. Red and blue dots correspond to significant log2 fold change (padj < 0.05, Wald test). The number in parenthesis indicates the number of misregulated genes ≥ 2-fold. The average from two biologically independent replicates is shown. d, Comparison between mRNA log2 fold change (y axis) by and 22G-RNA log2 fold change (x axis) as shown in Fig. 1a using piwi mutant and wild-type CRISPR-Cas9 lines at F9 (left) and F12 (right). e, Plot showing the number of individual up-regulated and down-regulated REs by RNA-seq (padj < 0.05, Wald test) in piwi mutant compared to wild-type CRISPR-Cas9 lines at F4, F9, and F12. Only uniquely mapped reads were considered for this analysis. Data shown represent average of two biologically independent replicates. f, Comparison between RNA log2 fold change (y axis) by and 22G-RNA log2 fold change (x axis) as shown in Fig. 1a using piwi mutant and wild-type CRISPR-Cas9 lines at F9 (left) and F12 (right). Significant misregulated RE families are indicated (padj < 0.05, Wald test). The average from two biologically independent replicates is shown. g, Average and standard deviation of H2B-mCherry quantification in 15 pachytene nuclei in each individual wild-type and piwi mutant worm. n = 15 animals. h, Visualization of chromosome compaction in pachytene nuclei using H2B-mCherry wild-type and piwi mutant worms. The arrow indicates an example of nucleus with defective chromosome compaction in sterile piwi mutant. The percentage of nuclei lacking chromosome compaction in piwi mutant is shown below the images and the number in parenthesis indicates the number of nuclei counted. The white bars indicate 20 µM size. The experiment was repeated twice with similar results. Statistical source data are available in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 WAGO-1 relocalize from piRNA targets to histone mRNAs.

a, RNA immunoprecipitation (RIP) experiments followed by RT-qPCR showing the log2 fold change of the WAGO-1-interacting mRNAs in piwi mutant vs. wild-type worms. The bars indicate the mean and error bars indicate the standard deviation. n = 4 biologically independent experiments. b, Co-IP experiments showing CSR-1 interactions with WAGO-1 in wild-type and piwi mutant worms. Presence (+) or absence (-) of the tagged proteins or piwi mutation are indicated. Immunoprecipitation was performed using α-FLAG antibody, and the blots were probed with α-CSR-1 or α-FLAG antibodies. c, Immunostaining with α-FLAG antibody showing WAGO-1-FLAG localization in wild-type and piwi mutant (green signal). DAPI signal is shown in blue. The white bars indicate 20µM size. The experiment was repeated independently twice with similar results. d, Live confocal images of WAGO-1-GFP, PGL-1-mCherry, and CSR-1-mCherry in sterile piwi mutant germlines. The white bars indicate 10µM size. The experiment was repeated independently three times with similar results. Source data are available in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 CSR-1 and CDL-1 contribute to the biogenesis of histone 22G-RNAs in piRNA mutant.

a, Co-IP experiments using α-FLAG antibody for IPs and α-PIWI, α-DEPS-1 or α-FLAG antibodies for blots. Presence (+) or absence (-) of the FLAG tagged proteins are indicated. The experiment was repeated independently twice with similar results. b, Co-IP experiments as in a, showing DEPS-1 interaction with PIWI and not with CSR-1. The blots were probed with α-DEPS-1 or α-FLAG antibodies. The experiment was repeated independently twice with similar results. c, Immunoblot showing CSR-1, PGL-1, DEPS-1, GAPDH from total protein lysate or FLAG immunoprecipitation in WT and piwi mutant worms. Blots for germline-enriched and ubiquitous proteins are shown in red and blue respectively. The experiment was repeated independently twice with similar results. d, Volcano plot showing enrichment values and corresponding significance levels for proteins co-purifying with CSR-1 (see also Supplementary Table 1c). Argonaute proteins, germ granule components, 22G-RNA and histone biogenesis factors are indicated. The size of the dots is proportional to the number of peptides used for the quantification. The linear model was used to compute protein quantification ratio and the red horizontal line indicates the two-tailed p value = 0.05. n = 4 biologically independent experiments. e, Co-IP experiments as in a, showing CSR-1 interaction with WAGO-1 in wild-type, piwi mutant and piwi mutant treated with mut-16 RNAi. The blots were probed with α-CSR-1 or α-FLAG antibodies. * The higher migration of this band is due by the GFP fused to WAGO-1-FLAG in this strain. The experiment was repeated independently twice with similar results. fh, Metaprofile analysis showing the distribution of normalized 22G-RNA reads (RPM) across histone genes in wild-type (blue line), piwi mutant (red line), and piwi mutant animals treated with control RNAi (blue line), csr-1 RNAi (light blue line) or cdl-1 RNAi (yellow line). The experiment was repeated independently twice with similar results. Statistical source data and unprocessed blots are available in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Depletion of MUT-16, PPW-1, PPW-2 and not HRDE-1 restores fertility in piwi mutant independently of REs silencing.

a, Schematic of the RNAi experiment using CRISPR-Cas9 piwi mutant worms grown immediately on plates seeded with E. coli expressing dsRNA targeting mut-16 or empty vector for 20 generations. b, Results from the experiments described in a. Each dot corresponds to the number of alive larvae from individual worms. The black lines indicate the mean and the error bars the standard deviation. Two-tailed p value calculated using the Mann-Whitney-Wilcoxon tests is shown. n = 15 animals. c, Schematic of the RNAi experiment using CRISPR-Cas9 piwi mutant worms grown for 10 generations on plates seeded with E. coli OP50 (standard maintenance food) and then shifted for two generations on plates seeded with E. coli expressing dsRNA targeting hrde-1, ppw-1, ppw-2, mut-16 or empty vector. d, Results from brood size assay of the experiment described in c. The brood size assay is performed as in b. hrde-1 RNAi and its own control has been performed independently from the other RNAi treatment. The black lines indicate the mean and the error bars the standard deviation. Two-tailed p value calculated using the Mann-Whitney-Wilcoxon tests is shown. n = 20 animals. e, Comparison similar to the one showed in Fig. 1a between mRNA log2 fold change (y axis) and 22G-RNA log2 fold change (x axis) in piwi mutant animals treated with mut-16 RNAi compared to control RNAi for protein-coding genes. Purple dots indicate the piRNA-dependent 22G-RNA targets. f, Plot showing the number of up-regulated and down-regulated individual REs by RNA-seq (padj < 0.05, Wald test) in piwi mutant animals treated with control or mut-16 RNAi. Only uniquely mapped reads were considered for this analysis. Data shown represent average of two biologically independent replicates. Source data available in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Decreased fertility in wild-type worms after crossing with piRNA mutants.

a, Brood size assay similar to the one showed in Fig. 6b of the outcross experiment described in Fig. 6a. One wild-type and two piwi mutants were selected from independent F2 heterozygote lines from cross number 4. The black lines indicate the mean, the error bars the standard deviation, and the n (animal number analyzed) is indicated in parenthesis. b, Genotyping results by electrophoresis gel analysis of the F3 lines derived from self-crossed F2 heterozygote lines. Genomic DNA were extracted from individual animals after they released their progeny and a region spanning prg-1 mutation was amplified by PCR and digested with a restriction enzyme. The expected mutant and wild-type pattern of digestion is indicated by the black arrows compared to the marker (M). The selected wild-type and piwi mutant F3 lines are indicated by arrows with different colors corresponding to the colors used in the brood size assay shown in a. The experiment was repeated independently twice with similar results. c, same analysis as in a, performed with the crossing experiment number 5. The black lines indicate the mean, the error bars the standard deviation, and the sample size (n) is indicated in parenthesis. d, same analysis as in b, performed with the crossing experiment number 5. The experiment was repeated independently twice with similar results. White cross marks in the upper two panels indicate some of the selected lines used) e, Genotyping results similar to the one described in b using F3 lines derived from the crossing experiment between CRISPR-Cas9 piwi mutant hermaphrodite and wild-type males. The experiment was repeated independently twice with similar results. f, Genotyping results similar to the one described in b. The experiment was repeated independently twice with similar results. Statistical source data are available in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 Model illustrating the molecular consequences in animals losing piRNAs.

a, PRDE-1 promotes the transcription of piRNAs from thousands of genomic loci. piRNAs are then loaded into PIWI, which triggers the biogenesis of WAGO-bound 22G-RNAs from thousands protein-coding genes and REs and keep the WAGO pathway in a paused state (left). In case of new genomic invasions, the piRNAs and WAGOs machineries promptly silence new REs at the transcriptional and the post-transcriptional levels (right). REs can be kept silenced at the chromatin level by H3K9 methyl transferases in a piRNA-dependent or piRNA-independent manner. b, In early generations of piRNA mutants (left), the PIWI-induced silencing complex is still maintained on piRNA targets thanks to the interaction with germ granule components. In late generations (right), the piRNA-induced silencing complex is disrupted and some of its components, including WAGO-1, relocalize to the cytoplasm where it interacts with CSR-1 on histone mRNAs to synthesize antisense 22G-RNAs in a CSR-1-dependent manner (right). The PIWI, the WAGO and the CSR-1 pathways share interactions with many RNAi factors and germ granule components in wild-type worms, and in late generations of piwi mutants WAGO-1 and possibly PPW-1 and PPW2 become preferentially loaded by CSR-1-dependent histone 22G-RNAs to silence histone mRNAs, which lead to sterility. We propose that the histone mRNA silencing acts as an evolutionary force to maintain a constant production of piRNAs ready to silence new genomic invasion.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: lists of significant interacting proteins (at least twofold with adjusted P < 0.05) identified by MS in PIWI (Supplementary Table 1a), WAGO-1 (Supplementary Table 1b) and CSR-1 (Supplementary Table 1c) IPs. The linear model was used to compute protein quantification ratio and their associated two-tailed P value; n = 4 biologically independent experiments. Supplementary Table 2: list of strains used in this study. Supplementary Table 3: table of the primers used in this study. Supplementary Table 4: table of the gRNAs used in this study. Supplementary Table 5: oligos used for rRNA depletion. Supplementary Table 6: gene lists used in this study.

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Barucci, G., Cornes, E., Singh, M. et al. Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants. Nat Cell Biol 22, 235–245 (2020). https://doi.org/10.1038/s41556-020-0462-7

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