Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Ozata, D. M., Gainetdinov, I., Zoch, A., O’Carroll, D. & Zamore, P. D. PIWI-interacting RNAs: small RNAs with big functions. Nat. Rev. Genet. 20, 89–108 (2019).

    Article  CAS  PubMed  Google Scholar 

  2. Thomson, T. & Lin, H. The biogenesis and function of PIWI proteins and piRNAs: progress and prospect. Annu. Rev. Cell Dev. Biol. 25, 355–376 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tóth, K. F., Pezic, D., Stuwe, E. & Webster, A. The piRNA pathway guards the germline genome against transposable elements. Adv. Exp. Med. Biol. 886, 51–77 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Wu, P. -H. et al. An evolutionarily conserved piRNA-producing locus required for male mouse fertility. Preprint at bioRxiv https://doi.org/10.1101/386201 (2018).

  5. Gou, L. T. et al. Ubiquitination-deficient mutations in human Piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis. Cell 169, 1090–1104 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Simon, M. et al. Reduced insulin/IGF-1 signaling restores germ cell immortality to Caenorhabditis elegans Piwi mutants. Cell Rep. 7, 762–773 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Katz, D. J., Edwards, T. M., Reinke, V. & KellyW. G. A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory. Cell 137, 308–320 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Buckley, B. A. et al. A nuclear argonaute promotes multigenerational epigenetic inheritance and germline immortality. Nature 489, 447–451 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Frézal, L., Demoinet, E., Braendle, C., Miska, E. & Félix, M.-A. Natural genetic variation in a multigenerational phenotype in C. elegans. Curr. Biol. 28, 2588–2596 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Spracklin, G. et al. The RNAi inheritance machinery of Caenorhabditis elegans. Genetics 206, 1403–1416 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Perez, M. F. & Lehner, B. Intergenerational and transgenerational epigenetic inheritance in animals. Nat. Cell Biol. 21, 143–151 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Batista, P. J. et al. PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. Mol. Cell 31, 67–78 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ruby, J. G. et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Das, P. P. et al. Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline. Mol. Cell 31, 79–90 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cecere, G., Zheng, G. X. Y., Mansisidor, A. R., Klymko, K. E. & Grishok, A. Promoters recognized by forkhead proteins exist for individual 21U-RNAs. Mol. Cell 47, 734–745 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gu, W. et al. CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors. Cell 151, 1488–1500 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shen, E. Z. et al. Identification of piRNA binding sites reveals the argonaute regulatory landscape of the C. elegans germline. Cell 172, 937–951 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang, D. et al. The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes. Science 359, 587–592 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee, H. C. et al. C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts. Cell 150, 78–87 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bagijn, M. P. et al. Function, targets, and evolution of Caenorhabditis elegans piRNAs. Science 337, 574–578 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Houri-Ze’evi, L. et al. A tunable mechanism determines the duration of the transgenerational small RNA inheritance in C. elegans. Cell 165, 88–99 (2016).

    Article  PubMed  CAS  Google Scholar 

  22. Spracklin, G. et al. The RNAi inheritance machinery of Caenorhabditis elegans. Genetics 206, 1403–1416 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wan, G. et al. Spatiotemporal regulation of liquid-like condensates in epigenetic inheritance. Nature 557, 679–683 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zeller, P. et al. Histone H3K9 methylation is dispensable for Caenorhabditis elegans development but suppresses RNA:DNA hybrid-associated repeat instability. Nat. Genet. 48, 1385–1395 (2016).

    Article  CAS  PubMed  Google Scholar 

  25. McMurchy, A. N. et al. A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress. eLife 6, e21666 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ni, J. Z., Chen, E. & Gu, S. G. Complex coding of endogenous siRNA, transcriptional silencing and H3K9 methylation on native targets of germline nuclear RNAi in C. elegans. BMC Genom. 15, 1157 (2014).

    Article  CAS  Google Scholar 

  27. Shirayama, M. et al. piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell 150, 65–77 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. de Albuquerque, B. F. M., Placentino, M. & Ketting, R. F. Maternal piRNAs are essential for germline development following de novo establishment of endo-siRNAs in Caenorhabditis elegans. Dev. Cell 34, 448–456 (2015).

    Article  PubMed  CAS  Google Scholar 

  29. Phillips, C. M., Brown, K. C., Montgomery, B. E., Ruvkun, G. & Montgomery, T. A. piRNAs and piRNA-dependent siRNAs protect conserved and essential C. elegans genes from misrouting into the RNAi pathway. Dev. Cell 34, 457–465 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Weick, E. M. et al. PRDE-1 is a nuclear factor essential for the biogenesis of ruby motif-dependent piRNAs in C. elegans. Genes Dev. 28, 783–796 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pettitt, J., Crombie, C., Schümperli, D. & Müller, B. The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division. J. Cell Sci. 115, 857–866 (2002).

  32. Kodama, Y., Rothman, J. H., Sugimoto, A. & Yamamoto, M. The stem-loop binding protein CDL-1 is required for chromosome condensation, progression of cell death and morphogenesis in Caenorhabditis elegans. Development 129, 187–196 (2002).

  33. Gu, W. et al. Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol. Cell 36, 231–244 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Spike, C. A., Bader, J., Reinke, V. & Strome, S. DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells. Development 135, 983–993 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Shirayama, M., Stanney, W., Gu, W., Seth, M. & Mello, C. C. The vasa homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans. Curr. Biol. 24, 845–851 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wedeles, C. J., Wu, M. Z. & Claycomb, J. M. Protection of germline gene expression by the C. elegans argonaute CSR-1. Dev. Cell 27, 664–671 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Seth, M. et al. The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression. Dev. Cell 27, 656–663 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Avgousti, D. C., Palani, S., Sherman, Y. & Grishok, A. CSR-1 RNAi pathway positively regulates histone expression in C. elegans. EMBO J. 31, 3821–3832 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Keall, R., Whitelaw, S., Pettitt, J. & Müller, B. Histone gene expression and histone mRNA 3′ end structure in Caenorhabditis elegans. BMC Mol. Biol. 8, 51 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Zhang, C. et al. mut-16 and other mutator class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 108, 1201–1208 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Heestand, B., Simon, M., Frenk, S., Titov, D. & Ahmed, S. Transgenerational sterility of Piwi mutants represents a dynamic form of adult reproductive diapause. Cell Rep. 23, 156–171 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gerson-Gurwitz, A. et al. A small RNA-catalytic argonaute pathway tunes germline transcript levels to ensure embryonic divisions. Cell 165, 396–409 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Seth, M. et al. The coding regions of germline mRNAs confer sensitivity to argonaute regulation in C. elegans. Cell Rep. 22, 2254–2264 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tang, W. et al. A sex chromosome piRNA promotes robust dosage compensation and sex determination in C. elegans. Dev. Cell 44, 762–770 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Barberán-Soler, S. et al. Co-option of the piRNA pathway for germline-specific alternative splicing of C. elegans TOR. Cell Rep. 8, 1609–1616 (2014).

    Article  PubMed  CAS  Google Scholar 

  46. Sarkies, P. et al. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages. PLoS Biol. 13, e1002061 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Tu, S. et al. Comparative functional characterization of the CSR-1 22G-RNA pathway in Caenorhabditis nematodes. Nucleic Acids Res. 43, 208–224 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Wyler-Duda, P., Bernard, V., Stadler, M., Suter, D. & Schümperli, D. Histone H4 mRNA from the nematode Ascaris lumbricoides is cis-spliced and polyadenylated. Biochim. Biophys. Acta 1350, 259–261 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Paix, A., Folkmann, A., Rasoloson, D. & Seydoux, G. High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics 201, 47–54 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Arribere, J. A. et al. Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198, 837–846 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Aoki, K., Moriguchi, H., Okawa, K. & Tabara, H. Biochemical genetic analyses of RdRP and Slicer activities related to RNAi in C. elegans. in International Worm Meeting (2007).

  53. Edgley, M. L., Baillie, D. L., Riddle, D. L. & Rose, A. M. in WormBook (The C. elegans Research Community, 2006).

  54. Kamath, R. S. & Ahringer, J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321 (2003).

    Article  CAS  PubMed  Google Scholar 

  55. Jayaprakash, A. D., Jabado, O., Brown, B. D. & Sachidanandam, R. Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res. 39, e141 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Cecere, G., Hoersch, S., O’Keeffe, S., Sachidanandam, R. & Grishok, A. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape. Nat. Struct. Mol. Biol. 21, 358–365 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Poullet, P., Carpentier, S. & Barillot, E. myProMS, a web server for management and validation of mass spectrometry-based proteomic data. Proteomics 7, 2553–2556 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Valot, B., Langella, O., Nano, E. & Zivy, M. MassChroQ: a versatile tool for mass spectrometry quantification. Proteomics 11, 3572–3577 (2011).

    Article  CAS  PubMed  Google Scholar 

  59. Cline, M. S. et al. Integration of biological networks and gene expression data using Cytoscape. Nat. Protoc. 2, 2366–2382 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Vizcaíno, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016).

    Article  PubMed  CAS  Google Scholar 

  61. Claycomb, J. M. et al. The argonaute CSR-1 and Its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell 139, 123–134 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Kawasaki, I. et al. The PGL family proteins associate with germ granules and function redundantly in Caenorhabditis elegans germline development. Genetics 167, 645–661 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Liao, Y., Smyth, G. K. & Shi, W. FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  PubMed  Google Scholar 

  65. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

  66. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).

    Article  Google Scholar 

  67. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Quinlan, A. R. BEDTools: the Swiss-army tool for genome feature analysis. Curr. Protoc. Bioinform. 47, 11.12.1–11.12.34 (2014).

    Article  Google Scholar 

  69. Neph, S. et al. BEDOPS: high-performance genomic feature operations. Bioinformatics 28, 1919–1920 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

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.

Author information

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.

Corresponding author

Correspondence to Germano Cecere.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

Source Data Fig. 1

Statistical source data

Source Data Fig. 1

Unprocessed immunoblots

Source Data Fig. 2

Statistical source data

Source Data Fig. 2

Unprocessed immunoblots

Source Data Fig. 3

Statistical source data

Source Data Fig. 4

Unprocessed immunoblots

Source Data Fig. 5

Statistical source data

Source Data Fig. 6

Statistical source data

Source Data Extended Data Fig. 1

Statistical source data

Source Data Extended Data Fig. 2

Statistical source data

Source Data Extended Data Fig. 3

Statistical source data

Source Data Extended Data Fig. 4

Unprocessed immunoblots

Source Data Extended Data Fig. 5

Statistical source data

Source Data Extended Data Fig. 6

Statistical source data

Source Data Extended Data Fig. 6

Unprocessed DNA gel stainings

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-020-0462-7

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing