In C. elegans, alterations to chromatin produce transgenerational effects, such as inherited increase in lifespan and gradual loss of fertility. Inheritance of histone modifications can be induced by double-stranded RNA-derived heritable small RNAs. Here, we show that the mortal germline phenotype, which is typical of met-2 mutants, defective in H3K9 methylation, depends on HRDE-1, an argonaute that carries small RNAs across generations, and is accompanied by accumulated transgenerational misexpression of heritable small RNAs. We discovered that MET-2 inhibits small RNA inheritance, and, as a consequence, induction of RNAi in met-2 mutants leads to permanent RNAi responses that do not terminate even after more than 30 generations. We found that potentiation of heritable RNAi in met-2 animals results from global hyperactivation of the small RNA inheritance machinery. Thus, changes in histone modifications can give rise to drastic transgenerational epigenetic effects, by controlling the overall potency of small RNA inheritance.

SummaryIn C. elegans nematodes, components of liquid-like germ granules were shown to be required for transgenerational small RNA inheritance. Surprisingly, we show here that mutants with defective germ granules can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated in pptr-1, which is required for stabilization of P granules in the early embryo, display extraordinarily strong heritable RNAi responses, lasting for tens of generations. Intriguingly, the RNAi capacity of descendants derived from mutants defective in the core germ granule proteins MEG-3 and MEG-4 is determined by the genotype of the ancestors and changes transgenerationally. Further, whether the meg-3/4 mutant alleles were present in the paternal or maternal lineages leads to different transgenerational consequences. Small RNA inheritance, rather than maternal contribution of the germ granules themselves, mediates the transgenerational defects in RNAi of meg-3/4 mutants and their progeny. Accordingly, germ granule defects lead to heritable genome-wide mis-expression of endogenous small RNAs. Upon disruption of germ granules, hrde-1 mutants can inherit RNAi, although HRDE-1 was previously thought to be absolutely required for RNAi inheritance. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance maintains this activity for multiple generations.

Highlights d A mutant defective in germ granule assembly is deficient in RNAi d RNAi-deficiency correlates with small RNA silencing of genes required for RNAi d Silencing requires the piRNA Argonaute PRG-1 and the nuclear Argonaute HRDE-1 d Localization in germ granules protects transcripts from piRNA-initiated silencing

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

15P granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for 16 self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not 17 assemble P granules in primordial germ cells loses competence for RNA-interference over several 18 generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the 19 RNA-interference genes rde-11 and sid-1. In wild-type, rde-11 and sid-1 transcripts are heavily targeted 20 by piRNAs, accumulate in P granules, but maintain expression. In the primordial germ cells of meg-3 21 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis 22 machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires 23 the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational 24 silencing of piRNA targets. These observations support a "safe harbor" model for P granules in 25 protecting germline transcripts from piRNA-initiated silencing. 26 27In the germ cells of animals, dense RNA-protein condensates accumulate on the cytoplasmic 29 face of the nuclear envelope. These condensates, collectively referred to as nuage, contain components 30 of the small RNA (sRNA) machinery that scan germline transcripts for foreign sequences. For example, in 31Drosophila, components of the piRNA machinery in nuage amplify small RNAs that target transcripts 32 from transposable elements for destruction (Huang et al., 2017). In C. elegans, the PIWI-class Argonaute 33 PRG-1 associates with ~15,000 piRNAs encoded in the genome that scan most, if not all, germline 34 1). Segregation of Argonautes and proteins required for 22G-RNA production into distinct nuage 60 compartments (P granules versus Z granules and mutator foci) could also play a role in sorting 22G-RNAs 61 or limiting their production (Wan et al., 2018). A direct test of these hypotheses, however, has been 62 difficult to obtain as complete loss of P granules causes sterility. 63We previously identified a mutant that affects P granule coalescence only during embryogenesis 64 (Wang et al., 2014). MEG-3 and MEG-4 are intrinsically-disordered proteins present in the germ plasm, a 65 specialized cytoplasm that is partitioned with the germ lineage during early embryonic cleavages (Wang 66 3 and Seydoux, 2013). MEG-3 and MEG-4 form gel-like scaffolds that recruit and stimulate the coalescence 67 of P granule proteins in germ plasm to ensure their partitioning to the embryonic germline and the 68 primordial germ cells Z2 and Z3 (Fig. 1;Putnam et al., 2019). In meg-3 meg-4 embryos, P granules do not 69 coalesce in germ plasm, causing granule components to be partitioned equally to all cells and turned 70 over ( Fig. 1; Wang et al., 2014). Despite lacking P granules during embryogenesis, meg-3 meg-4 71 assemble P granules de novo when the primordial germ cells resume divisions in the first larval stage to 72 generate the ~ 2000 germ cells that constitu...