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.

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.

Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or "slicing". Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous proteincoding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are de-repressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans.The Piwi/piRNA pathway has an evolutionarily conserved role in germline transposon silencing in animals. C. elegans encodes two Piwi family proteins, PRG-1 and PRG-2, although PRG-2 has likely little or no function (1, 2). PRG-1 and piRNA expression is restricted to the male and female germline. The piRNAs of C. elegans are 21 nucleotides in length with a 5′ uracil (21U-RNAs) (1-4). In C. elegans piRNAs have a sequence motif, situated ~40 bp upstream of each piRNA locus, that is thought to be required for piRNA biogenesis (2, 3). A challenge in the field is to understand the mechanism(s) by which piRNAs act on their targets. Proposed functions for Piwi/piRNA complexes include the RNAi-like slicing of RNA transcripts (5-7), transcript deadenylation (8) and de novo DNA methylation (9, 10). Here we identify the targeting mechanism and the endogenous targets of C. elegans piRNAs.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Here we show that Sp3 is a target for SUMO modi®cation in vivo and in vitro. SUMO modi®cation of Sp3 occurs at a single lysine located between the second glutamine-rich activation domain and the DNA-binding domain. Mutational analyses identi®ed the sequence IKXE as essential for SUMO conjugation to Sp3. We identi®ed the protein inhibitor of activated STAT1 (PIAS1) as an interaction partner of Sp3 and Ubc9. Moreover, PIAS1 strongly stimulated SUMO conjugation to Sp3, thus acting as an E3 ligase for SUMO conjugation to Sp3. All mutations that prevented SUMO modi®cation in vitro strongly enhanced the transcriptional activity of Sp3, showing that SUMO modi®cation silences Sp3 activity. SUMOmodi®ed Sp3 bound to DNA with similar speci®city and af®nity as unmodi®ed Sp3. However, DNA-bound Sp3 did not act as a substrate for SUMO modi®cation.

In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood. Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. This work uncovers a multi-step siRNA amplification pathway that promotes germline integrity via epigenetic silencing of endogenous and invading genetic elements. In addition, the same pathway can be engaged in environmentally induced heritable gene silencing and could therefore promote the inheritance of acquired traits.

Repetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among factors and pathways underlies the importance of safeguarding the genome through multiple means.DOI: http://dx.doi.org/10.7554/eLife.21666.001

SUMO modification of many transcription factors is linked to transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription are largely unknown. Here we report a genome-wide RNA interference screen in Drosophila melanogaster cells for components regulating and mediating SUMO-dependent transcriptional repression. Analysis of >21,000 double-stranded RNAs (dsRNAs) identified 120 genes whose dsRNA-mediated knockdowns impaired SUMO-dependent transcriptional repression. Several of these genes encode chromatin-associated proteins, including the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1, and the polycomb protein Sfmbt. Knockdown of these proteins did not impair SUMO conjugation, demonstrating that they act downstream of SUMO attachment. Biochemical analyses revealed that MEP-1, Mi-2, and Sfmbt interact with each other, bind to SUMO, and are recruited to promoters in a SUMOylation-dependent manner. Our results suggest that MEP-1, Mi-2, and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Both proteins contain a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich domains in the N-terminal moiety. Immunoblot analyses of Sp3 reveal a striking complex protein pattern of up to eight distinct species. This pattern is not observed in Sp3-deficient cell lines showing that all signals reflect Sp3 antigen. In this study, we have unraveled the complexity of Sp3 expression. We show that four isoforms of Sp3 that retain different parts of the N terminus are expressed in vivo. The four isoforms derive from alternative translational start sites at positions 1, 37, 856, and 907. An upstream open reading frame located at position ؊47 to ؊18 regulates expression of the two long isoforms. Unlike Sp1, none of the Sp3 isoforms is glycosylated. However, all four isoforms become SUMO-modified in vivo and in vitro specifically and exclusively at lysine residue 551. The transcriptional activity of the two long isoforms strongly depends on the promoter settings, whereas the small isoforms appear to be inactive. The transcriptional activity of all the Sp3 isoforms is regulated by SUMO modification. Our results demonstrate that Sp3 has many unique features and is not simply a functional equivalent of Sp1.The transcription factor Sp3 is a ubiquitously expressed member of the Sp family of transcription factor that is involved in the expression and regulation of many genes, including housekeeping genes, tissue-specifically expressed genes, viral genes, and cell cycle-regulated genes (1, 2). Sp3 contains a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich activation domains in the N-terminal moiety. The expression pattern, the structure, and the DNAbinding properties of Sp3 are very similar to Sp1, which suggested originally that these two proteins exert similar functions. The physiological roles of Sp1 and Sp3, however, appear to be significantly different. Sp1 knock-out mouse embryos are severely retarded in growth, and die after day 10 of embryonic development (3). Sp3-deficient embryos develop until birth, but die invariably of respiratory failure immediately after birth (4). In addition, late tooth and bone developmental processes are impaired in Sp3Ϫ/Ϫ mice (4, 5).Functional analyses of the transcriptional properties of Sp1 and Sp3 also revealed significant differences between these two transcription factors (6). On many reporter constructs containing multiple Sp-binding sites Sp3 is, unlike Sp1, inactive or acts only as a weak activator (7). The molecular basis for the inactivity of Sp3 under these conditions has been mapped to an inhibitory domain located between the second glutamine-rich activation domain and the zinc finger region (8). More recently, it was shown that Sp3 is post-translationally modified by the small ubiquitin-like modifier (SUMO) 1 within its inhibitory domain and that SUMO modification leads to inactivation (9, 10).All previously published studies with Sp3 (more than 500 ...