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• W2154222068 abstract "Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish “self” from “nonself” nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of “self” and “nonself” RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression. Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish “self” from “nonself” nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of “self” and “nonself” RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression. Epigenetic silencing triggered by piRNA-mediated recognition of nonself RNA piRNAs scan using imperfect base pairing to initiate gene silencing Maintenance of silencing requires chromatin factors and RdRP-generated small RNAs Activating and silencing signals may compete in self versus nonself discrimination All organisms balance the need to maintain genetic variation against the danger of accumulating potentially deleterious genes or pathogenic sequences (Antonovics et al., 2011Antonovics J. Boots M. Abbate J. Baker C. McFrederick Q. Panjeti V. Biology and evolution of sexual transmission.Ann. N Y Acad. Sci. 2011; 1230: 12-24Crossref PubMed Scopus (28) Google Scholar). The experimental introduction of DNA (transgenes) into the germline provides an opportunity to probe an organism's response to foreign DNA (Rülicke and Hübscher, 2000Rülicke T. Hübscher U. Germ line transformation of mammals by pronuclear microinjection.Exp. Physiol. 2000; 85: 589-601Crossref PubMed Google Scholar) and has revealed that organisms use a variety of mechanisms to silence transgenes in the germline (Birchler et al., 2003Birchler J. Pal-Bhadra M. Bhadra U. Transgene cosuppression in animals.in: Hannon G. RNAi: A guide to gene silencing. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2003: 23-42Google Scholar, Brodersen and Voinnet, 2006Brodersen P. Voinnet O. The diversity of RNA silencing pathways in plants.Trends Genet. 2006; 22: 268-280Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). Interestingly, some mutants that disrupt transgene silencing also desilence endogenous genes, including self-replicating elements called transposons (Ketting et al., 1999Ketting R.F. Haverkamp T.H. van Luenen H.G. Plasterk R.H. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.Cell. 1999; 99: 133-141Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, Tabara et al., 1999Tabara H. Sarkissian M. Kelly W.G. Fleenor J. Grishok A. Timmons L. Fire A. Mello C.C. The rde-1 gene, RNA interference, and transposon silencing in C. elegans.Cell. 1999; 99: 123-132Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). Thus, the mechanisms involved in transgene silencing protect the genome from invasive DNA elements. In many organisms, transgene silencing has been linked to factors that are also required for the RNAi pathway (Bosher and Labouesse, 2000Bosher J.M. Labouesse M. RNA interference: genetic wand and genetic watchdog.Nat. Cell Biol. 2000; 2: E31-E36Crossref PubMed Scopus (261) Google Scholar). RNAi was first identified as a sequence-specific response triggered by double-stranded RNA (dsRNA) (Fire et al., 1998Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (11739) Google Scholar). During RNAi, dsRNA is processed by the RNase III-related protein, Dicer, into ∼21 nucleotide (nt) short-interfering RNAs (siRNAs) (Bernstein et al., 2001Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001; 409: 363-366Crossref PubMed Scopus (3776) Google Scholar, Carmell and Hannon, 2004Carmell M.A. Hannon G.J. RNase III enzymes and the initiation of gene silencing.Nat. Struct. Mol. Biol. 2004; 11: 214-218Crossref PubMed Scopus (311) Google Scholar, Zamore et al., 2000Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar), which are loaded onto Argonaute (AGO) proteins to form the key effectors of RNA-induced silencing complexes (Hammond et al., 2001Hammond S.M. Boettcher S. Caudy A.A. Kobayashi R. Hannon G.J. Argonaute2, a link between genetic and biochemical analyses of RNAi.Science. 2001; 293: 1146-1150Crossref PubMed Scopus (1180) Google Scholar, Liu et al., 2004Liu J. Carmell M.A. Rivas F.V. Marsden C.G. Thomson J.M. Song J.J. Hammond S.M. Joshua-Tor L. Hannon G.J. Argonaute2 is the catalytic engine of mammalian RNAi.Science. 2004; 305: 1437-1441Crossref PubMed Scopus (2035) Google Scholar, Meister et al., 2004Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar). AGOs are RNase H-related proteins that use the base-pairing potential of small RNA cofactors to guide sequence-specific binding to target sequences (Song et al., 2004Song J.J. Smith S.K. Hannon G.J. Joshua-Tor L. Crystal structure of Argonaute and its implications for RISC slicer activity.Science. 2004; 305: 1434-1437Crossref PubMed Scopus (1092) Google Scholar). In some cases, AGOs directly cleave their targets; in other cases, AGOs recruit cofactors that direct mRNA destruction or other modes of regulation. Despite a clear overlap between the mechanisms that mediate RNAi and the silencing of transposons and transgenes, several findings point to distinct triggering mechanisms. For example, the AGO protein RDE-1 is essential for the dsRNA response in C. elegans but is not required for transposon or transgene silencing (Tabara et al., 1999Tabara H. Sarkissian M. Kelly W.G. Fleenor J. Grishok A. Timmons L. Fire A. Mello C.C. The rde-1 gene, RNA interference, and transposon silencing in C. elegans.Cell. 1999; 99: 123-132Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). RDE-1 engages siRNAs produced by Dicer and mediates the initial search for target RNAs in the cell (Parrish and Fire, 2001Parrish S. Fire A. Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.RNA. 2001; 7: 1397-1402PubMed Google Scholar, Yigit et al., 2006Yigit E. Batista P.J. Bei Y. Pang K.M. Chen C.C. Tolia N.H. Joshua-Tor L. Mitani S. Simard M.J. Mello C.C. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.Cell. 2006; 127: 747-757Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). RDE-1 is thought to recruit a cellular RNA-dependent RNA polymerase (RdRP), which then utilizes the target mRNA as a template for the production of secondary siRNAs, termed 22G-RNAs (Gu et al., 2009Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, Pak and Fire, 2007Pak J. Fire A. Distinct populations of primary and secondary effectors during RNAi in C. elegans.Science. 2007; 315: 241-244Crossref PubMed Scopus (447) Google Scholar, Sijen et al., 2001Sijen T. Fleenor J. Simmer F. Thijssen K.L. Parrish S. Timmons L. Plasterk R.H. Fire A. On the role of RNA amplification in dsRNA-triggered gene silencing.Cell. 2001; 107: 465-476Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar, Sijen et al., 2007Sijen T. Steiner F.A. Thijssen K.L. Plasterk R.H. Secondary siRNAs result from unprimed RNA synthesis and form a distinct class.Science. 2007; 315: 244-247Crossref PubMed Scopus (312) Google Scholar, Yigit et al., 2006Yigit E. Batista P.J. Bei Y. Pang K.M. Chen C.C. Tolia N.H. Joshua-Tor L. Mitani S. Simard M.J. Mello C.C. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.Cell. 2006; 127: 747-757Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). The 22G-RNAs are loaded onto members of an expanded, partially redundant, group of worm-specific AGOs (WAGOs). WAGOs that localize to the cytoplasm are thought to mediate mRNA turnover, whereas WAGOs that localize to the nucleus mediate transcriptional silencing (Gu et al., 2009Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, Guang et al., 2008Guang S. Bochner A.F. Pavelec D.M. Burkhart K.B. Harding S. Lachowiec J. Kennedy S. An Argonaute transports siRNAs from the cytoplasm to the nucleus.Science. 2008; 321: 537-541Crossref PubMed Scopus (241) Google Scholar). Many components of the RNAi pathway that function downstream of RDE-1 are required for transposon and transgene silencing, including the RdRP system (Gu et al., 2009Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, Smardon et al., 2000Smardon A. Spoerke J.M. Stacey S.C. Klein M.E. Mackin N. Maine E.M. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans.Curr. Biol. 2000; 10: 169-178Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), the polynucleotide polymerase RDE-3 (Chen et al., 2005Chen C.C. Simard M.J. Tabara H. Brownell D.R. McCollough J.A. Mello C.C. A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans.Curr. Biol. 2005; 15: 378-383Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), the nuclease MUT-7 (Ketting et al., 1999Ketting R.F. Haverkamp T.H. van Luenen H.G. Plasterk R.H. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.Cell. 1999; 99: 133-141Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar), and the WAGO proteins (Yigit et al., 2006Yigit E. Batista P.J. Bei Y. Pang K.M. Chen C.C. Tolia N.H. Joshua-Tor L. Mitani S. Simard M.J. Mello C.C. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.Cell. 2006; 127: 747-757Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar), among others (Robert et al., 2004Robert V.J. Vastenhouw N.L. Plasterk R.H. RNA interference, transposon silencing, and cosuppression in the Caenorhabditis elegans germ line: similarities and differences.Cold Spring Harb. Symp. Quant. Biol. 2004; 69: 397-402Crossref PubMed Scopus (19) Google Scholar). The fact that RDE-1 is not required for transposon and transgene silencing suggests that features unique to transposons and transgenes underlie the initial recruitment of RdRP to these targets and that dsRNA is unlikely to be the trigger. In the germline, RdRPs not only produce 22G-RNAs that interact with WAGOs, but also produce 22G-RNAs that interact with a distinct AGO, CSR-1, required for fertility and chromosome segregation (Claycomb et al., 2009Claycomb J.M. Batista P.J. Pang K.M. Gu W. Vasale J.J. van Wolfswinkel J.C. Chaves D.A. Shirayama M. Mitani S. Ketting R.F. et al.The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.Cell. 2009; 139: 123-134Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, Yigit et al., 2006Yigit E. Batista P.J. Bei Y. Pang K.M. Chen C.C. Tolia N.H. Joshua-Tor L. Mitani S. Simard M.J. Mello C.C. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.Cell. 2006; 127: 747-757Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). However, some factors, including RDE-3 and MUT-7, are only required for WAGO 22G-RNA accumulation (Gu et al., 2009Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), indicating that the CSR-1 and WAGO 22G pathways also involve distinct mechanisms. Indeed, the WAGO and CSR-1 22G pathways together target virtually all germline-expressed mRNAs; however, their targets are largely nonoverlapping (Gu et al., 2009Gu W. Shirayama M. Conte Jr., D. Vasale J. Batista P.J. Claycomb J.M. Moresco J.J. Youngman E.M. Keys J. Stoltz M.J. et al.Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.Mol. Cell. 2009; 36: 231-244Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Furthermore, unlike the WAGO pathway, the CSR-1 22G pathway does not appear to silence its targets (Claycomb et al., 2009Claycomb J.M. Batista P.J. Pang K.M. Gu W. Vasale J.J. van Wolfswinkel J.C. Chaves D.A. Shirayama M. Mitani S. Ketting R.F. et al.The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.Cell. 2009; 139: 123-134Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Instead, the CSR-1 pathway may help to define and maintain euchromatic regions along the holocentric chromosomes in order to support the proper assembly of kinetochores. In most animals, the Piwi family AGOs are required for fertility and transposon silencing (Cox et al., 1998Cox D.N. Chao A. Baker J. Chang L. Qiao D. Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal.Genes Dev. 1998; 12: 3715-3727Crossref PubMed Scopus (802) Google Scholar, Juliano et al., 2011Juliano C. Wang J. Lin H. Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms.Annu. Rev. Genet. 2011; 45: 447-469Crossref PubMed Scopus (277) Google Scholar). In C. elegans, however, the Piwi-related gene product PRG-1 has only been linked to the silencing of one transposon family, Tc3 (Batista et al., 2008Batista P.J. Ruby J.G. Claycomb J.M. Chiang R. Fahlgren N. Kasschau K.D. Chaves D.A. Gu W. Vasale J.J. Duan S. et al.PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.Mol. Cell. 2008; 31: 67-78Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, Das et al., 2008Das P.P. Bagijn M.P. Goldstein L.D. Woolford J.R. Lehrbach N.J. Sapetschnig A. Buhecha H.R. Gilchrist M.J. Howe K.L. Stark R. et al.Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline.Mol. Cell. 2008; 31: 79-90Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Interestingly, PRG-1 appears to recruit RdRP and the WAGO 22G pathway to maintain Tc3 silencing. Piwi-interacting RNAs (piRNAs) (21U-RNAs in C. elegans) are genomically encoded and appear to be expressed as Pol II transcripts whose single-stranded products are processed and loaded onto Piwi (Aravin and Hannon, 2008Aravin A.A. Hannon G.J. Small RNA silencing pathways in germ and stem cells.Cold Spring Harb. Symp. Quant. Biol. 2008; 73: 283-290Crossref PubMed Scopus (95) Google Scholar, Kim et al., 2009Kim V.N. Han J. Siomi M.C. Biogenesis of small RNAs in animals.Nat. Rev. Mol. Cell Biol. 2009; 10: 126-139Crossref PubMed Scopus (2581) Google Scholar). More than 15,000 distinct piRNA species exist in C. elegans, and millions of species are expressed in the testes of mammals (Aravin et al., 2006Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. et al.A novel class of small RNAs bind to MILI protein in mouse testes.Nature. 2006; 442: 203-207Crossref PubMed Scopus (1126) Google Scholar, Batista et al., 2008Batista P.J. Ruby J.G. Claycomb J.M. Chiang R. Fahlgren N. Kasschau K.D. Chaves D.A. Gu W. Vasale J.J. Duan S. et al.PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.Mol. Cell. 2008; 31: 67-78Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, Das et al., 2008Das P.P. Bagijn M.P. Goldstein L.D. Woolford J.R. Lehrbach N.J. Sapetschnig A. Buhecha H.R. Gilchrist M.J. Howe K.L. Stark R. et al.Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline.Mol. Cell. 2008; 31: 79-90Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, Girard et al., 2006Girard A. Sachidanandam R. Hannon G.J. Carmell M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins.Nature. 2006; 442: 199-202Crossref PubMed Scopus (1245) Google Scholar, Grivna et al., 2006Grivna S.T. Beyret E. Wang Z. Lin H. A novel class of small RNAs in mouse spermatogenic cells.Genes Dev. 2006; 20: 1709-1714Crossref PubMed Scopus (654) Google Scholar, Lau et al., 2006Lau N.C. Seto A.G. Kim J. Kuramochi-Miyagawa S. Nakano T. Bartel D.P. Kingston R.E. Characterization of the piRNA complex from rat testes.Science. 2006; 313: 363-367Crossref PubMed Scopus (751) Google Scholar). The majority of these piRNAs map uniquely to the genome and lack obvious targets. As such, their function remains entirely unknown. Here, we use a homologous gene-targeting method called “Mos1-mediated single-copy insertion” (MosSCI; Frøkjaer-Jensen et al., 2008Frøkjaer-Jensen C. Davis M.W. Hopkins C.E. Newman B.J. Thummel J.M. Olesen S.P. Grunnet M. Jorgensen E.M. Single-copy insertion of transgenes in Caenorhabditis elegans.Nat. Genet. 2008; 40: 1375-1383Crossref PubMed Scopus (714) Google Scholar) to show that strains bearing identical single-copy transgenes inserted at the same chromosomal site can exhibit opposite and remarkably stable epigenetic fates, either expressed or silenced. Transgenes consisting of an endogenous germline-expressed gene fused to a relatively long foreign sequence (e.g., gfp) were prone to silencing. By contrast, otherwise identical transgenes fused to a short foreign sequence (e.g., flag) were always expressed. Our genetic and molecular analyses reveal that silencing is dependent on nuclear and cytoplasmic WAGOs and is correlated with the accumulation of 22G-RNAs targeting the foreign portion of the transgene. Importantly, PRG-1 is required to initiate, but not to maintain, silencing. We propose that PRG-1 and its 21U-RNA cofactors scan for foreign RNA sequences and initiate WAGO-maintained gene silencing, and endogenous mRNAs are protected from silencing, perhaps by the CSR-1/22G-RNA pathway. Single-copy insertions can overcome barriers to transgene expression in the germline (Rieckher et al., 2009Rieckher M. Kourtis N. Pasparaki A. Tavernarakis N. Transgenesis in Caenorhabditis elegans.Methods Mol. Biol. 2009; 561: 21-39Crossref PubMed Scopus (22) Google Scholar). Indeed, the single-copy insertion of transgenes at a defined chromosomal locus via the recently developed MosSCI approach reproducibly achieves germline expression (Frøkjaer-Jensen et al., 2008Frøkjaer-Jensen C. Davis M.W. Hopkins C.E. Newman B.J. Thummel J.M. Olesen S.P. Grunnet M. Jorgensen E.M. Single-copy insertion of transgenes in Caenorhabditis elegans.Nat. Genet. 2008; 40: 1375-1383Crossref PubMed Scopus (714) Google Scholar). However, while using MosSCI, we were surprised to find that not all single-copy transgenes were expressed in the germline (Figures 1A–1C ). The failure to express was only common for transgene fusions to lengthy foreign sequences, gfp (Figure 1A); transgenes with the flag epitope sequences were nearly always fully expressed (Figure 1A). Furthermore, we observed that transgenes in which gfp was inserted at the 5′ (rather than 3′) end of the construct were much less likely to be expressed (Figure 1A). PCR and sequence analyses indicated that nonexpressed transgenes are structurally identical to expressed transgenes, suggesting that the former are actively silenced. We next crossed a silent line to an expressing line to see which phenotype dominates. Strikingly, we found that 100% of the F1 cross-progeny (n = 12) and F2 self-progeny (n = 24) failed to express gfp in the germline (Figure 1D). Identical results were obtained even when the silent and active alleles were inserted on separate chromosomes (Figure 1E), suggesting that chromosomal pairing is not required for transfer of the silent state. Although transgenes with 3′ gfp insertions were less prone to silencing during transgene formation, they were fully silenced when crossed to a silent line (Figure 3J and data not shown). We found that either parent could contribute the dominant silencing signal. However, when the silent allele was male derived, it took more than one generation to completely silence the active allele. For example, silencing was observed in 67% (n = 15) of F1 progeny when the silent allele was paternally derived, whereas 100% (n = 12) of F1 progeny were silenced when maternally derived. Nevertheless, regardless of the parent of origin, in the F3 and subsequent generations, 100% of the descendants were GFP negative (n > 100). The silent phenotype was fully penetrant, with no evidence of expression or reversion even after the formerly active allele was resegregated as a homozygote (Figure 1E). These results clearly indicate that the failure to express these single-copy transgenes represents an active silencing process that involves a dominant trans-acting silencing signal. We first observed this dominant silencing activity in crosses with gfp::csr-1, which raised a concern because CSR-1 is an Argonaute that is potentially involved in silencing mechanisms. However, identical results were obtained in crosses with cdk-1 transgenes (data not shown), indicating that there is nothing unusual about the csr-1 transgenic lines. We refer to this phenomenon as RNA-induced epigenetic silencing (RNAe) because the silent state is stable indefinitely (without evidence of reversion), and (as shown below) maintenance of silencing involves a small RNA silencing signal that is epigenetically programmed (not genomically encoded). We identify transgenes exhibiting this type of silencing by including the term “(RNAe)” after the transgene name (e.g., neSi11 gfp::cdk-1(RNAe)). For clarity, active versions of the same alleles are referred to using (+), e.g., neSi11 gfp::cdk-1(+). High-copy transgenes in C. elegans can induce cosuppression of endogenous homologous genes (Dernburg et al., 2000Dernburg A.F. Zalevsky J. Colaiácovo M.P. Villeneuve A.M. Transgene-mediated cosuppression in the C. elegans germ line.Genes Dev. 2000; 14: 1578-1583PubMed Google Scholar, Ketting and Plasterk, 2000Ketting R.F. Plasterk R.H. A genetic link between co-suppression and RNA interference in C. elegans.Nature. 2000; 404: 296-298Crossref PubMed Scopus (164) Google Scholar). Several of the transgenes we analyzed are fusion constructs with essential genes (e.g., gfp::cdk-1) and should result in obvious visible phenotypes if the corresponding endogenous locus was cosuppressed. However, no phenotypic evidence of cosuppression was observed in the silent lines analyzed (data not shown), suggesting that, despite the dominant nature of the silencing signal, silencing does not spread to the endogenous locus. To ask whether there is a partial suppression of the endogenous locus, we performed western blot analysis to determine the relative expression of the transgene and endogenous protein products in both active and silent lines. Consistent with the lack of phenotypic evidence for cosuppression, we observed identical levels of endogenous protein expression in both the active and silent transgenic lines (Figure 2A ). To ask whether silencing is regulated transcriptionally or posttranscriptionally, we isolated total RNA from otherwise identical silent and active gfp::csr-1 strains and measured the abundance of pre-mRNAs and mRNAs by real-time quantitative PCR (qPCR). We found that both the pre-mRNA and mRNA levels were significantly reduced in the silent line compared to the active line (Figures 2B and 2D). Moreover, although a reduction at the pre-mRNA level appeared to account for the majority of silencing, a further reduction was evident at the mRNA level, suggesting that silencing is achieved at both transcriptional and posttranscriptional levels (Figures 2B and 2D). Previous work has shown that the methylation of lysine 9 on histone H3 (H3K9me), a histone modification associated with silent chromatin, is enriched on high-copy number transgenes in the germline (Bessler et al., 2010Bessler J.B. Andersen E.C. Villeneuve A.M. Differential localization and independent acquisition of the H3K9me2 and H3K9me3 chromatin modifications in the Caenorhabditis elegans adult germ line.PLoS Genet. 2010; 6: e1000830Crossref PubMed Scopus (81) Google Scholar, Kelly et al., 2002Kelly W.G. Schaner C.E. Dernburg A.F. Lee M.H. Kim S.K. Villeneuve A.M. Reinke V. X-chromosome silencing in the germline of C. elegans.Development. 2002; 129: 479-492PubMed Google Scholar). Furthermore, germline silencing of high-copy transgenes is dependent on a number of chromatin-associated factors, including the Polycomb group complex (MES-2/-3/-6), a Trithorax-related protein (MES-4), and the heterochromatin proteins (HPL-1 and -2) (Couteau et al., 2002Couteau F. Guerry F. Muller F. Palladino F. A heterochromatin protein 1 homologue in Caenorhabditis elegans acts in germline and vulval development.EMBO Rep. 2002; 3: 235-241Crossref PubMed Scopus (94) Google Scholar, Grishok et al., 2005Grishok A. Sinskey J.L. Sharp P.A. Transcriptional silencing of a transgene by RNAi in the soma of C. elegans.Genes Dev. 2005; 19: 683-696Crossref PubMed Scopus (131) Google Scholar, Kelly and Fire, 1998Kelly W.G. Fire A. Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans.Development. 1998; 125: 2451-2456PubMed Google Scholar, Kelly et al., 1997Kelly W.G. Xu S. Montgomery M.K. Fire A. Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene.Genetics. 1997; 146: 227-238Crossref PubMed Google Scholar). Consistent with these previous findings, we found that transgene sequences from a silent MosSCI allele, but not an active MosSCI allele, were enriched in chromatin immunoprecipitation (ChIP) experiments using antibodies specific for H3K9me3 (Figures 2C and 2D). The lysates used were from whole worms; therefore, only a portion of the chromatin present in the total lysate corresponds to germline chromatin, perhaps accounting for the relatively weak 2-fold enrichment observed. Finally, we found that mes-3, mes-4, and hpl-2 mutants all desilenced the gfp::csr-1 and gfp::cdk-1 transgenes (Table 1). These findings suggest that the maintenance of single-copy transgene silencing involves a chromatin component.Table 1Genetic Test for Maintenance of Gene SilencingGene(Allele)Gene FunctionTransgene Expressiongfp::csr-1gfp::cdk-1rde-1(ne300)Argonaute in RNAi−−prg-1(tm872)Piwi homolog−−rde-3(ne3370)Poly(A) polymerase++mut-7(ne4255)3′-to-5′ exonuclease++hpl-1(tm1624)HP1 homolog−−hpl-2(tm1489)HP1 homolog+cGFP is desilenced in fraction of germline in the same worm.+bGFP is partially desilenced (GFP signal is weak in each worm).hpl-1(tm1624)HP1 homologs++hpl-2(tm1489)met-1(n4337)methyltransferases−NAmet-2(n4256)mes-3(bn35)aScored in sterile M−Z− mutants.Polycomb complex+bGFP is partially desilenced (GFP signal is weak in each worm).+bGFP is partially desilenced (GFP signal is weak in each worm).mes-4(bn23)aScored in sterile M−Z− mutants.Trithorax complex+bGFP is partially desilenced (GFP signal is weak in each worm" @default.
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• W2154222068 title "piRNAs Initiate an Epigenetic Memory of Nonself RNA in the C. elegans Germline" @default.
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• W2154222068 doi "https://doi.org/10.1016/j.cell.2012.06.015" @default.
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