piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans.

Transgenerational 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 meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.

Telomeric small RNAs in the genus Caenorhabditis.

Telomeric DNA is composed of simple tandem repeat sequences and has a G-rich strand that runs 5' to 3' toward the chromosome terminus. Small RNAs with homology to telomeres have been observed in several organisms and could originate from telomeres or from interstitial telomere sequences (ITSs), which are composites of degenerate and perfect telomere repeat sequences found on chromosome arms. We identified Caenorhabditis elegans small RNAs composed of the Caenorhabditis telomere sequence (TTAGGC)n with up to three mismatches, which might interact with telomeres. We rigorously defined ITSs for genomes of C. elegans and for two closely related nematodes, Caenorhabditis briggsae and Caenorhabditis remanei Most telomeric small RNAs with mismatches originated from ITSs, which were depleted from mRNAs but were enriched in introns whose genes often displayed hallmarks of genomic silencing. C. elegans small RNAs composed of perfect telomere repeats were very rare but their levels increased by several orders of magnitude in C. briggsae and C. remanei Major small RNA species in C. elegans begin with a 5' guanine nucleotide, which was strongly depleted from perfect telomeric small RNAs of all three Caenorhabditis species. Perfect G-rich or C-rich telomeric small RNAs commonly began with 5' UAGGCU and 5' UUAGGC or 5' CUAAGC, respectively. In contrast, telomeric small RNAs with mismatches had a mixture of all four 5' nucleotides. We suggest that perfect telomeric small RNAs have a mechanism of biogenesis that is distinct from known classes of small RNAs and that a dramatic change in their regulation occurred during recent Caenorhabditis evolution.

Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans.

Single-copy transgenes in Caenorhabditis elegans can be subjected to a potent, irreversible silencing process termed small RNA-induced epigenetic silencing (RNAe). RNAe is promoted by the Piwi Argonaute protein PRG-1 and associated Piwi-interacting RNAs (piRNAs), as well as by proteins that promote and respond to secondary small interfering RNA (siRNA) production. Here we define a related siRNA-mediated silencing process, termed "multigenerational RNAe," which can occur for transgenes that are maintained in a hemizygous state for several generations. We found that transgenes that contain either GFP or mCherry epitope tags can be silenced via multigenerational RNAe, whereas a transgene that possesses GFP and a perfect piRNA target site can be rapidly and permanently silenced via RNAe. Although previous studies have shown that PRG-1 is typically dispensable for maintenance of RNAe, we found that both initiation and maintenance of multigenerational RNAe requires PRG-1 and the secondary siRNA biogenesis protein RDE-2. Although silencing via RNAe is irreversible, we found that transgene expression can be restored when hemizygous transgenes that were silenced via multigenerational RNAe become homozygous. Furthermore, multigenerational RNAe was accelerated when meiotic pairing of the chromosome possessing the transgene was abolished. We propose that persistent lack of pairing during meiosis elicits a reversible multigenerational silencing response, which can lead to permanent transgene silencing. Multigenerational RNAe may be broadly relevant to single-copy transgenes used in experimental biology and to shaping the epigenomic landscape of diverse species, where genomic polymorphisms between homologous chromosomes commonly result in unpaired DNA during meiosis.

Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.

Caenorhabditis elegans POT-2 telomere protein represses a mode of alternative lengthening of telomeres with normal telomere lengths.

Canonical telomere repeats at chromosome termini can be maintained by a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). Human cancers that survive via ALT can exhibit long and heterogeneous telomeres, although many telomerase-negative tumors possess telomeres of normal length. Here, we report that Caenorhabditis elegans telomerase mutants that survived via ALT possessed either long or normal telomere lengths. Most ALT strains displayed end-to-end chromosome fusions, suggesting that critical telomere shortening occurred before or concomitant with ALT. ALT required the 9-1-1 DNA damage response complex and its clamp loader, HPR-17. Deficiency for the POT-2 telomere binding protein promoted ALT in telomerase mutants, overcame the requirement for the 9-1-1 complex in ALT, and promoted ALT with normal telomere lengths. We propose that telomerase-deficient human tumors with normal telomere lengths could represent a mode of ALT that is facilitated by telomere capping protein dysfunction.

The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans.

The telomerase reverse transcriptase adds de novo DNA repeats to chromosome termini. Here we define Caenorhabditis elegans MRT-1 as a novel factor required for telomerase-mediated telomere replication and the DNA-damage response. MRT-1 is composed of an N-terminal domain homologous to the second OB-fold of POT1 telomere-binding proteins and a C-terminal SNM1 family nuclease domain, which confer single-strand DNA-binding and processive 3'-to-5' exonuclease activity, respectively. Furthermore, telomerase activity in vivo depends on a functional MRT-1 OB-fold. We show that MRT-1 acts in the same telomere replication pathway as telomerase and the 9-1-1 DNA-damage response complex. MRT-1 is dispensable for DNA double-strand break repair, but functions with the 9-1-1 complex to promote DNA interstrand cross-link (ICL) repair. Our data reveal MRT-1 as a dual-domain protein required for telomerase function and ICL repair, which raises the possibility that telomeres and ICL lesions may share a common feature that plays a critical role in de novo telomere repeat addition.

The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53-mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

Nematode chromosomes.

The nematode Caenorhabditis elegans has shed light on many aspects of eukaryotic biology, including genetics, development, cell biology, and genomics. A major factor in the success of C. elegans as a model organism has been the availability, since the late 1990s, of an essentially gap-free and well-annotated nuclear genome sequence, divided among 6 chromosomes. In this review, we discuss the structure, function, and biology of C. elegans chromosomes and then provide a general perspective on chromosome biology in other diverse nematode species. We highlight malleable chromosome features including centromeres, telomeres, and repetitive elements, as well as the remarkable process of programmed DNA elimination (historically described as chromatin diminution) that induces loss of portions of the genome in somatic cells of a handful of nematode species. An exciting future prospect is that nematode species may enable experimental approaches to study chromosome features and to test models of chromosome evolution. In the long term, fundamental insights regarding how speciation is integrated with chromosome biology may be revealed.

Genetic and Epigenetic Inheritance at Telomeres.

Transgenerational inheritance can occur at telomeres in distinct contexts. Deficiency for telomerase or telomere-binding proteins in germ cells can result in shortened or lengthened chromosome termini that are transmitted to progeny. In human families, altered telomere lengths can result in stem cell dysfunction or tumor development. Genetic inheritance of altered telomeres as well as mutations that alter telomeres can result in progressive telomere length changes over multiple generations. Telomeres of yeast can modulate the epigenetic state of subtelomeric genes in a manner that is mitotically heritable, and the effects of telomeres on subtelomeric gene expression may be relevant to senescence or other human adult-onset disorders. Recently, two novel epigenetic states were shown to occur at C. elegans telomeres, where very low or high levels of telomeric protein foci can be inherited for multiple generations through a process that is regulated by histone methylation.Together, these observations illustrate that information relevant to telomere biology can be inherited via genetic and epigenetic mechanisms, although the broad impact of epigenetic inheritance to human biology remains unclear.

Gametes deficient for Pot1 telomere binding proteins alter levels of telomeric foci for multiple generations.

Deficiency for telomerase results in transgenerational shortening of telomeres. However, telomeres have no known role in transgenerational epigenetic inheritance. C. elegans Protection Of Telomeres 1 (Pot1) proteins form foci at the telomeres of germ cells that disappear at fertilization and gradually accumulate during development. We find that gametes from mutants deficient for Pot1 proteins alter levels of telomeric foci for multiple generations. Gametes from pot-2 mutants give rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci throughout development, which persists for six generations. In contrast, gametes from pot-1 mutants or pot-1; pot-2 double mutants induce diminished Pot1 foci for several generations. Deficiency for MET-2, SET-25, or SET-32 methyltransferases, which promote heterochromatin formation, results in gametes that induce diminished Pot1 foci for several generations. We propose that C. elegans POT-1 may interact with H3K9 methyltransferases during pot-2 mutant gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that causes constitutively high levels of heterochromatic Pot1 foci.

Germ granule dysfunction is a hallmark and mirror of Piwi mutant sterility.

In several species, Piwi/piRNA genome silencing defects cause immediate sterility that correlates with transposon expression and transposon-induced genomic instability. In C. elegans, mutations in the Piwi-related gene (prg-1) and other piRNA deficient mutants cause a transgenerational decline in fertility over a period of several generations. Here we show that the sterility of late generation piRNA mutants correlates poorly with increases in DNA damage signaling. Instead, sterile individuals consistently exhibit altered perinuclear germ granules. We show that disruption of germ granules does not activate transposon expression but induces multiple phenotypes found in sterile prg-1 pathway mutants. Furthermore, loss of the germ granule component pgl-1 enhances prg-1 mutant infertility. Environmental restoration of germ granule function for sterile pgl-1 mutants restores their fertility. We propose that Piwi mutant sterility is a reproductive arrest phenotype that is characterized by perturbed germ granule structure and is phenocopied by germ granule dysfunction, independent of genomic instability.

Engineering threshold-based selection systems.

Using model organisms to identify novel therapeutic targets is frequently constrained by pre-existing genetic toolkits. To expedite positive selection for identification of novel downstream effectors, we engineered conditional expression of activated CED-10/Rac to disrupt Caenorhabditis elegans embryonic morphogenesis, titrated to 100% lethality. The strategy of engineering thresholds for positive selection using experimental animals was validated with pharmacological and genetic suppression and is generalizable to diverse molecular processes and experimental systems.

End joining at Caenorhabditis elegans telomeres.

Critically shortened telomeres can be subjected to DNA repair events that generate end-to-end chromosome fusions. The resulting dicentric chromosomes can enter breakage-fusion-bridge cycles, thereby impeding elucidation of the structures of the initial fusion events and a mechanistic understanding of their genesis. Current models for the molecular basis of fusion of critically shortened, uncapped telomeres rely on PCR assays that typically capture fusion breakpoints created by direct ligation of chromosome ends. Here we use independent approaches that rely on distinctive features of Caenorhabditis elegans to study the frequency of direct end-to-end chromosome fusion in telomerase mutants: (1) holocentric chromosomes that allow for genetic isolation of stable end-to-end fusions and (2) unique subtelomeric sequences that allow for thorough PCR analysis of samples of genomic DNA harboring multiple end-to-end fusions. Surprisingly, only a minority of end-to-end fusion events resulted from direct end joining with no additional genome rearrangements. We also demonstrate that deficiency for the C. elegans Ku DNA repair heterodimer does not affect telomere length or cause synthetic effects in the absence of telomerase.

trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.

Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen-like sliding clamp. Thus, the 9-1-1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1.

Maternal Inheritance: Longevity Programs Nourish Progeny via Yolk.

Epigenetic effects can be mediated by changes in chromatin state that are transmitted from parent to child via gametes, but support is gathering for maternal yolk, which is deposited into ooctyes, as an extranuclear epigenetic factor that can contribute to phenotypic plasticity across generations in Caenorhabditis elegans.

The Caenorhabditis elegans Rad17 homolog HPR-17 is required for telomere replication.

Subunits of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen (PNCA)-like sliding clamp are required for DNA damage responses and telomerase-mediated telomere replication in the nematode Caenorhabditis elegans. PCNA sliding clamps are loaded onto DNA by a replication factor C (RFC) clamp loader. The C. elegans Rad17 RFC clamp loader homolog, hpr-17, functions in the same pathway as the 9-1-1 complex with regard to both the DNA damage response and telomerase-mediated telomere elongation. Thus, hpr-17 defines an RFC-like complex that facilitates telomerase activity in vivo in C. elegans.

Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause.

Environmental stress can induce adult reproductive diapause, a state of developmental arrest that temporarily suspends reproduction. Deficiency for C. elegans Piwi protein PRG-1 results in strains that reproduce for many generations but then become sterile. We found that sterile-generation prg-1/Piwi mutants typically displayed pronounced germ cell atrophy as L4 larvae matured into 1-day-old adults. Atrophied germlines spontaneously reproliferated across the first days of adulthood, and this was accompanied by fertility for day 2-4 adults. Sterile day 5 prg-1 mutant adults remained sterile indefinitely, but providing an alternative food source could restore their fertility. Our data imply that late-generation prg-1 mutants experience a dynamic form of adult reproductive diapause, promoted by stress response, cell death, and RNAi pathways, where delayed fertility and reproductive quiescence represent parallel adaptive developmental outcomes. This may occur in response to a form of "heritable stress" that is transmitted by gametes and epigenetic in nature.

Developmental modulation of nonhomologous end joining in Caenorhabditis elegans.

Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.