End-of-life targeted degradation of DAF-2 insulin/IGF-1 receptor promotes longevity free from growth-related pathologies.

Preferably, lifespan-extending therapies should work when applied late in life without causing undesired pathologies. Reducing insulin/insulin-like growth factor (IGF)-1 signaling (IIS) increases lifespan across species, but the effects of reduced IIS interventions in extreme geriatric ages remains unknown. Using the nematode Caenorhabditis elegans, we engineered the conditional depletion of the DAF-2/insulin/IGF-1 transmembrane receptor using an auxin-inducible degradation (AID) system. This allowed for the temporal and spatial reduction in DAF-2 protein levels at time points after which interventions such as RNAi become ineffective. Using this system, we found that AID-mediated depletion of DAF-2 protein surpasses the longevity of daf-2 mutants. Depletion of DAF-2 during early adulthood resulted in multiple adverse phenotypes, including growth retardation, germline shrinkage, egg retention, and reduced brood size. By contrast, AID-mediated depletion of DAF-2 post-reproduction, or specifically in the intestine in early adulthood, resulted in an extension of lifespan without these deleterious effects. Strikingly, at geriatric ages, when 75% of the population had died, AID-mediated depletion of DAF-2 protein resulted in a doubling in lifespan. Thus, we provide a proof-of-concept that even close to the end of an individual's lifespan, it is possible to slow aging and promote longevity.

The goal of geroscience, or research into old age, is to promote health during old age, and thus, to increase lifespan. In the body, the groups of biochemical reactions, or 'pathways', that allow an organism to sense nutrients, and regulate growth and stress, play major roles in ensuring healthy aging. Indeed, organisms that do not produce a working version of the insulin/IGF-1 receptor, a protein involved in one such pathway, show increased lifespan. In the worm Caenorhabditis elegans, mutations in the insulin/IGF-1 receptor can even double their lifespan. However, it is unclear whether this increase can be achieved once the organism has reached old age. To answer this question, Venz et al. genetically engineered the nematode worm C. elegans so that they could trigger the rapid degradation of the insulin/IGF-1 receptor either in the entire organism or in a specific tissue. Venz et al. started by aging several C. elegans worms for three weeks, until about 75% had died. At this point, they triggered the degradation of the insulin/IGF-1 receptor in some of the remaining worms, keeping the rest untreated as a control for the experiment. The results showed that the untreated worms died within a few days, while worms in which the insulin/IGF-1 receptor had been degraded lived for almost one more month. This demonstrates that it is possible to double the lifespan of an organism at the very end of life. Venz et al.'s findings suggest that it is possible to make interventions to extend an organism's lifespan near the end of life that are as effective as if they were performed when the organism was younger. This sparks new questions regarding the quality of this lifespan extension: do the worms become younger with the intervention, or is aging simply slowed down?

Synergistic lethality between BRCA1 and H3K9me2 loss reflects satellite derepression.

Caenorhabditis elegans has two histone H3 Lys9 methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/SUV39H1 related). In worms, we found simple repeat sequences primarily marked by H3K9me2, while transposable elements and silent tissue-specific genes bear H3K9me3. RNA sequencing (RNA-seq) in histone methyltransferase (HMT) mutants shows that MET-2-mediated H3K9me2 is necessary for satellite repeat repression, while SET-25 silences a subset of transposable elements and tissue-specific genes through H3K9me3. A genome-wide synthetic lethality screen showed that RNA processing, nuclear RNA degradation, the BRCA1/BARD1 complex, and factors mediating replication stress survival are necessary for germline viability in worms lacking MET-2 but not SET-25. Unlike set-25 mutants, met-2-null worms accumulated satellite repeat transcripts, which form RNA:DNA hybrids on repetitive sequences, additively with the loss of BRCA1 or BARD1. BRCA1/BARD1-mediated H2A ubiquitination and MET-2 deposited H3K9me2 on satellite repeats are partially interdependent, suggesting both that the loss of silencing generates BRCA-recruiting DNA damage and that BRCA1 recruitment by damage helps silence repeats. The artificial induction of MSAT1 transcripts can itself trigger damage-induced germline lethality in a wild-type background, arguing that the synthetic sterility upon BRCA1/BARD1 and H3K9me2 loss is directly linked to the DNA damage provoked by unscheduled satellite repeat transcription.

Heterochromatic foci and transcriptional repression by an unstructured MET-2/SETDB1 co-factor LIN-65.

The segregation of the genome into accessible euchromatin and histone H3K9-methylated heterochromatin helps silence repetitive elements and tissue-specific genes. In Caenorhabditis elegans, MET-2, the homologue of mammalian SETDB1, catalyzes H3K9me1 and me2, yet like SETDB1, its regulation is enigmatic. Contrary to the cytosolic enrichment of overexpressed MET-2, we show that endogenous MET-2 is nuclear throughout development, forming perinuclear foci in a cell cycle-dependent manner. Mass spectrometry identified two cofactors that bind MET-2: LIN-65, a highly unstructured protein, and ARLE-14, a conserved GTPase effector. All three factors colocalize in heterochromatic foci. Ablation of lin-65, but not arle-14, mislocalizes and destabilizes MET-2, resulting in decreased H3K9 dimethylation, dispersion of heterochromatic foci, and derepression of MET-2 targets. Mutation of met-2 or lin-65 also disrupts the perinuclear anchoring of genomic heterochromatin. Loss of LIN-65, like that of MET-2, compromises temperature stress resistance and germline integrity, which are both linked to promiscuous repeat transcription and gene expression.

NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans.

Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity.

Structural basis and function of XRN2 binding by XTB domains.

The RNase XRN2 is essential in RNA metabolism. In Caenorhabditis elegans, XRN2 functions with PAXT-1, which shares a putative XRN2-binding domain (XTBD) with otherwise unrelated mammalian proteins. Here, we characterize the structure and function of an XTBD-XRN2 complex. Although XTBD stably interconnects two XRN2 domains through numerous interacting residues, mutation of a single critical residue suffices to disrupt XTBD-XRN2 complexes in vitro and to recapitulate paxt-1-null mutant phenotypes in vivo. Demonstrating conservation of function, vertebrate XTBD-containing proteins bind XRN2 in vitro, and human CDKN2AIPNL (HsC2AIL) can substitute for PAXT-1 in vivo. In vertebrates, which express three distinct XTBD-containing proteins, XRN2 may partition into distinct stable heterodimeric complexes, which probably differ in subcellular localization or function. In C. elegans, complex formation with PAXT-1, the sole XTBD protein, serves to preserve the stability of XRN2 in the absence of substrate.

CRISPR/Cas9 Genome Editing in Caenorhabditis elegans: Evaluation of Templates for Homology-Mediated Repair and Knock-Ins by Homology-Independent DNA Repair.

Precise genome editing by the Cas9 nuclease depends on exogenously provided templates for homologous recombination. Here, we compare oligonucleotides with short homology and circular DNA molecules with extensive homology to genomic targets as templates for homology-based repair of CRISPR/Cas9 induced double-strand breaks. We find oligonucleotides to be templates of choice for introducing small sequence changes into the genome based on editing efficiency and ease of use. We show that polarity of oligonucleotide templates greatly affects repair efficiency: oligonucleotides in the sense orientation with respect to the target gene are better templates. In addition, combining a gene loss-of-function phenotype screen with detection of integrated fluorescent markers, we demonstrate that targeted knock-ins in Caenorhabditis elegans also can be achieved by homology-independent repair.

Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes.

The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY proteins.

Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans.

We have achieved targeted heritable genome modification in Caenorhabditis elegans by injecting mRNA of the nuclease Cas9 and Cas9 guide RNAs. This system rapidly creates precise genomic changes, including knockouts and transgene-instructed gene conversion.

Insulin/Insulin-like growth factor signaling controls non-Dauer developmental speed in the nematode Caenorhabditis elegans.

Identified as a major pathway controlling entry in the facultative dauer diapause stage, the DAF-2/Insulin receptor (InsR) signaling acts in multiple developmental and physiological regulation events in Caenorhabditis elegans. Here we identified a role of the insulin-like pathway in controlling developmental speed during the C. elegans second larval stage. This role relies on the canonical DAF-16/FOXO-dependent branch of the insulin-like signaling and is largely independent of dauer formation. Our studies provide further evidence for broad conservation of insulin/insulin-like growth factor (IGF) functions in developmental speed control.

Mos1 transposition as a tool to engineer the Caenorhabditis elegans genome by homologous recombination.

Gene knockouts and knock-ins have emerged as powerful tools to study gene function in model organisms. The construction of such engineered alleles requires that homologous recombination between a transgenic fragment carrying the modifications desired in the genome and the locus to engineer occurs at high frequencies. Homologous recombination frequency is significantly increased in the vicinity of a DNA double-strand break. Based on this observation, a new generation of transgene-instructed genome engineering protocols was developed. Here, we present MosTIC (for "Mos1 excision-induced transgene-instructed gene conversion"), a new technique that provides a means to engineer the Caenorhabditis elegans genome. MosTIC is initiated by the mobilization of Mos1, a Drosophila transposon experimentally introduced in C. elegans. During MosTIC, a Mos1 insertion localized in the genomic region to engineer is mobilized after germline expression of the Mos transposase. Mos1 excision generates a DNA double-strand break, which is repaired by homologous recombination using a transgenic repair template. This results in the transfer of information from the transgene into the genome. Depending on the method used to trigger Mos1 excision, two alternative MosTIC protocols are available, which are presented here in detail. This technique can be used for a wide range of applications, such as structure-function analysis, protein localization and purification, genetic screens or generation of single copy transgenes at a defined locus in the genome.

EMB-4: a predicted ATPase that facilitates lin-12 activity in Caenorhabditis elegans.

The sel-6 gene was previously identified in a screen for suppressors of the egg-laying defect associated with hypermorphic alleles of lin-12 (Tax et al. 1997). Here we show that sel-6 and two other previously defined genes, mal-2 and emb-4, are the same gene, now called "emb-4." We perform a genetic and molecular characterization of emb-4 and show that it functions cell autonomously as a positive regulator of lin-12 activity. Viable alleles identified as suppressors of lin-12 are partial loss-of-function mutations, whereas the null phenotype encompasses a range of lethal terminal phenotypes that apparently are not related to loss of lin-12/Notch signaling. emb-4 encodes a large nuclearly localized protein containing a predicted ATPase domain and has apparent orthologs in fission yeast, plants, and animals.

New positive regulators of lin-12 activity in Caenorhabditis elegans include the BRE-5/Brainiac glycosphingolipid biosynthesis enzyme.

Screens for suppressors of lin-12 hypermorphic alleles in C. elegans have identified core components and modulators of the LIN-12/Notch signaling pathway. Here we describe the recovery of alleles of six new genes from a screen for suppressors of the egg-laying defect associated with elevated lin-12 activity. The molecular identification of one of the new suppressor genes revealed it as bre-5, which had previously been identified in screens for mutations that confer resistance to Bt toxin in C. elegans. bre-5 is the homolog of D. melanogaster brainiac. BRE-5/Brainiac catalyzes a step in the synthesis of glycosphingolipids, components of lipid rafts that are thought to act as platforms for association among certain kinds of membrane-bound proteins. Reducing the activity of several other genes involved in glycosphingolipid biosynthesis also suppresses the effects of constitutive lin-12 activity. Genetic analysis and cell ablation experiments suggest that bre-5 functions prior to ligand-induced ectodomain shedding that activates LIN-12 for signal transduction.