The superoxide radical (O 2 − ) has long been considered a major cause of aging. O 2 − in cytosolic, extracellular, and mitochondrial pools is detoxified by dedicated superoxide dismutase (SOD) isoforms. We tested the impact of each SOD isoform in Caenorhabditis elegans by manipulating its five sod genes and saw no major effects on life span. sod genes are not required for daf-2 insulin/ IGF-1 receptor mutant longevity. However, loss of the extracellular Cu/ZnSOD sod-4 enhances daf-2 longevity and constitutive diapause, suggesting a signaling role for sod-4. Overall, these findings imply that O 2 − is not a major determinant of aging in C. elegans.Supplemental material is available at http://www.genesdev.org.Received April 11, 2008; revised version accepted September 29, 2008. Many forms of pathology lead to elevated levels of damage to biological macromolecules (Halliwell and Gutteridge 2007). This is also true of aging, the poorly understood biological process that leads to progressive deterioration and death. One strategy to discover the underlying mechanisms of aging has been to seek the causes of its associated molecular damage. An important early theory, proposed by Harman (1956), postulates that the cause might be oxygen free radicals. Harman later developed the theory, proposing a central role for the superoxide (O 2 − ) radical, issuing from the mitochondrial electron transport chain (Harman 1972). During the last few decades, much effort has been invested in tests of this nexus of theories (for review, see Muller et al. 2007 (Fujii et al. 1998). Two mitochondrial MnSOD isoforms are encoded by sod-2 and sod-3 (Giglio et al. 1994;Suzuki et al. 1996;Hunter et al. 1997).This superabundance of SOD isoforms has been a technical hurdle to investigations of the role of SOD and O 2 − in aging in C. elegans, and some of the sod genes have been barely studied. In situ gel SOD activity assays of a sod-1 deletion mutant imply that this gene encodes the major cytosolic Cu/ZnSOD (Jensen and Culotta 2005), leaving the function of sod-5 unclear. sod-3 mRNA levels are elevated in the dauer larva (Honda and Honda 1999), suggesting that this gene may play a special role in antioxidant defense in this long-lived, stress-resistant diapausal stage, but the role of sod-2 has remained obscure. In this study, we describe in detail the function of each of the five sod genes, characterizing their expression, and the phenotypic effects of manipulating their expression. This has allowed us to assess the effect on life history, especially aging, of each of the three major O 2 − pools, thereby critically testing the role of SOD and, by inference, O 2 − , in longevity assurance and aging. O 2 − can affect living organisms in a variety of ways. It can cause molecular damage that might contribute to aging; thus, one expectation of our study was that lowering SOD activity and increasing O 2 − levels might accelerate aging, and vice versa. H 2 O 2 derived from O 2 − can also act a secondary messenger-for example, in receptor tyrosine ...

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Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e−11). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16∷DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF ‘parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.