The SKN‐1/Nrf2 transcription factor can protect against oxidative stress and increase lifespan in C. elegans by distinct mechanisms

Summary In C. elegans, the skn‐1 gene encodes a transcription factor that resembles mammalian Nrf2 and activates a detoxification response. skn‐1 promotes resistance to oxidative stress (Oxr) and also increases lifespan, and it has been suggested that the former causes the latter, consistent with the theory that oxidative damage causes aging. Here, we report that effects of SKN‐1 on Oxr and longevity can be dissociated. We also establish that skn‐1 expression can be activated by the DAF‐16/FoxO transcription factor, another central regulator of growth, metabolism, and aging. Notably, skn‐1 is required for Oxr but not increased lifespan resulting from over‐expression of DAF‐16; concomitantly, DAF‐16 over‐expression rescues the short lifespan of skn‐1 mutants but not their hypersensitivity to oxidative stress. These results suggest that SKN‐1 promotes longevity by a mechanism other than protection against oxidative damage.

Heat stress assays were carried out in liquid culture using death fluorescence as a readout of mortality (Coburn et al. 2013); a detailed account of the methodology used will appear shortly (A. Benedetto and D. Gems in preparation).

Protein oxidation measurements
Protein oxidation was measured using the OxyBlot Protein Oxidation Detection Kit (Merck Millipore). For analysis of mutant lines, 20 worms were harvested in 20μl cell lytic (Sigma) supplemented with 50mM DTT and protease inhibitor cocktail (Roche). For analysis of RNAi samples, protein extracts were made from synchronised plates of worms fed control or skn-1 RNAi. Adult worms on day 1 of adulthood were used in both cases. Samples were kept on ice. Samples were sonicated for 5 minutes at 30 second intervals using a Bioruptor sonicator (Diagenode), debris spun down and 5μl protein extract transferred to a new tube.
The carbonyl groups in each sample were derivatized to 2,4-dinitrophenylhydrazone (DNPhydrazone), the modified samples separated by SDS PAGE, transferred to a membrane and western blotted using a primary antibody specific to the DNP moiety of the proteins. After analysis, blots were stripped and re-probed with a β-actin antibody (Santa Cruz) as a loading control. Imaging and quantification of bands was carried out using the ImageQuant LAS4000 imaging system and software (GE Healthcare).

RT qPCR and chromatin immunoprecipitation
RNA was isolated from adult worms after transfer of the worms to an unseeded NGM plate to remove E. coli. 50 -100 worms were used for each assay. RNA was extracted using Trizol (Sigma) and cDNA synthesized using SuperScript II reverse transcriptase with oligo dT (Invitrogen). qRT-PCR was carried out using Fast SYBR Green Master Mix (Applied Biosystems) and the 7900 HT Fast PCR system (Applied Biosystems). Normalization of transcript quantity was carried out using the geometric mean of three stably expressed reference genes Y45F10D.4, pmp-3, and cdc-42 in order to control for cDNA input, as previously described (Hoogewijs et al. 2008). Primer sequences to detect skn-1 by qPCR were designed by Primerdesign (sequences available on request). Statistical analysis was preformed using a student t-test. The protocol for chromatin immunoprecipitation PCR was as described ) and the primer sequences as follows: skn-1b/c promoter F: gcgcgccgatagagtagatc R: ccctgcgtgtctacagtttcag; skn-1b promoter F: gcacgcctccttcattagtc R: gctggttgcactttctcctc; control region F: tgtatgggggtgaacaggat R: cccggagctcagactacatc.

Epifluorescence microscopy
Worms were raised at 15˚C, picked at L4 stage, and shifted to 25˚C for 24 hr to increase transgenic extrachromosomal array expression and to induce the daf-2 phenotype in the daf-2(e1370) 1 day old adults. For each slide, 30-40 worms were mounted in M9 + 0.2% levamisole on a 2% agarose pad and imaged within 30 min. Quantification of GFP expression from transgenic strains was carried out using a Leica DMRXA2 microscope with a GFP filter cube (excitation: 470/40 nm; emission: 525/50 nm), an Orca C10600 digital camera (Hamamatsu) and Volocity image analysis software (Improvision).    Table S1). FUDR caused a slight increase in the mean lifespan of skn-1(zu135) mutants (p=0.02) but this strain was still markedly short lived compared to wild type. No lifespan extension by FUDR was observed in other skn-1 mutants (Table S1). (B) The effect of skn-1 on daf-16(oe) longevity is not affected by daf-2 RNAi (trial 1 in Table S1). Trials conducted at 20˚C, with 40M