{'fraction_non_alphanumeric': 0.03985969387755102, 'fraction_numerical': 0.024744897959183672, 'mean_word_length': 4.712568306010929, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Absence of effects of Sir2 over-expression on lifespan in C. elegans and Drosophila

Over-expression of sirtuins (NAD+-dependent protein deacetylases) has been reported to increase lifespan in budding yeast, Caenorhabditis elegans and Drosophila melanogaster1-3. Studies of gene effects on ageing are vulnerable to confounding effects of genetic background4. We re-examined the reported effects of sirtuin over-expression on ageing and found that standardisation of genetic background and use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high level sir-2.1 over-expression1 abrogated the longevity increase, but not sir-2.1 over-expression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low copy number sir-2.1 over-expression2 also abrogated longevity. A Drosophila strain with ubiquitous over-expression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported3, but not relative to the appropriate transgenic controls, and nor was a new line with stronger over-expression of dSir2. These findings underscore the importance of controlling for genetic background and the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life extending effect of dietary restriction (DR) on ageing in Drosophila has also been reported to be dSir2 dependent3. We found that DR increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects on lifespan in C. elegans and Drosophila.

with stronger over-expression of dSir2. These findings underscore the importance of controlling for genetic background and the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life extending effect of dietary restriction (DR) on ageing in Drosophila has also been reported to be dSir2 dependent 3 . We found that DR increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects on lifespan in C. elegans and Drosophila.

Keywords
Ageing; C. elegans; Drosophila; sirtuin; genetic background The role of sirtuins in ageing was discovered in budding yeast (Saccharomyces cerevisiae), where over-expression of SIR2 increases replicative lifespan 5 . It was then reported that elevated sirtuin levels increase lifespan in the nematode C. elegans 1,2,6 and the fruitfly Drosophila 3 , suggesting an evolutionarily ancient role of sirtuins in longevity assurance 7 .
Dietary restriction (DR), reduced food intake short of starvation, extends lifespan in organisms ranging from yeast to mammals 8 , and initial studies suggested that DR increases lifespan by activating sirtuins in yeast 9 , C. elegans 10 and Drosophila 3 . Pharmacological activation of sirtuins has thus been widely promulgated as a potential means to mimic DR and slow ageing in humans 11 . However, several aspects of the role of sirtuins in ageing have proved controversial 12 . Subsequent studies have suggested that sirtuins do not mediate DR effects on ageing, at least in budding yeast and C. elegans 13,14 . The plant-derived polyphenol resveratrol and other compounds have been reported to activate sirtuins and extend lifespan 15,16 . More recent findings have challenged both effects [17][18][19][20] . We therefore re-examined the effects of sirtuin over-expression on lifespan in C. elegans and Drosophila.
In particular, we wished to exclude the possibility that the increased longevity observed in strains with sirtuin gene over-expression are caused by differences in genetic background, or by the mutagenic effects of transgene insertion, which frequently confound studies of the genetics of ageing 4 .
LG100 exhibited a neuronal dye-filling (Dyf) defect 22 that did not segregate with the transgene upon outcrossing (Fig. S2A). Dyf mutants often exhibit extended lifespan 23 . To determine whether the longevity of LG100 might be attributable to a dyf mutation, we derived from it three Dyf, non-Rol lines (lacking geIn3) and three non-Dyf, Rol lines (carrying geIn3). Dyf, non-Rol lines were long-lived and showed wild-type SIR-2.1 protein levels ( Fig. 1c,d, Table S3). Non-Dyf, Rol lines showed elevated SIR-2.1 protein levels but had wild-type lifespans. Dyf mutant longevity appeared to be partially daf-16 dependent ( Fig. S2B), as seen previously for other Dyf mutants 23 . The co-segregation of longevity with this dyf mutation but not geIn3 was previously noted by another research team (S.S. Lee, Cornell University, personal communication). Furthermore, knock-down of sir-2.1 expression in LG100 using RNA-mediated interference did not suppress longevity, despite lowering SIR-2.1 protein to wild-type levels (Fig. 1e,f; Table S4). Taken together, these results imply that the longevity of LG100 is attributable to an unidentified dyf mutation (or possibly another mutation closely-linked to the dyf locus), and that high level overexpression of sir-2.1 is not sufficient to increase lifespan in these strains.
The duplication mDp4 includes the sir-2.1 locus, and the mDp4-containing strain DR1786 is long lived 1 . We too found that DR1786 is long-lived, and also shows elevated sir-2.1 expression. However, longevity was not suppressed by sir-2.1 RNAi (Fig. S3, Table S7) suggesting causation by factors other than sir-2.1, either on mDp4 or elsewhere in the genome.
In Drosophila over-expression of dSir2 reportedly increases lifespan relative to wild-type controls 3 . Over-expression was achieved using the GAL4, UAS binary system 24 , with the largest increases in lifespan produced by combination of EP-UAS-dSir2 (dSir2 EP2300 ) with a ubiquitously-expressed tubulin-GAL4 driver. We outcrossed these two transgenes (x6) into the control white Dahomey (w Dah ) background. Assayed on a medium similar to that used in the original study, EP-UAS-dSir2/tubulin-GAL4 flies were longer lived than wild-type controls, as previously reported 3 (Fig. 2a). However, they were not longer lived than the tubulin-GAL4/+ control flies (Fig. 2a). This implies that life extension is due to transgenelinked genetic effects other than over-expression of dSir2. Lifespan was assayed on a range of food media (see Methods for details) to test for nutrient-dependence of any effect. However, in no case were EP-UAS-dSir2/tubulin-GAL4 flies longer lived than one or both transgenic controls (Fig. S4).
Lack of an observable effect on lifespan could reflect the relatively modest increase in dSir2 expression in EP-UAS-dSir2/tubulin-GAL4 flies, both in terms of levels of mRNA ( Fig. S5) and protein (+35% relative to wild type, Fig. S6). We therefore created lines with a higher level of over-expression of dSir2 (UAS-dSir2-Myc9/tubulin-GAL4). Here dSir2 mRNA and protein levels were robustly increased relative to wild type (+318% relative to wild-type protein levels; Fig. S5, S6). We examined recombinant protein raised in E. coli to check that the presence of the Myc tag did not interfere with dSir2 histone deacetylase (HDAC) activity, as measured by deacetylation of the fluorophore-containing p53 (Fluor de Lys) or native acetylated histone H4 substrates, and it did not (Fig. S7). We also found that dSir2 HDAC activity was unaffected by addition of resveratrol in either assay (Fig. S7). We saw no increase in lifespan in UAS-dSir2-Myc/tubulin-GAL4 flies relative to tubulin-GAL4/+ controls, on food medium similar to that used in the original study (Fig. 2b) or relative to either control on a range of other media (Fig. S4b,c,f). An independent research team also saw no increase in lifespan in tubulin-GAL4/UAS-dSir2-Myc9 flies (Fig. S8). These results suggest that the previously observed longevity of EP-UAS-dSir2/tubulin-GAL4 flies was not attributable to elevated expression of dSir2, and that stronger, ubiquitous overexpression of dSir2 also does not extend fly lifespan.
The role of sirtuins in the extension of lifespan by DR in yeast and C. elegans is controversial, with multiple groups reporting that sirtuins are not required for life span extension from DR in both organisms 8 . In Drosophila, it was reported that DR does not increase lifespan in dSir2 deletion mutant flies 3 . We tested this too, using the deletion alleles dSir2 4.5 (tested previously 3 ) and dSir2 17 . We first outcrossed these alleles (Fig. S9a) into the Canton S wild type (see Methods), used in the previous DR study 3 . We then checked the effect of each allele on dSir2 gene expression. dSir2 17 abrogated dSir2 mRNA, implying that this is a null allele. By contrast, dSir2 4.5 , which contains a relatively small deletion at the 5′ end of the gene, did not reduce dSir2 mRNA levels (Fig. S9b,c).
To reassess the role of dSir2 in DR in Drosophila, we compared lifespans of wild-type (Canton S), dSir2 4.5 and dSir2 17 homozygotes. All genotypes responded similarly and normally to DR in trials conducted by two independent research teams (Fig. 2c, Fig. S10), hence the effect of DR on lifespan did not require dSir2.
In this study, we were unable to verify the effect of sirtuin over-expression on lifespan in either C. elegans or Drosophila. Increased lifespan was seen in two C. elegans lines with elevated sir-2.1 expression derived from independent studies, as previously reported, but in each case this was abrogated by outcrossing. sir-2.1 over-expression does exert effects on traits other than lifespan. For example, geIn3 is neuroprotective in a worm model of neuron dysfunction in Huntington's disease 25 and, importantly, this effect is not attributable to the dyf mutation (Fig. S11). Moreover, both NL3909 and its outcrossed derivative are thermotolerant (M. Somogyvári, C. Sőti, unpublished data). In Drosophila, lines overexpressing dSir2 were longer-lived than wild type controls, as previously reported, but they were not longer lived than lines containing the appropriate transgenic controls. That all transgenic lines were longer lived than the Dahomey wild type into which they had been outcrossed could reflect heterosis in the vicinity of the transgene inserts, or a mutagenic effect of the GAL4 insert.
Lifespan was not increased by over-expression either of sir-2.1 from its own promoter in C. elegans, or dSir2 ubiquitously from a heterologous promoter in Drosophila. Our findings call into question the robustness of earlier reports of a role of sirtuins in longevity-assurance based upon over-expression in C. elegans and Drosophila, and also on the role of dSir2 in the response to DR in Drosophila. However, sirtuins can affect lifespan in animals under certain conditions: C. elegans daf-2(e1370) mutants are hypersensitive to genetic effects on lifespan 26 , and here deletion of sir-2.1 reproducibly increases lifespan 6 (Fig. S12).
Our finding that resveratrol does not activate HDAC activity of dSir2 using a native histone H4 peptide is consistent with earlier findings with yeast SIR2 and mammalian SirT1 (Ref. 17,18). Resveratrol increased Drosophila lifespan in one study 27 but not another 21 . In principle, this could reflect sensitivity of resveratrol effects to subtle differences in culture conditions. If this were the case, our findings would imply that such effects are not attributable to direct activation of dSir2 by resveratrol.

Nematode lifespan measurements
These were performed as described 28 , at 20°C. To prevent progeny production, 5-fluoro-2′deoxyuridine (FUdR) was added to seeded plates, to a final concentration of 10, 40 or 50 μM. Before testing effects of RNA-mediated interference (RNAi) on lifespan, worms were kept for 2 generations on the RNAi bacteria. Statistical significance of effects on lifespan was estimated using the log rank test, performed using JMP, Version 7 (SAS Institute).

Fly stocks and maintenance
tubulin-GAL4 and dSir2 EP2300 were obtained from the Bloomington Stock Center. dSir2-Myc2 and dSir2-Myc9 lines were generated by germ-line transformation into strain w 04 . dSir2 4.5 /SM6B, dSir2 17 /Cyo and Canton S were gifts from S. Pletcher, J. Rine and S.
Helfand. All lines were outcrossed at least 6 times into the relevant controls. Experiments were performed at 25°C on a 12h : 12 h light-dark cycle at constant humidity.

Fly lifespan assays
Flies were bred at standard density, allowed to mate for 48 hours after emerging then sorted into 10 females per vial. Vials were changed every 48 hours, and deaths per vial scored until all flies were dead. Over-expression studies n=200. dSir2 mutant studies n=100. For statistical methodology, see above.

dSir2 deacetylation assays
We used both the SirT1 Fluorimetric Drug Discovery Kit (Enzo Life Sciences) and an HPLC-based acetyl-histone H4 deacetylation assay 29 . dSir2 and dSir2-Myc were cloned into pET SUMO (Invitrogen) and purified on HisPur cobalt spin columns (Thermo Scientific).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Isolation of Dyf, non-Rol and non-Dyf, Rol lines-LG100 was crossed with N2, and lines established from individual F2 animals exhibiting Dyf, non-Rol or non-Dyf, Rol phenotypes. The Dyf phenotype was identified by staining with the dye DiI, and looking for absence of dye uptake into the amphid and phasmid neurons. Non-Dyf, Rol F2 animals that were heterozygous for the geIn3 transgene array (the rol-6 marker is dominant) were identified by the presence of non-Rol animals in the F3, and excluded.

RNA-mediated interference (RNAi)-Animals
were fed E. coli containing the HT115 vector either with or without a portion of the sir-2.1 gene cloned into it. The sir-2.1 feeding strain was obtained from the Ahringer RNAi library 33 . Worms were maintained on RNAi feeding strains for two generations prior to lifespan measurements. One day before starting measurements, 5-fluoro-2′-deoxyuridine (FUDR) was applied to seeded plates to 10 μM to prevent progeny production.
Analysis of SIR-2.1 protein levels-Protein was prepared from synchronous nematode cultures (L4 larvae and young adults) raised on E. coli OP50 or RNAi bacteria for two generations. Western blots were performed with anti-actin monoclonal antibodies (Santa-Cruz Biotechnology), anti-SIR-2.1 polyclonal antibody (kindly provided by A. Gartner 34 ). For all assays, 3-5 replicate worm cultures were used.
Neuroprotection assays-To test for sirtuin protection from expanded polyglutamines (polyQs), we crossed GA919 (geIn3 dissociated from dyf-?(wu250)) to strains carrying integrated polyQ arrays. These polyQ strains co-express the first 57 amino acids of human huntingtin (htt) with either 19 or 128 Glns fused to CFP, expressed from the mec-3 promoter, and YFP expressed from the mec-7 promoter in touch receptor neurons 25 . Response to touch at the tail was tested as described 25 . Three trials were performed and 150-200 animals/genotype tested.
Lifespan analysis-Lifespans of synchronized population cohorts were measured as previously described 28 . FUDR was applied to the plates, to 10, 40 or 50 μM (see Supplemental Tables). Lifespan experiments were performed at 20°C. A small proportion of animals were censored, usually due to uterine rupture, which mainly occurred at midadulthood (~day 9-11).
Statistical analysis-Statistical significance of effects on lifespan were estimated using the log rank test, performed using JMP, Version 7 (SAS Institute).