Sirt6 regulates lifespan in Drosophila melanogaster
Edited by Nancy Bonini, Department of Biology, University of Pennsylvania, Philadelphia, PA; received June 17, 2021; accepted December 1, 2021
Significance
Sirt6 is well known for its role in regulating the aging process, particularly for its ability to extend lifespan in mice when overexpressed. However, the underlying molecular mechanisms responsible for lifespan regulation by Sirt6 are not well understood. Here, we characterized dSirt6 in fruit flies (Drosophila melanogaster). We found that dSirt6 functions very similarly to mammalian Sirt6 at the molecular and biochemical levels. Furthermore, overexpressing dSirt6 increased lifespan in flies. dSirt6 overexpression extends lifespan, in part, by opposing the activity of Myc, a master regulator of protein synthesis, which is associated with decreased protein synthesis. These findings have relevance for the treatment of age-related disease by modulating Sirt6 activity.
Abstract
Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. Here, we characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD+-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. Our results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.
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Advanced age is the leading risk factor for many chronic diseases. Interventions which target the aging process itself may be useful to delay or prevent multiple age-related diseases simultaneously (1). At the cellular and molecular level, primary drivers of the aging process include genome instability, epigenetic alterations, telomere attrition, and loss of proteostasis (2). Enhancing the activity of genes which oppose these processes may therefore be an effective strategy to combat aging and age-related disease.
Sirtuins are a family of NAD+-dependent protein deacylases well known for their role in regulating metabolism, genomic stability, and aging. Mammalian genomes contain seven sirtuins, and sirtuin overexpression (OE) has been shown to extend lifespan in yeast (3), worms (4), flies (5, 6), and mice (7).
Over the last 15 y, increasing evidence has defined Sirt6 as a key regulator of longevity with diverse antiaging functions across multiple cellular pathways (8). Most notably, Sirt6 OE extends lifespan in mice (9, 10) and ameliorates some aging phenotypes, while Sirt6 knockout mice are short lived and present an accelerated aging-like phenotype (11). Sirt6 is a nuclear-localized sirtuin that associates tightly with nucleosomes and chromatin (11, 12). In vitro, Sirt6 has weak deacetylase activity but is an efficient deacylase of long-chain fatty acids (13). Despite this, ample evidence suggests that Sirt6 functions in vivo as a transcriptional corepressor via deacetylation of H3K9ac near gene promoters (8). This discrepancy may be explained by the finding that Sirt6 deacetylase activity is enhanced by binding to free fatty acids (13), as well as association with nucleosomes (12). In addition, Sirt6 has mono-adenosine 5′-diphosphate (mono-ADP) ribosylation activity (14), which promotes DNA repair via PARP1 (15), retrotransposon (RTE) silencing via KAP1 (16), and activation of NRF2 target genes via BAF170 (17).
In mammalian systems, Sirt6 has been found to regulate an extensive list of cellular processes in a manner which may enhance longevity. Sirt6 plays a major role in DNA repair, rapidly localizing to damage sites and recruiting DNA repair factors and chromatin remodelers (15, 18, 19). In addition, Sirt6 enhances telomere stability via H3K9ac deacetylation (20). Sirt6 also opposes numerous proaging processes, including NF-κB signaling (21), RTE activation (16, 22), ribosome biogenesis (23), protein synthesis (24), and tumor growth (23). Although it is clear that Sirt6 possesses many functions relevant to aging, the precise mechanisms by which it is able to extend lifespan via OE are unclear.
Drosophila melanogaster represents an excellent model for investigating genetic regulation of longevity. Drosophila possess five sirtuin genes, orthologous to mammalian Sirt1, Sirt2, Sirt4, Sirt6, and Sirt7. Despite extensive study of Sirt6 in mammals, very little is known about the function of the Drosophila ortholog of Sirt6 (herein called “dSirt6”). Here we characterize the D. melanogaster dSirt6 gene and investigate its role in regulating longevity.
Results
Drosophila Sirt6 Is a Nuclear Protein with NAD+-Dependent Histone Deacetylase Function.
To examine Sirt6 function in Drosophila, we cloned dSirt6 into several different expression constructs and created transgenic flies with transgenes for wild-type dSirt6 (UAS-dSirt6) and dSirt6 with a C-terminal GFP-fusion protein (UAS-dSirt6-GFP), under the control of UAS-regulatory sequences. Driving expression of UAS-dSirt6-GFP in the whole fly with daughterless-GAL4 (da-GAL4), we observed a strong nuclear GFP signal in all tissues of larvae (salivary gland shown in Fig. 1A). In addition, dSirt6-GFP protein was strongly associated with salivary gland polytene chromosomes (Fig. 1B), as evident by immunofluorescent staining. We next quantified NAD+-dependent deacetylase activity of dSirt6 using an in vitro deacetylase assay. dSirt6 deacetylated H3K9ac peptide substrate in an NAD+-dependent fashion and at a rate very similar to human SIRT6 (Fig. 1C). Furthermore, we found that adult dSirt6 OE flies have reduced levels of H3K9ac in vivo, at both young (10 d old) (Fig. 1D) and old (40 d old) (SI Appendix, Fig. S1 ) ages, compared to wild-type w1118 controls. The observed reduction of H3K9ac during dSirt6 OE was consistent with previous findings (25). We also found reduced levels of H3K56ac in dSirt6 OE flies (SI Appendix, Figs. S1 and S2 ). Overall, these results indicate that, like mammalian SIRT6, dSirt6 is a predominantly nuclear and chromatin-associated protein that functions as an NAD+-dependent histone deacetylase.
Fig. 1.

Modulating dSirt6 Levels Influences Organismal Lifespan.
Having established the conserved cellular localization and histone deacetylase activity of dSirt6 in flies, we next tested whether its role in lifespan regulation was also conserved. We began by overexpressing dSirt6 in the whole body using tubulin-GAL4 (tub-GAL4) to drive UAS-Sirt6, and measuring lifespan. Strikingly, whole-body dSirt6 OE increased median lifespan by 38% in females and 33% in males (Fig. 2 A and B), compared to tub-GAL4>w1118 controls. We repeated these experiments twice more and consistently found that dSirt6 OE extends lifespan, ranging from 25 to 38% in females, and 10 to 33% in males (Dataset S1 ). A second UAS-dSirt6 line, created using site-specific integration of the UAS-dSirt6 transgene into an attP landing site, also reproducibly extended lifespan when crossed to tub-GAL4 flies, compared to controls (SI Appendix, Fig. S3 and Dataset S1 ). Whole-body OE of the native dSirt6 gene, achieved using an EP-dSirt6 line (which contains a GAL4 activation site inserted ∼250 bp upstream of the dSirt6 5′ untranslated region), also extended lifespan, by 14% in males and 17% in females (Fig. 2 C and D). In the experiments in Fig. 2 C and D, we activated EP-dSirt6 using the mifepristone (RU486) inducible tubulin-GeneSwitch (tubGS) driver, which activates the UAS-transgene upon flies being fed RU486. Flies fed diluent (EtOH) control serve as genetically identical controls from the same cohort (26). Together, these results indicate that OE of both transgenic and the native dSirt6 gene extends lifespan in both male and female flies. Conversely, reducing dSirt6 expression levels by ∼40% by RNA interference (RNAi) in adult flies (SI Appendix, Fig. S4 ) using the tubGS system shortened median lifespan by 12% and 10% in males and females, respectively (Fig. 2 E and F). As an additional control, we found that expressing mCherry RNAi in this same system did not shorten lifespan in either sex (SI Appendix, Fig. S3 and Dataset S1 ). Last, we found that overexpressing dSirt6 in the fat body alone, using the fat body–specific GeneSwitch driver S106, was sufficient to extend lifespan by 8% in male and 14% female flies (Fig. 2 G and H).
Fig. 2.

Longevity-Associated Phenotypes in dSirt6 OE Flies.
We next asked whether lifespan extension in dSirt6 OE flies was associated with reduced nutrient intake or reduced fertility, as either of these phenotypes can lead to increased longevity (27, 28). Food consumption was not altered in dSirt6 OE flies, as measured by Capillary Feeder (CAFE) assay (SI Appendix, Fig. S5 ). Similarly, we did not observe any difference in the fertility of dSirt6 OE females (SI Appendix, Fig. S6 ), compared to controls. We then examined phenotypes previously shown to be impacted by Sirt6 levels in mammalian systems that can also impact longevity, specifically, oxidative stress resistance (15) and protein synthesis rate (29). dSirt6 OE flies survived longer than controls when treated with the oxidative stress-inducing agent paraquat (Fig. 3 A–D), particularly in old (40 d old) flies (Fig. 3 B and D), indicating dSirt6 OE conveys increased resistance to oxidative stress. In addition, we found that protein synthesis is reduced by ∼35% in dSirt6 OE flies compared to controls, as measured by surface sensing of translation (SUnSET) assay (30, 31) (Fig. 3 E and F). Given the reduction in protein synthesis, we also examined body weight and found that both male and female dSirt6 OE flies weigh ∼5% less than genetically matched controls (Fig. 3G). Finally, we also examined the impact of dSirt6 OE on physical function in aged flies by measuring their climbing ability. As expected, control flies showed a significant decline in climbing performance from day 10 to day 30 (SI Appendix, Fig. S7 ); however, dSirt6 OE flies had preserved climbing ability at day 30. Together, these data indicate that dSirt6 OE may extend lifespan and health span in flies via increased oxidative stress resistance and/or reduced protein synthesis.
Fig. 3.

dSirt6 OE Reduces Expression of dMyc Target Genes.
Given Sirt6’s role as a histone deacetylase and major regulator of gene expression in mammals, we next sought to determine whether dSirt6 OE altered expression of genes involved in stress resistance and/or growth and protein synthesis. To address this, we performed RNA sequencing (RNA-seq) of fat body tissue from young (10 d old) and old (40 d old) control and dSirt6 OE flies. dSirt6 OE had a significant impact on gene expression, resulting in 130 up- and 242 down-regulated genes in 10-d-old flies, and 413 up- and 204 down-regulated genes in 40-d-old flies (false discovery rate [FDR] <0.05 and log2 fold change [FC] > 0.585 or < −0.585 [1.5× FC]), versus age-matched controls (Fig. 4 A and B and Dataset S2 ). Pathway analysis of genes significantly down-regulated by dSirt6 OE revealed a strong enrichment for endoplasmic reticulum and protein processing genes, as well as previously described functions of mammalian Sirt6, such as negative regulation of ribosomal and glycolysis genes (Dataset S3 ). Although many genes were also up-regulated in dSirt6 OE flies, these genes showed minimal enrichment for specific pathways in young animals (Dataset S3 and SI Appendix, Fig. S8 ). Old dSirt6 OE flies had increased expression of immune response genes as well as lipid metabolism genes (Dataset S3 ).
Fig. 4.

We also conducted gene set enrichment analysis (GSEA) (32) to identify pathways affected by dSirt6 OE. GSEA identified pathways similar to those described above (Fig. 4 C and D), as well as several additional pathways. Notably, dSirt6 OE in young flies was associated with decreased expression of genes in multiple annotated datasets related to ribosome biogenesis and protein synthesis, including “transfer RNA (tRNA) metabolic processes,” “ribosome biogenesis,” “noncoding RNA metabolic processes,” and “nucleolus” (Fig. 4C). In old flies, dSirt6 OE was associated with decreased expression of cytosolic ribosomal protein genes and translation genes, as well as decreased expression of mitochondrial matrix and oxidative phosphorylation genes, compared to age-matched controls (Fig. 4D).
dMyc [the Drosophila ortholog of mammalian Myc genes (33)] is a master regulator of ribosome biogenesis genes in Drosophila, and, in mammals, Sirt6 acts as a negative regulator of Myc-induced transcriptional activation of ribosomal protein genes (23, 34). Our RNA-seq results, along with these previous findings, led us to ask whether dSirt6 OE may be acting as a negative regulator of dMyc target genes. To test this, we created two custom gene sets for GSEA: one of genes up-regulated by dMyc OE in Drosophila [“dMyc Induced” (35)], and the other of genes with high-confidence dMyc binding sites near their TSS (determined by chromatin immunoprecipitation sequencing [ChIP-seq]) and whose expression is also reduced by dMyc RNAi [“dMyc Bound” (36)] (Dataset S4 ), and examined whether expression of these genes was altered by dSirt6 OE. In young flies, the “dMyc Induced” gene set showed the strongest enrichment (based on normalized enrichment score [NES]) of any gene set for down-regulation by dSirt6 OE (Fig. 4 C and E), and was also among the top gene sets down-regulated by dSirt6 OE in old flies (Fig. 4 D and F). The “dMyc Bound” gene set was also among the top sets of genes down-regulated by dSirt6 OE in both young and old flies (Fig. 4 C–F). Expression of dMyc itself was moderately elevated in young dSirt6 OE flies (1.58 FC, FDR = 0.007) vs. control flies, possibly reflecting a cellular compensation for reduced expression of dMyc target genes, but did not differ vs. controls in old dSirt6 OE flies. Together, these data strongly indicate that dSirt6 OE reduces the expression of dMyc target genes, which are primarily involved in ribosome biogenesis.
Mammalian Sirt6 has been previously shown to repress target genes via deacetylation of H3K9ac at promoter-proximal regions and gene bodies (34). We examined whether dMyc target genes with reduced transcript levels in dSirt6 OE flies also had lower levels of H3K9 acetylation. Using ChIP-qPCR on chromatin from fat body tissue, we found that H3K9ac was strongly enriched near the transcription start site (TSS) of selected dMyc target genes in control flies (SI Appendix, Fig. S9 ). These regions had reduced levels of H3K9ac in dSirt6 OE flies, suggesting epigenetic repression of these dMyc target genes by dSirt6.
dSirt6 and dMyc Are Epistatic.
Reducing Myc expression extends lifespan in both mice (37) (c-Myc) and flies (38) (dMyc). Given the connection between reduced Myc activity and longevity, along with our results that dSirt6 OE reduces expression of dMyc target genes, we sought to further explore the role of dSirt6 in repressing dMyc function. We used a well-characterized UAS-dMyc line (39) to OE dMyc in adulthood (“dMyc OE”), and tested whether co-OE of dSirt6 could alter or reverse dMyc-induced phenotypes. dMyc OE in Drosophila causes endoreplication and increased transcription of nucleolar and ribosome biogenesis genes, leading to increased nuclear and nucleolar size (35). Thus, we asked whether co-OE of dSirt6 together with dMyc (“dMyc+dSirt6 co-OE”) could also attenuate the increase in nuclear size caused by dMyc OE. dMyc OE led to a 2.2-fold increase in median nuclear cross-sectional area in the adult fat body (24.4 μM2 in controls vs. 53.3 μM2 in dMyc OE, P < 0.0001) (Fig. 5 A and B), a size increase consistent with previous results in larvae (40). Co-OE of dSirt6 together with dMyc attenuated this size increase by 0.59-fold (median size: 39.3 μM2 in dMyc+dSirt6 co-OE vs. 53.3 μM2 in dMyc single-OE, P < 0.0001 [Fig. 5 A and B]). In addition to increased nuclear size, dMyc OE is also associated with increased protein synthesis. Since we had already observed that dSirt6 OE flies have reduced protein synthesis, we also tested whether dSirt6 OE could block increased protein synthesis caused by dMyc OE. As expected, we observed increased protein synthesis rates (approximately twofold) in dMyc OE flies, compared to controls. However, co-OE of dSirt6 almost completely blocked this increase (SI Appendix, Fig. S10 ). Together, these data suggest dSirt6 OE directly counteracts the effects of dMyc OE at the cellular level.
Fig. 5.

Next, we performed RNA-seq on fat body tissue from control, dMyc OE, and dMyc+dSirt6 co-OE flies (Fig. 5C and Dataset S5 ). We used a stringent FC and FDR cutoff (FDR < 0.01, FC > 2) to select the genes most strongly up-regulated by dMyc OE alone, and asked whether their expression was lower when dMyc OE was accompanied by dSirt6 OE. Of the 627 genes up-regulated by dMyc OE alone under these significance criteria, 398 (64%) had considerably lower expression in dMyc+dSirt6 co-OE flies (FDR 0.05, log2 FC < −0.263; median log2 FC = −0.59). Gene ontology analysis of dMyc-induced genes which are significantly down-regulated by dSirt6 co-OE revealed that most of these genes are involved with various aspects of ribosome biogenesis (Fig. 5D), in agreement with these groups of genes being induced by dMyc (35, 36) and also in agreement with our previous results (Fig. 4 C and D) that dSirt6 OE reduces ribosome biogenesis genes. Interestingly, the 229 dMyc-induced genes that were not statistically significantly attenuated by dSirt6 co-OE were enriched for DNA replication and DNA repair genes (Fig. 5E); these two categories were not enriched in the 398 dMyc-induced genes reduced by dSirt6 co-OE. Importantly, dMyc protein levels are not decreased by dSirt6 co-OE, compared to dMyc OE alone (SI Appendix, Fig. S11 ). Finally, we used the 627 genes up-regulated by dMyc OE in adult fat body to create a custom gene set, and used this to run GSEA on our previous RNA-seq data from control and dSirt6 OE flies. This gene set had significantly lower expression in dSirt6 OE flies versus controls (FDR = 0.0027, NES = 2.05) (SI Appendix, Fig. S12 ), further supporting the idea that dSirt6 OE represses dMyc target genes.
Having established that dSirt6 OE opposes dMyc activity at the cellular and molecular levels, we next examined the interplay between these two genes in regulating lifespan. We first measured lifespan in the same three groups used above: control, dMyc OE, and dMyc+dSirt6 co-OE. As expected based on previous work (38), dMyc OE shortened lifespan considerably, by 16% in male (Fig. 5F) and 36% in female (Fig. 5G) flies. dSirt6 co-OE partially rescued this effect, with dMyc+dSirt6 co-OE flies having a median lifespan 10% longer than dMyc OE flies in both males (Fig. 5F) and females (Fig. 5G) (P < 0.005).
Given our findings that dSirt6 OE reduces dMyc activity, and previous results indicating that reducing dMyc expression extends lifespan, we next tested whether lifespan extension by dSirt6 OE was mediated by decreased dMyc activity. In agreement with previous findings, dMyc heterozygous flies, containing a null mutation in a single copy of dMyc (“dMyc4”) (38), were long-lived (median lifespan: 63 d vs. 71 d; 13% increase, adjusted [adj] P value < 0.00001) compared to dMyc homozygous sibling controls. dSirt6 OE also extended median lifespan at levels consistent with our earlier findings (median lifespan: 63 d vs. 75 d; 19% increase, adj P value < 0.00001). However, overexpressing dSirt6 in dMyc heterozygous flies conveyed only a very mild additional lifespan benefit (median lifespan: 75 d vs. 78 d; 4% increase, adj P value > 0.05), versus dSirt6 OE alone. These findings suggest that dSirt6 OE and dMyc haploinsufficiency may act within the same lifespan-extending pathway and that reducing dMyc activity is sufficient to account for a significant portion of the lifespan extension gained by dSirt6 OE.
Discussion
Mammalian SIRT6/Sirt6 has been extensively studied and is appreciated for its important roles in multiple cellular functions, disease, and longevity. Despite this, less is known about the Drosophila ortholog, dSirt6. We have performed an initial characterization of dSirt6 in Drosophila, particularly in regard to its molecular function and role in aging and longevity. We found that the ability of Sirt6 OE to extend lifespan is conserved in lower eukaryotes, and we identified repression of dMyc transcriptional activity as a key mechanism for lifespan extension by dSirt6 OE (SI Appendix, Fig. S13 ).
Like mammalian SIRT6, we found that dSirt6 is a chromatin-associated protein with NAD+-dependent histone deacetylase activity. Specifically, dSirt6 exhibits in vitro deacetylase activity toward H3K9ac, and dSirt6 OE leads to a strong in vivo reduction of H3K9ac. This reduction was observed both at the total protein level and near the TSS of dMyc target genes.
One of the most exciting discoveries made about Sirt6 is that increasing its expression levels extends lifespan in mice (9, 10). The current study provides important confirmation that Sirt6 OE extends organismal lifespan in other species. Additionally, by using the GeneSwitch-GAL4 system, we have demonstrated that dSirt6 OE starting in adult life (i.e., not during development) is sufficient to extend lifespan. A recent report found that OE of mouse and beaver Sirt6 using the GeneSwitch system also extended lifespan in flies (41), supporting the notion that adult-specific OE is sufficient for lifespan extension. Finally, fat body–specific dSirt6 OE was sufficient to convey some lifespan extension. Drosophila fat body is analogous to adipose, liver, and immune tissues of mammals. Interestingly, long-lived Sirt6 OE mice have reduced AKT phosphorylation in liver and white adipose tissue (9). A recent report also found that Sirt6 OE protects against age-related decline in hepatic glucose output and homeostasis, in part by enhancing glycerol release from adipose tissue (10). Together, these findings implicate the importance of these tissues in mediating lifespan extension by Sirt6 OE.
A major goal of our studies was to better understand the molecular mechanisms by which dSirt6 OE extends lifespan. Our transcriptomic analysis indicates that dSirt6 is a strong negative regulator of ribosome biogenesis and protein synthesis genes, which are also dMyc target genes. dMyc has been well studied in the context of transcriptional activation, with the core set of dMyc transcriptional targets consistently including ribosomal protein, ribosomal RNA processing, tRNA processing, and translation factor genes (35, 36). Importantly, our GSEA results showed that dSirt6 OE led to decreased expression of two independent dMyc target gene sets, which are largely distinct from one another (only 16 common genes between 230 genes total; Dataset S4 ). This result was observed in both young and old flies. Mammalian Sirt6 was previously reported to act as a repressor of Myc transcriptional activity, although primarily in tumor cells (23). Our findings suggest that this function of Sirt6 is conserved in flies, and is a function of dSirt6 in healthy tissue. Myc does not appear to be deacetylated by Sirt6. Rather, the current model proposes that Sirt6 represses glycolysis and ribosomal protein gene transcription by binding to Pol II and blocking the recruitment of transcription elongation factors such as Myc, and deacetylating H3K9ac and H3K56ac, thereby promoting transcriptional pausing (34). Consistent with this, we found reduced ribosomal protein and glycolysis gene expression and reduced levels of H3K9ac in dSirt6 OE flies, both at the protein level and specifically at the promoter and/or gene body of dMyc target genes that had reduced transcript levels in dSirt6 OE flies. The requirement of dSirt6 deacetylase function for lifespan extension is still an important question and should be addressed in future studies.
Expression levels of Myc and ribosome biogenesis genes are major determinants of protein synthesis rates (29, 31, 37). Our initial phenotypic characterization of dSirt6 OE flies revealed that they have reduced protein synthesis, a phenotype strongly associated with increased longevity (29, 42). A recent report demonstrated that protein synthesis is significantly elevated in mice with reduced Sirt6 levels (Sirt6+/− mice) (24). Importantly, this study also found that Sirt6 OE reduces protein synthesis in cell culture. Our in vivo studies in flies corroborate these results indicating that dSirt6 is a negative regulator of protein synthesis and suggest possible conservation of this function in mammals.
Inhibition of mTOR is a well-established means to extend lifespan. Similar to dSirt6 OE, inhibition of mTOR by rapamycin treatment also reduces protein synthesis and extends lifespan in Drosophila (43), raising the question of whether there may be overlap in these pathways. In particular, it would be interesting, in future studies, to examine the combined effects of dSirt6 OE and rapamycin treatment on lifespan and protein synthesis.
Our lifespan epistasis experiments indicate that the magnitude of lifespan extension granted by dSirt6 OE alone receives little if any additional benefit from reducing dMyc expression, suggesting that lifespan extension by dSirt6 OE is mediated, in part, through reducing dMyc activity. We found that dSirt6 OE + dMyc heterozgyote flies lived longer than dMyc heterozygote flies, suggesting that dSirt6 OE extends lifespan through additional mechanisms beyond reducing dMyc activity. A likely possibility is that dSirt6 OE flies have enhanced DNA repair and/or reduced RTE expression, two additional functions of Sirt6 that are highly connected to aging and longevity. Our observation that dSirt6 OE improves survival in flies treated with paraquat is in line with previous reports that SIRT6 OE enhances DNA double-strand break repair in human fibroblasts treated with paraquat (15).
Several small-molecule activators of Sirt6 have been identified in recent years (44, 45), and our results highlight the potential for these compounds for treating age-related disease and extending health span in humans. Drosophila may offer a useful model to study these compounds on the aging process and age-related diseases.
Materials and Methods
Fly Stocks and Husbandry.
dSirt6 lines were made by cloning the coding DNA sequence of Drosophila Sirt6 (CG6284) into the following constructs: a pUASt-based pTW vector (Drosophila Genomics Resource Center) for “UAS-Sirt6,” pTGW for “UAS-Sirt6-GFP,” and pUASg.attB for “UAS-Sirt6-3R.” UAS-Sirt6 and UAS-Sirt6-GFP were injected (Best Gene) into w1118 flies to create transgenic lines, with insertions on the second chromosome, while UAS-Sirt6-3R was injected into M{3xP3-RFP.attP}ZH-86Fb flies (Bloomington Drosophila Stock Center [BDSC] #24749) to generate a site-specific insertion on the third chromosome. The following additional stocks were obtained from the BDSC: UAS-dMyc (BDSC #9674), dMyc4 (BDSC #64769), tubulin-GAL4 (BDSC #5138), EP-Sirt6 (BDSC #30115), UAS-Sirt6-RNAi (BDSC #34530), s106 (BDSC #8151), and daughterless-GAL4 (BDSC #55850). The tubulinGeneSwitch (“tubGS”) was a kind gift from S. Pletcher, University of Michigan, Ann Arbor, MI.
For all experiments, flies were aged and maintained on 15% dextrose/15% yeast/2% agar food at 25 °C on a 12-h light/dark cycle at 60% relative humidity. For experiments using GeneSwitch drivers, either RU486 dissolved in EtOH at the indicated concentration (Dataset S1 ) or EtOH alone (for controls) was added to the food at 20 mL/L.
Lifespan Assays.
For all experiments comparing a GAL4-driven UAS transgene to a w1118 control line (i.e., all except +/−RU486 experiments), the UAS line was backcrossed to the w1118 line for at least eight generations. Flies were sorted under brief CO2 anesthesia and placed in food vials at a density of 15 males and 15 females per vial, with a total of at least 150 flies per sex for most conditions. Flies were passed to fresh food every other day, and dead flies were scored and counted. Lifespan statistics and log-rank P values were determined using the web-based Online Application for Survival Analysis (OASIS) tool (46).
Western Blotting and SUnSET Assay.
SUnSET assay was performed as described (31) using eviscerated adult abdomens. Histone Western blotting was performed as described (47). The following antibodies were used: anti-puromycin (Developmental Studies Hybridoma Bank, monoclonal [PMY-2A4]), anti-Actin (Abcam, ab3280, monoclonal [C4]), anti-H3K9ac (Active Motif, polyclonal [39917]), anti-H3K56ac (Epitomics, 2134-1, monoclonal [EPR996Y]), anti-histone H3 (Abcam, ab1791, polyclonal), and anti-dMyc (DSHB, monoclonal [P4C4-B10]). Total protein for SUnSET assay was visualized using the Pierce Reversible Protein Stain Kit for PVDF Membranes (Thermo Fisher).
Deacetylase Assay.
Deacetylation assay was performed as described (45). Full details are provided in SI Appendix, Supplementary Methods .
Imaging.
Polytene chromosomes preparations were performed as described (48) and stained with anti-GFP (Thermo Fisher Scientific, A-11120, monoclonal [3E6]) and Hoechst 33342 (Fisher Scientific). For the nuclear size assay, female flies were sectioned at 10 μM on a cryostat and stained and imaged as described (49) with Nile Red (Invitrogen, N1142) and Hoechst 33342 (Fisher Scientific). Fat body cells were identified based on positive Nile Red staining, peripheral location, and large cytoplasm. All images were taken on a Zeiss AxioImager.Z1 ApoTome epifluorescence microscope and processed, and the cross-sectional area was analyzed using ZEN software (Zeiss) with an experimenter blinded to sample identities.
RNA-seq and Bioinformatics.
For all groups, total RNA was collected from ∼10 dissected female fat bodies, using the RNaqueous Micro Kit with DNase digestions step (Ambion). For dSirt6 OE and controls, RNA libraries were made using the Ovation Universal RNA-seq kit (Tecan Genomics). For dMyc OE rescue experiments, RNA libraries were made using the TruSeq RNA Library Prep Kit v2. Libraries were sequenced by GENEWIZ on an Illumina HiSeq, in 2 × 150 bp mode.
Transcript abundance was quantified using Salmon (50), and differential expression was calculated using the DESeq2 function in DEBrowser (51) using default parameters. Genes were considered differentially expressed with an FDR of < 0.05 and log2 FC of ±0.585 (1.5×-fold). Gene ontology and pathway analysis were done with Flymine (52). GSEA (32) was performed using normalized count data. Redundant terms were removed using REVIGO (53).
Egg Lay Assay.
Female flies were allowed to mate in groups of 10 males and 10 females for 3 d, then transferred as single flies to individual food vials, which were passed daily. The number of eggs laid by each individual fly over a 24-h period was quantified at day 7 and day 14. Egg lays from 10 individual females were quantified.
CAFE Assay.
Data Availability
RNA-seq FASTQ files have been deposited in GEO Omnibus (GSE191320) and can be accessed at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE191320 (56).
Acknowledgments
We thank Will Lightfoot and Christoph Schorl and the Brown University Genomics Core Facility for technical assistance. This work was supported, in part, by National Institute on Aging (NIA) Grant K99AG057812 to J.R.T., NIA grants to V.G. and A.S., NIA Grants AG016694 and AG051449 to J.M.S., and NIA Grants AG024353, AG051449, and AG067306 to S.L.H.
Supporting Information
Materials/Methods, Supplementary Text, Tables, Figures, and/or References
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References
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Copyright © 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data Availability
RNA-seq FASTQ files have been deposited in GEO Omnibus (GSE191320) and can be accessed at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE191320 (56).
Submission history
Received: June 17, 2021
Accepted: December 1, 2021
Published online: January 28, 2022
Published in issue: February 1, 2022
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Acknowledgments
We thank Will Lightfoot and Christoph Schorl and the Brown University Genomics Core Facility for technical assistance. This work was supported, in part, by National Institute on Aging (NIA) Grant K99AG057812 to J.R.T., NIA grants to V.G. and A.S., NIA Grants AG016694 and AG051449 to J.M.S., and NIA Grants AG024353, AG051449, and AG067306 to S.L.H.
Notes
This article is a PNAS Direct Submission.
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Competing Interests
Competing interest statement: J.D.B. is a founder and Director of CDI Labs, Inc., is a founder of Neochromosome, Inc., is a founder and Scientific Advisory Board (SAB) member of ReOpen Diagnostics, and serves or served on the SAB of the following: Sangamo, Inc., Modern Meadow, Inc., Sample6, Inc., and the Wyss Institute. J.M.S. is a cofounder and SAB chair of Transposon Therapeutics and consults for Atropos Therapeutics, Gilead Sciences, and Oncolinea.
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Sirt6 regulates lifespan in Drosophila melanogaster, Proc. Natl. Acad. Sci. U.S.A.
119 (5) e2111176119,
https://doi.org/10.1073/pnas.2111176119
(2022).
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