Sirt6 regulates lifespan in Drosophila melanogaster

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.

Keywords: Myc; Sirt6; aging; histone; lifespan.

Fig. 1.

Drosophila Sirt6 is a chromatin associated protein with NAD⁺-dependent histone deacetylase activity. (A) da-GAL4>dSirt6-GFP immunofluorescence (green) in live, whole-mount larvae salivary gland. (Scale bar, 50 μM.) (B) Polytene chromosome preparation from salivary glands of da-GAL4>w¹¹¹⁸ (Top; “control”) and da-GAL4>UAS-dSirt6-GFP (Bottom; “Sirt6-GFP”) third instar larvae. As GFP fluorescence is quenched by polytene preparation, dSirt6-GFP was visualized by staining with fluorescent antibody to GFP (green). In both A and B, DNA was labeled by Hoechst dye and shown in blue. Images were taken at 20× magnification. (C) H3K9ac in vitro deacetylation assay with human SIRT6 and dSirt6, in the presence or absence of NAD⁺. Rate of formation of the deacetylated H3K9 peptide is quantified; n = 3 independent replicates. Gray bars, without NAD⁺; blue bars, with NAD⁺. Error bars represent the SEM. (D) Western blot of H3K9ac from control (tub-GAL4>w¹¹¹⁸) and dSirt6 OE (tub-GAL4>UAS-dSirt6) eviscerated abdomen lysates from 10-d-old female flies (n = 20 flies per group). Histone H3 and actin were used as loading controls.

Fig. 2.

Effect of dSirt6 on lifespan. Flies overexpressing dSirt6 in whole body (tub-GAL4>UAS-dSirt6; blue lines) are long-lived compared with tub-GAL4>w¹¹¹⁸ controls (black lines), showing a 33% median lifespan increase in males (A) and a 38% increase in females (B). OE of the native dSirt6 gene using tubGS>EP-dSirt6 fed RU486 (blue lines) extends median lifespan by 14% in males (C) and 17% in females (D), compared to diluent-fed controls (tubGS>EP-dSirt6 -RU; black lines). Knockdown of dSirt6 by RNAi (tubGS>dSirt6-RNAi +RU486; red lines) shortens median lifespan by 12% in males (E) and 10% in females (F), compared to diluent-fed controls (tubGS>dSirt6-RNAi -RU; black lines). Fat body–specific dSirt6 OE (S106>UAS-dSirt6 +RU486; blue lines) extends median lifespan by 8% in males (G) and 14% in females (H), compared to diluent-fed controls (S106>UAS-dSirt6 -RU; black lines). Log-rank P value < 0.0001 for all lifespans is shown. Full statistics, including number of individuals assayed and median lifespan values for all experiments, are presented in Dataset S1.

Fig. 3.

dSirt6 OE Flies have increased resistance to oxidative stress, decreased protein synthesis, and reduced body weight. (A–D) Survival of w¹¹¹⁸ control (black bars) and dSirt6 OE (red bars) flies maintained on 20 mM paraquat. dSirt6 OE improves survival in young (10 d old) male (A) and female flies (C), and strongly improves survival in old (40 d old) male (B) and female (D) flies. (E) Western blot analysis depicting reduced puromycin incorporation into eviscerated abdomen tissue (enriched for fat body), a measurement of protein synthesis rate (SUnSET assay), in female dSirt6 OE flies compared to controls. Total protein and actin were used as loading controls. (F) Quantification of SUnSET assay results, normalized to total protein; n = 3 replicates of 10 flies each. (G) dSirt6 OE flies have reduced body weight compared to controls; n = 3 replicates of 10 pooled flies each. In all experiments, the genotype of control flies was tub-GAL4>w¹¹¹⁸, and the genotype of dSirt6 OE flies was tub-GAL4>UAS-dSirt6. n.s., not significant; *P < 0.05, **P < 0.005, ***P < 0.0005, unpaired two-tailed t test. Error bars represent SEM.

Fig. 4.

dSirt6 OE reduces ribosome biogenesis and expression of Myc target genes. (A and B) Volcano plots showing genes significantly up-regulated (red) and down-regulated (blue) by dSirt6 OE in young, 10-d-old (A) and 40-d-old (B) fat body tissue, as determined by RNA-seq. Dotted lines indicate FDR cutoff of <0.05 (y axis) and log₂ FC cutoff of ±0.585 (1.5× FC) (x axis). Genes that did not meet FDR and FC cutoff are colored gray. (C and D) Top GSEA terms identified in genes down-regulated by dSirt6 OE in young (C) and old (D) flies; “dMyc Induced Genes” is a custom gene set of previously published genes up-regulated upon UAS-dMyc activation in Drosophila (35); “dMyc Bound Genes” is a custom gene set of previously published genes bound by dMyc in ChIP-seq experiments in Drosophila (36). GSEA reports FDR values < 0.00001 as zero, thus a –log10[FDR] of five means FDR ≤ 0.00001. NES reflects the degree to which a gene set is overrepresented, and is superimposed on top of −log10[FDR]. (E and F) Representative GSEA plots showing that most dMyc-induced and dMyc-bound genes exhibit higher expression in control genotype in young (E) and old (F) flies, compared to dSirt6 OE flies (i.e., expression is lower in dSirt6 OE flies). Green lines indicate enrichment score. Female flies were used for RNA-seq experiments, the genotype of control flies was tub-GAL4>w¹¹¹⁸, and the genotype of dSirt6 OE flies was tub-GAL4>UAS-dSirt6.

Fig. 5.

dSirt6 OE attenuates the effects of dMyc OE. (A) Representative images of fat body nuclei from abdominal cross-sections of adult control flies (tubGS>w¹¹¹⁸ +RU486, “w¹¹¹⁸ Control”) and flies overexpressing dMyc (tubGS>UAS-Myc +RU486, “dMyc OE”) or flies overexpressing both dMyc and dSirt6 (tubGS>UAS-Myc; UAS-dSirt6 +RU486, “dMyc + dSirt6 OE”). Fat body tissue was identified by peripheral location in abdomen and presence of lipid droplets stained by Nile Red (red); nuclei are stained with Hoechst (blue). White arrowheads indicate enlarged nuclei induced by dMyc OE. (Scale bar, 50 μM.) (B) Quantification of nuclei cross-sectional area shown in A; n = 200, 137, and 224 individual nuclei for control, dMyc OE, and dMyc+dSirt6 co-OE, respectively. Dots represent individual nuclei, bars are interquartile range, *** = adjusted P value < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (C) RNA-seq heatmap of 627 genes most strongly up-regulated (FC > 2, FDR < 0.01) in fat body of adult dMyc OE flies (Middle) compared to controls (Left), and relative expression (Z score) of these same genes in dMyc+dSirt6 co-OE flies (Right). The top portion of the heatmap represents 398 genes which were statistically significantly lower in dMyc+dSirt6 co-OE flies vs. dMyc OE alone; the bottom portion represents 229 genes that were not statistically significantly lower in double-OE flies vs. dMyc OE alone; n = 2 replicates of 10 fat bodies each per group. (D) Top pathways (FDR < 0.0005) identified by gene ontology (GO) term analysis of 398 genes significantly lower in double-OE flies vs. dMyc OE alone; (E) top pathways identified by GO term analysis of 229 genes that were not significantly lower in double-OE flies vs. dMyc OE alone. Genotypes for RNA-seq experiment are the same as listed in A. (F and G) Lifespan curves of control (solid black line), dMyc OE (solid blue line), and dMyc+dSirt6 co-OE (dashed green line) male (F) and female (G) flies, using same genotypes listed in A. (H) Lifespan curve for control (tub-GAL4>w¹¹¹⁸; solid black line), dSirt6 OE (tub-GAL4>UAS-dSirt6; solid blue line), dMyc heterozygote (tub-GAL4>w¹¹¹⁸;dMyc⁴ dashed gray line), and dSirt6 OE + dMyc Heterozygote (tub-GAL4>UAS-dSirt6;Myc⁴; dashed green line) flies. Log-rank adjusted P value < 0.05 for all comparisons in H, except for dSirt6 OE vs. dSirt6 OE + dMyc Heterozygote (adjusted P value > 0.05, with Bonferroni correction). Full statistics, including n (number of individuals assayed), median, mean, and maximum lifespan values for all experiments, are presented in Dataset S1.