Sirt6 regulates lifespan in Drosophila melanogaster.

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.

Sirt4 is a mitochondrial regulator of metabolism and lifespan in Drosophila melanogaster.

Sirtuins are an evolutionarily conserved family of NAD+-dependent deacylases that control metabolism, stress response, genomic stability, and longevity. Here, we show the sole mitochondrial sirtuin in Drosophila melanogaster, Sirt4, regulates energy homeostasis and longevity. Sirt4 knockout flies have a short lifespan, with increased sensitivity to starvation and decreased fertility and activity. In contrast, flies overexpressing Sirt4 either ubiquitously or specifically in the fat body are long-lived. Despite rapid starvation, Sirt4 knockout flies paradoxically maintain elevated levels of energy reserves, including lipids, glycogen, and trehalose, while fasting, suggesting an inability to properly catabolize stored energy. Metabolomic analysis indicates several specific pathways are affected in Sirt4 knockout flies, including glycolysis, branched-chain amino acid metabolism, and impaired catabolism of fatty acids with chain length C18 or greater. Together, these phenotypes point to a role for Sirt4 in mediating the organismal response to fasting, and ensuring metabolic homeostasis and longevity.

A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan.

In gonadal tissues, the Piwi-interacting (piRNA) pathway preserves genomic integrity by employing 23-29 nucleotide (nt) small RNAs complexed with argonaute proteins to suppress parasitic mobile sequences of DNA called transposable elements (TEs). Although recent evidence suggests that the piRNA pathway may be present in select somatic cells outside the gonads, the role of a non-gonadal somatic piRNA pathway is not well characterized. Here we report a functional somatic piRNA pathway in the adult Drosophila fat body including the presence of the piRNA effector protein Piwi and canonical 23-29 nt long TE-mapping piRNAs. The piwi mutants exhibit depletion of fat body piRNAs, increased TE mobilization, increased levels of DNA damage and reduced lipid stores. These mutants are starvation sensitive, immunologically compromised and short-lived, all phenotypes associated with compromised fat body function. These findings demonstrate the presence of a functional non-gonadal somatic piRNA pathway in the adult fat body that affects normal metabolism and overall organismal health.

Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend life span in Drosophila.

Transposable elements (TEs) are mobile genetic elements, highly enriched in heterochromatin, that constitute a large percentage of the DNA content of eukaryotic genomes. Aging in Drosophila melanogaster is characterized by loss of repressive heterochromatin structure and loss of silencing of reporter genes in constitutive heterochromatin regions. Using next-generation sequencing, we found that transcripts of many genes native to heterochromatic regions and TEs increased with age in fly heads and fat bodies. A dietary restriction regimen, known to extend life span, repressed the age-related increased expression of genes located in heterochromatin, as well as TEs. We also observed a corresponding age-associated increase in TE transposition in fly fat body cells that was delayed by dietary restriction. Furthermore, we found that manipulating genes known to affect heterochromatin structure, including overexpression of Sir2, Su(var)3-9, and Dicer-2, as well as decreased expression of Adar, mitigated age-related increases in expression of TEs. Increasing expression of either Su(var)3-9 or Dicer-2 also led to an increase in life span. Mutation of Dicer-2 led to an increase in DNA double-strand breaks. Treatment with the reverse transcriptase inhibitor 3TC resulted in decreased TE transposition as well as increased life span in TE-sensitized Dicer-2 mutants. Together, these data support the retrotransposon theory of aging, which hypothesizes that epigenetically silenced TEs become deleteriously activated as cellular defense and surveillance mechanisms break down with age. Furthermore, interventions that maintain repressive heterochromatin and preserve TE silencing may prove key to preventing damage caused by TE activation and extending healthy life span.

Trimethylation of Lys36 on H3 restricts gene expression change during aging and impacts life span.

Functional data indicate that specific histone modification enzymes can be key to longevity in Caenorhabditis elegans, but the molecular basis of how chromatin structure modulates longevity is not well understood. In this study, we profiled the genome-wide pattern of trimethylation of Lys36 on histone 3 (H3K36me3) in the somatic cells of young and old Caenorhabditis elegans. We revealed a new role of H3K36me3 in maintaining gene expression stability through aging with important consequences on longevity. We found that genes with dramatic expression change during aging are marked with low or even undetectable levels of H3K36me3 in their gene bodies irrespective of their corresponding mRNA abundance. Interestingly, 3' untranslated region (UTR) length strongly correlates with H3K36me3 levels and age-dependent mRNA expression stability. A similar negative correlation between H3K36me3 marking and mRNA expression change during aging was also observed in Drosophila melanogaster, suggesting a conserved mechanism for H3K36me3 in suppressing age-dependent mRNA expression change. Importantly, inactivation of the methyltransferase met-1 resulted in a decrease in global H3K36me3 marks, an increase in mRNA expression change with age, and a shortened life span, suggesting a causative role of the H3K36me3 marking in modulating age-dependent gene expression stability and longevity.

Chromatin structure and transposable elements in organismal aging.

Epigenetic regulatory mechanisms are increasingly appreciated as central to a diverse array of biological processes, including aging. An association between heterochromatic silencing and longevity has long been recognized in yeast, and in more recent years evidence has accumulated of age-related chromatin changes in Caenorhabditis elegans, Drosophila, and mouse model systems, as well as in the tissue culture-based replicative senescence model of cell aging. In addition, a number of studies have linked expression of transposable elements (TEs), as well as changes in the RNAi pathways that cells use to combat TEs, to the aging process. This review summarizes the recent evidence linking chromatin structure and function to aging, with a particular focus on the relationship of heterochromatin structure to organismal aging.

Dietary and genetic effects on age-related loss of gene silencing reveal epigenetic plasticity of chromatin repression during aging.

During aging, changes in chromatin state that alter gene transcription have been postulated to result in expression of genes that are normally silenced, leading to deleterious age-related effects on cellular physiology. Despite the prevalence of this hypothesis, it is primarily in yeast that loss of gene silencing with age has been well documented. We use a novel position effect variegation (PEV) reporter in Drosophila melanogaster to show that age-related loss of repressive heterochromatin is associated with loss of gene silencing in metazoans and is affected by Sir2, as it is in yeast. The life span-extending intervention, calorie restriction (CR), delays the age-related loss of gene silencing, indicating that loss of gene silencing is a component of normal aging. Diet switch experiments show that such flies undergo a rapid change in their level of gene silencing, demonstrating the epigenetic plasticity of chromatin during aging and highlighting the potential role of diet and metabolism in chromatin maintenance, Thus, diet and related interventions may be of therapeutic importance for age-related diseases, such as cancer.

Increased expression of Drosophila Sir2 extends life span in a dose-dependent manner.

Sir2, a member of the sirtuin family of protein acylases, deacetylates lysine residues within many proteins and is associated with lifespan extension in a variety of model organisms. Recent studies have questioned the positive effects of Sir2 on lifespan inDrosophila. Several studies have shown that increased expression of the Drosophila Sir2 homolog (dSir2) extends life span while other studies have reported no effect on life span or suggested that increased dSir2 expression was cytotoxic. To attempt to reconcile the differences in these observed effects of dSir2 on Drosophila life span, we hypothesized that a critical level of dSir2 may be necessary to mediate life span extension. Using approaches that allow us to titrate dSir2 expression, we describe here a strong dose-dependent effect of dSir2 on life span. Using the two transgenic dSir2 lines that were reported not to extend life span, we are able to show significant life span extension when dSir2 expression is induced between 2 and 5-fold. However, higher levels decrease life span and can induce cellular toxicity, manifested by increased expression of the JNK-signaling molecule Puc phosphatase and induction of dnaJ-H. Our results help to resolve the apparently conflicting reports by demonstrating that the effects of increased dSir2 expression on life span in Drosophila are dependent upon dSir2 dosage.

Genetic and biochemical tools for investigating sirtuin function in Drosophila melanogaster.

Drosophila melanogaster is one of the most widely used genetic model systems in biology. The ease of working in an invertebrate model system allows the design and execution of many experiments that would be infeasible in a vertebrate model. Although the strength of the fly as a model system lies primarily in the ease of genetic manipulation, it is flexible enough that biochemical and proteomic approaches can also be used to build a more comprehensive study. Here we present a pair of complementary protocols that we have used to examine sirtuin biology in Drosophila. First, we describe our protocol for measuring lifespan in flies expressing a gene of interest under the inducible control of the Gene-Switch system. Finally, we describe a method for performing chromatin immunoprecipitation on adult flies, including some of the difficulties associated with using this technique in chitinous tissue.

Chromatin remodeling in the aging genome of Drosophila.

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

dSir2 and Dmp53 interact to mediate aspects of CR-dependent lifespan extension in D. melanogaster.

Calorie Restriction (CR) is a well established method of extending life span in a variety of organisms. In the fruit fly D. melanogaster, CR is mediated at least in part by activation of dSir2. In mammalian systems, one of the critical targets of Sir2 is the tumor suppressor p53. This deacetylation of p53 by Sir2 leads to inhibition of p53's transcriptional activity. We have recently shown that inhibition of Dmp53 activity in the fly brain through the use of dominant-negative (DN) constructs that inhibit DNA-binding can extend life span. This life span extension appears to be related to CR, as CR and DN-Dmp53 donot display additive effects on life span. Here we report that life span extension by DN-Dmp53 expression is highly dynamic and can be achieved even when DN-Dmp53 is expressed later in life. In addition, we demonstrate that life span extension by activation of dSir2 and DN-Dmp53 expression are not additive. Furthermore, we show that dSir2 physically interacts with Dmp53 and can deacetylate Dmp53-derived peptides. Taken together, our data demonstrate that Dmp53 is a down stream target of dSir2 enzymatic activity and mediates some aspects of the life span extending effects of CR.

Small molecules that regulate lifespan: evidence for xenohormesis.

Barring genetic manipulation, the diet known as calorie restriction (CR) is currently the only way to slow down ageing in mammals. The fact that CR works on most species, even microorganisms, implies a conserved underlying mechanism. Recent findings in the yeast Saccharomyces cerevisiae indicate that CR extends lifespan because it is a mild biological stressor that activates Sir2, a key component of yeast longevity and the founding member of the sirtuin family of deacetylases. The sirtuin family appears to have first arisen in primordial eukaryotes, possibly to help them cope with adverse conditions. Today they are found in plants, yeast, and animals and may underlie the remarkable health benefits of CR. Interestingly, a class of polyphenolic molecules produced by plants in response to stress can activate the sirtuins from yeast and metazoans. At least in the case of yeast, these molecules greatly extend lifespan by mimicking CR. One explanation for this surprising observation is the 'xenohormesis hypothesis', the idea that organisms have evolved to respond to stress signalling molecules produced by other species in their environment. In this way, organisms can prepare in advance for a deteriorating environment and/or loss of food supply.

Sirtuin activators mimic caloric restriction and delay ageing in metazoans.

Caloric restriction extends lifespan in numerous species. In the budding yeast Saccharomyces cerevisiae this effect requires Sir2 (ref. 1), a member of the sirtuin family of NAD+-dependent deacetylases. Sirtuin activating compounds (STACs) can promote the survival of human cells and extend the replicative lifespan of yeast. Here we show that resveratrol and other STACs activate sirtuins from Caenorhabditis elegans and Drosophila melanogaster, and extend the lifespan of these animals without reducing fecundity. Lifespan extension is dependent on functional Sir2, and is not observed when nutrients are restricted. Together these data indicate that STACs slow metazoan ageing by mechanisms that may be related to caloric restriction.

Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan.

In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases. Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor. Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.

Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae.

Calorie restriction extends lifespan in a broad range of organisms, from yeasts to mammals. Numerous hypotheses have been proposed to explain this phenomenon, including decreased oxidative damage and altered energy metabolism. In Saccharomyces cerevisiae, lifespan extension by calorie restriction requires the NAD+-dependent histone deacetylase, Sir2 (ref. 1). We have recently shown that Sir2 and its closest human homologue SIRT1, a p53 deacetylase, are strongly inhibited by the vitamin B3 precursor nicotinamide. Here we show that increased expression of PNC1 (pyrazinamidase/nicotinamidase 1), which encodes an enzyme that deaminates nicotinamide, is both necessary and sufficient for lifespan extension by calorie restriction and low-intensity stress. We also identify PNC1 as a longevity gene that is responsive to all stimuli that extend lifespan. We provide evidence that nicotinamide depletion is sufficient to activate Sir2 and that this is the mechanism by which PNC1 regulates longevity. We conclude that yeast lifespan extension by calorie restriction is the consequence of an active cellular response to a low-intensity stress and speculate that nicotinamide might regulate critical cellular processes in higher organisms.

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels.

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.