Gene name - Sirtuin 1
Synonyms - Sir2
Cytological map position - 34A7
Keywords - chromatin modification, gene silencing, master metabolic sensor
Symbol - Sirtuin 1
FlyBase ID: FBgn0024291
Genetic map position -
Classification - NAD-dependent histone deacetylase
Cellular location - cytoplasmic and nuclear
|Recent literature||Slade, J. D. and Staveley, B. E. (2016).. Extended longevity and survivorship during amino-acid starvation in a Drosophila Sir2 mutant heterozygote. Genome [Epub ahead of print] PubMed ID: 27074822
The regulation of energy homeostasis is pivotal to survive periods of inadequate nutrition. The sirtuin deacetylase Sir2 is well conserved from single-celled yeast to mammals, and it controls a number of downstream targets that are active during periods of extreme stress. Overexpression of Sir2 has been established to enhance survival of a number of model organisms undergoing calorie restriction, during which insulin receptor signalling (IRS) is reduced, a condition that itself can enhance survivorship during starvation. Increased Sir2 expression and reduced IRS result in an increase in the activity of the transcription factor foxo, an advantageous activation during stress but lethal when overly active. This study found that a lowered gene dosage of Sir2, in mutant heterozygotes, can extend normal longevity and greatly augment survivorship during amino-acid starvation in Drosophila. Additionally, these mutants, in either heterozygous or homozygous form, do not appear to have any disadvantageous effects upon development or cell growth of the organism unlike IRS mutants. These results may advance the understanding of the biological response to starvation and allow for the development of a model organism to mimic the ability of individuals to tolerate nutrient deprivation.
|Engel, G. L., Marella, S., Kaun, K. R., Wu, J., Adhikari, P., Kong, E. C. and Wolf, F. W. (2016). Sir2/Sirt1 links acute inebriation to presynaptic changes and the development of alcohol tolerance, preference, and reward. J Neurosci 36: 5241-5251. PubMed ID: 27170122
Acute ethanol inebriation causes neuroadaptive changes in behavior that favor increased intake. Ethanol-induced alterations in gene expression, through epigenetic and other means, are likely to change cellular and neural circuit function. Ethanol markedly changes histone acetylation, and the sirtuin Sir2/SIRT1 that deacetylates histones and transcription factors is essential for the rewarding effects of long-term drug use. This study found that Sir2 in the mushroom bodies of the fruit fly Drosophila promotes short-term ethanol-induced behavioral plasticity by allowing changes in the expression of presynaptic molecules. Acute inebriation strongly reduces Sir2 levels and increases histone H3 acetylation in the brain. Flies lacking Sir2 globally, in the adult nervous system, or specifically in the mushroom body α/β-lobes show reduced ethanol sensitivity and tolerance. Sir2-dependent ethanol reward is also localized to the mushroom bodies, and Sir2 mutants prefer ethanol even without a priming ethanol pre-exposure. Transcriptomic analysis reveals that specific presynaptic molecules, including the synaptic vesicle pool regulator Synapsin, depend on Sir2 to be regulated by ethanol. Synapsin is required for ethanol sensitivity and tolerance. It is proposed that the regulation of Sir2/SIRT1 by acute inebriation forms part of a transcriptional program in mushroom body neurons to alter presynaptic properties and neural responses to favor the development of ethanol tolerance, preference, and reward.
|Horvath, M., Mihajlovic, Z., Slaninova, V., Perez Gomez, R., Moshkin, Y. and Krejci, A. (2016). The silent information regulator 1 (Sirt1) is a positive regulator of the Notch pathway in Drosophila. Biochem J [Epub ahead of print]. PubMed ID: 27623778
The silent information regulator 1 (Sirt1) has previously been shown to have negative effects on the Notch pathway in several contexts. This study brings evidence that Sirt1 has a positive effect on Notch activation in Drosophila, in the context of sensory organ precursor specification and during wing development. The phenotype of Sirt1 mutant resembles weak Notch loss of function phenotypes and genetic interactions of Sirt1 with the components of the Notch pathway also suggest a positive role of Sirt1 in Notch signalling. Sirt1 is necessary for the efficient activation of E(spl) genes by Notch in S2N cells. Additionally, the Notch dependent response of several E(spl) genes is sensitive to metabolic stress caused by 2-deoxyglucose treatment, in a Sirt1 dependent manner. Sirt1 associates with several proteins involved in Notch repression as well as activation, including the cofactor exchange factor Ebi (TBL1), the RLAF/LAF histone chaperon complex and the Tip60 acetylation complex. Moreover, Sirt1 participates in the deacetylation of the CSL transcription factor Su(H). The role of Sirt1 in Notch signalling is therefore more complex than previously recognised and its diverse effects may be explained by a plethora of Sirt1 substrates involved in the regulation of Notch signalling.
|Wood, J.G., Jones, B.C., Jiang, N., Chang, C.,
Hosier, S., Wickremesinghe, P., Garcia, M., Hartnett, D.A., Burhenn, L.,
Neretti, N. and Helfand, S.L. (2016). Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend life span in Drosophila. Proc
Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27621458
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, this study 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, represses the age-related increased expression of genes located in heterochromatin, as well as TEs. A corresponding age-associated increase in TE transposition in fly fat body cells was also observed that was delayed by dietary restriction. Furthermore, 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, mitigate age-related increases in expression of TEs. Increasing expression of either Su(var)3-9 or Dicer-2 also leads to an increase in life span. Mutation of Dicer-2 leads to an increase in DNA double-strand breaks. Treatment with the reverse transcriptase inhibitor 3TC results 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.
|Banerjee, K. K., Deshpande, R. S., Koppula, P., Ayyub, C. and Kolthur-Seetharam, U. (2017). Central metabolic-sensing remotely controls nutrient -sensitive endocrine response in Drosophila via Sir2/Sirt1-upd2-IIS axis. J Exp Biol [Epub ahead of print]. PubMed ID: 28104798
Endocrine signaling is central in coupling organismal nutrient status with maintenance of systemic metabolic homeostasis. While local nutrient sensing within the insulinogenic tissue is well-studied, distant mechanisms that relay organismal nutrient status in controlling metabolic-endocrine signaling are less understood. This study reports a novel mechanism underlying the distant regulation of metabolic endocrine response in Drosophila melanogaster. The communication between fat-body and insulin producing cells (IPCs), important for the secretion of dILPs, is regulated by the master metabolic sensor Sir2/Sirt1. This communication involves a fat body-specific direct regulation of the JAK/STAT cytokine upd2, by Sir2/Sirt1. This study also uncovered the importance of this regulation in coupling nutrient-inputs with dILP-secretion, and distantly controlling intestinal insulin signaling. These results provide fundamental mechanistic insights into the top-down control involving tissues that play key roles in metabolic sensing, endocrine signaling and nutrient uptake.
|Wen, D. T., Zheng, L., Yang, F., Li, H. Z. and Hou, W. Q. (2018). Endurance exercise prevents high-fat-diet induced heart and mobility premature aging and dsir2 expression decline in aging Drosophila. Oncotarget 9(7): 7298-7311. PubMed ID: 29484111
High-Fat-Diet (HFD)-induced obesity is a major contributor to heart and mobility premature aging and mortality in both Drosophila and humans. The dSir2 genes are closely related to aging, but there are few directed reports showing that whether HFD could inhibit the expression dSir2 genes. Endurance exercise can prevent fat accumulation and reverse HFD-induced cardiac dysfunction. Endurance also delays age-relate functional decline. It is unclear whether lifetime endurance exercise can combat lifetime HFD-induced heart and mobility premature aging, and relieve the harmful HFD-induced influence on the dSir2 gene and lifespan yet. In this study, flies are fed a HFD and trained from when they are 1 week old until they are 5 weeks old. Then, triacylglycerol levels, climbing index, cardiac function, lifespan, and dSir2 mRNA expressions are measured. Endurance exercise was shown to improve climbing capacity, cardiac contraction, and dSir2 expression, and it reduces body and heart triacylglycerol levels, heart fibrillation, and mortality in both HFD and aging flies. So, lifelong endurance exercise delays HFD-induced accelerated age-related locomotor impairment, cardiac dysfunction, death, and dSir2 expression decline, and prevents HFD-induced premature aging in Drosophila.
|Wen, D. T., Zheng, L., Li, J. X., Lu, K. and Hou, W. Q. (2019). The activation of cardiac dSir2-related pathways mediates physical exercise resistance to heart aging in old Drosophila. Aging (Albany NY) 11. PubMed ID: 31503544
Cardiac aging is notably characterized by increased diastolic dysfunction, lipid accumulation, oxidative stress, and contractility debility. The Sir2/Sirt1 gene overexpression delays cell aging and reduces obesity and oxidative stress. Exercise improves heart function and delays heart aging. However, it remains unclear whether exercise delaying heart aging is related to cardiac Sir2/Sirt1-related pathways. In this study, cardiac dSir2 overexpression or knockdown was regulated using the UAS/hand-Gal4 system in Drosophila. Flies underwent exercise interventions from 4 weeks to 5 weeks old. Results showed that either cardiac dSir2 overexpression or exercise remarkably increased the cardiac period, systolic interval, diastolic interval, fractional shortening, SOD activity, dSIR2 protein, Foxo, dSir2, Nmnat, and bmm expression levels in the aging flies; they also notably reduced the cardiac triacylglycerol level, malonaldehyde level, and the diastolic dysfunction index. Either cardiac dSir2 knockdown or aging had almost opposite effects on the heart as those of cardiac dSir2 overexpression. Therefore, this study claims that cardiac dSir2 overexpression or knockdown delayed or promoted heart aging by reducing or increasing age-related oxidative stress, lipid accumulation, diastolic dysfunction, and contractility debility. The activation of cardiac dSir2/Foxo/SOD and dSir2/Foxo/Bmm pathways may be two important molecular mechanisms through which exercise works against heart aging in Drosophila.
|Wen, D. T., Zheng, L., Li, J. X., Cheng, D., Liu, Y., Lu, K. and Hou, W. Q. (2019). Endurance exercise resistance to lipotoxic cardiomyopathy is associated with cardiac NAD(+)/dSIR2/PGC-1alpha pathway activation in old Drosophila. Biol Open 8(10). PubMed ID: 31624074
Lipotoxic cardiomyopathy is caused by excessive lipid accumulation in myocardial cells and it is a form of cardiac dysfunction. Cardiac PGC-1alpha overexpression prevents lipotoxic cardiomyopathy induced by a high-fat diet (HFD). The level of NAD(+) and Sir2 expression upregulate the transcriptional activity of PGC-1alpha. Exercise improves cardiac NAD(+) level and PGC-1alpha activity. However, the relationship between exercise, NAD(+)/dSIR2/PGC-1alpha pathway and lipotoxic cardiomyopathy remains unknown. In this study, flies were fed a HFD and exercised. The heart dSir2 gene was specifically expressed or knocked down by UAS/hand-Gal4 system. The results showed that either a HFD or dSir2 knockdown remarkably increased cardiac TG level and dFAS expression, reduced heart fractional shortening and diastolic diameter, increased arrhythmia index, and decreased heart NAD(+) level, dSIR2 protein, dSir2 and PGC-1alpha expression levels. Contrarily, either exercise or dSir2 overexpression remarkably reduced heart TG level, dFAS expression and arrhythmia index, and notably increased heart fractional shortening, diastolic diameter, NAD(+) level, dSIR2 level, and heart dSir2 and PGC-1alpha expression. Therefore, exercise training could improve lipotoxic cardiomyopathy induced by a HFD or cardiac dSir2 knockdown in old Drosophila. The NAD(+)/dSIR2/PGC-1alpha pathway activation was an important molecular mechanism of exercise resistance against lipotoxic cardiomyopathy.
Yeast SIR2 (Silent Information Regulator 2) is a nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylase required for heterochromatic silencing at telomeres, rDNA, and mating-type loci. The Drosophila Sir2 also encodes deacetylase activity and is required for heterochromatic silencing, but unlike ySir2, is not required for silencing at telomeres. Drosophila Sir2 interacts genetically and physically with members of the Hairy/Deadpan/E(Spl) family of bHLH euchromatic repressors, key regulators of Drosophila development. Drosophila Sir2 is an essential gene whose loss of function results in both segmentation defects and skewed sex ratios, associated with reduced activities of the Hairy and Deadpan bHLH repressors. These results indicate that Sir2 in higher organisms plays an essential role in both euchromatic repression and heterochromatic silencing (Rosenberg, 2002).
Histone deacetylases (HDACs) act as cofactors that are recruited to promoters by sequence-specific DNA binding factors resulting in the local modification of histones to promote chromatin compaction with subsequent inhibition of gene transcription. HDACs have been divided into classes based on their similarity to known yeast factors: class I HDACs are similar to yRPD3, while class II HDACs are related to yHDA1. Class III HDACs, exemplified by ySIR2, have NAD+-dependent HDAC activity and are not sensitive to inhibitors of class I HDACs, such as trichostatin A (TSA; Bernstein, 2000). SIR2 also has ADP-ribosylase activity (Tanny, 1999). While SIR2's HDAC activity is essential for silencing in yeast, its ADP-ribosylase activity is not essential for silencing (Imai, 2000), and no biological function has yet been assigned to this activity. ySIR2 acts as a dedicated silencing protein that deacetylates histones at heterochromatic targets, including the mating-type loci, telomeres, and rDNA repeats (reviewed in Gottschling, 2000; Guarente, 2000). ySIR2 plays an important role in aging, but is not an essential gene. There are four other SIR2-like proteins (or sirtuins) in yeast, however, none can compensate for all of the functions of ySIR2 and the yeast quintuple mutant is viable (Brachmann, 1995). Like other HDACs, ySIR2 is recruited to DNA by DNA binding factors (Rosenberg, 2002 and references therein).
Cofactor recruitment by DNA bound factors is an important feature of transcriptional repression mechanisms used to establish complex patterns of gene expression during development. A number of developmentally regulated repressors are transcription factors that recruit HDACs as cofactors to bring about repression. In Drosophila, the sequence-specific DNA binding repressor Hairy has been studied extensively in this context. hairy is a member of the pair-rule class of genes that is essential for the proper establishment of segmentation in the developing embryo. hairy encodes a bHLH transcription factor that belongs to the Hairy/Enhancer of Split/Deadpan (or HES) family of proteins. Drosophila HES family proteins are key repressors in the developmental processes of segmentation, neurogenesis, and sex determination. All members of this repressor family possess (1) a highly conserved bHLH domain, required for protein dimerization and DNA binding; (2) an adjacent Orange domain, which confers specificity among family members, and (3) a C-terminal tetrapeptide motif, WRPW, which has been shown to be necessary and sufficient for the recruitment of the corepressor, Groucho. Groucho has in turn been proposed to recruit the class I HDAC, Rpd3, suggesting a mechanism by which HES repressors use Groucho and Rpd3 to create a chromatin environment that is repressive of transcription. In some assays, however, Hairy can function in the absence of its WRPW motif, indicating that in the absence of Groucho and presumably Rpd3, Hairy can still repress transcription. This repression may be achieved through other mechanisms, such as the recruitment of other cofactors (Rosenberg, 2002).
A Drosophila homolog of the yeast histone deacetylase SIR2 was identified by sequence homology (CG5216). There are five sirtuins in Drosophila, with Sir2 (CG5216) sharing the highest degree of sequence similarity to ySir2. Drosophila Sir2 is an essential gene that is dynamically expressed throughout development. Sir2 is required for position effect variegation, suggesting a role for Sir2 similar to that of its yeast counterpart in maintaining heterochromatic silencing. Sir2 also has a strong maternal component such that progeny from mothers with reduced Sir2 exhibit segmentation defects. A direct physical and genetic interaction is observed between Sir2 and Hairy, suggesting this as a basis for the segmentation defects. In addition, a direct physical interaction has been detected between Sir2 and Deadpan (Dpn), but not with other HES family proteins. Consistent with this and with a role for Sir2 in Dpn repression, progeny with altered gene dosage of Sir2 exhibit skewed sex ratios. These results indicate that Sir2 interacts directly with members of the HES bHLH class of euchromatic transcriptional repressors in mediating processes essential for the early development of the embryo (Rosenberg, 2002).
In addition to its conserved role in heterochromatic silencing, examination of loss-of-function mutants reveals a significant role for Sir2 in Drosophila embryogenesis. Sir2 is essential for zygotic function since progeny that are homozygous for Sir205327 die as embryos. Loss of zygotic Sir2 function does not affect early embryo patterning since the dead homozygous mutant embryos exhibit cuticle phenotypes indistinguishable from wild-type. Sir2 also has a strong maternal effect. A Sir2 mutation was initially identified in a genetic screen for maternal genes essential for embryonic development. In this screen, a change-of-function mutation (called wimp) in the second largest subunit of RNA polymerase II was used to reduce, but not eliminate, maternal Sir2 contribution. Embryos derived from mothers trans-heterozygous for wimp and the Sir2 allele die and exhibit anterior-posterior patterning defects, including loss of segments and pairwise denticle band fusions, as compared to wild-type or wimp/+, demonstrating a role for maternally contributed Sir2 in the establishment of body pattern (Rosenberg, 2002).
To identify the earliest stage at which segmentation is affected, the expression of genes at different tiers of the segmentation gene hierarchy were examined. Protein expression patterns of the gap genes Krüppel and knirps are unaffected in progeny from females with reduced maternal Sir2 (Sir205327/+;wimp/+ or Sir2ex10/+;wimp/+ transheterozygous mothers). Sir2 is first required for regulation of segmentation at the level of pair rule gene expression. Pair rule genes can be separated into two classes: primary pair rule genes establish double segment periodicity, whereas secondary pair rule genes respond to this pattern. Pair rule gene products are expressed as a series of seven transverse stripes in wild-type or wimp/+ embryos. Stripes of the secondary pair rule gene fushi tarazu (ftz) are severely derepressed (stripes are broadened) in embryos from mothers with reduced Sir2 expression. Aberrant regulation of Ftz stripe expression in Sir2 mutant embryos is consistent with reduced function of the primary pair rule gene, Hairy, which behaves genetically as a repressor of ftz. Hairy expression was examined in Sir2 mutant embryos: in contrast to Ftz expression, which is significantly altered, Hairy is largely unaffected in these embryos (Rosenberg, 2002).
The Ftz derepression phenotype in Sir2 embryos is reminiscent of the Ftz expression pattern seen in hairy mutants. Sir2 was examined for genetic interaction with hairy; these mutations exhibit a dominant genetic interaction. Progeny from either hairy heterozygous mothers or Sir2 heterozygous mothers mated to wild-type males are viable and exhibit wild-type cuticle phenotypes. In contrast, embryos derived from mothers heterozygous for both Sir2 and hairy (Sir2/+; hairy/+ trans-heterozygous mothers) mated to wild-type males exhibit moderate to severe cuticle abnormalities. Consistent with this segmentation cuticle phenotype, Ftz is derepressed in these embryos, with a reduction in expression of stripes 4, 6, and 7, suggesting that these segmentation defects are largely mediated by interaction of Sir2 with Hairy. Interestingly, Hairy stripes 3 and 4 are also affected in progeny from mothers trans-heterozygous for Sir2 and hairy (Sir2/+; hairy/+ females), suggesting interactions between Sir2 and other developmental regulators. Sir2 was tested for interaction with repression cofactors groucho (gro) and dCtBP, as well as the other primary pair rule genes, even skipped (eve), and runt (run). No dominant synthetic lethal interactions were detected between Sir2 and any of these mutations. Hairy was tested for genetic interaction with the class I HDAC, Rpd3, which has been proposed to be recruited to Hairy via the corepressor Groucho. However, no genetic interaction was detected between Rpd3 and hairy (Rosenberg, 2002).
In yeast, SIR2 is required for silencing at heterochromatic loci (including mating-type loci, rDNA arrays, and telomeres; Rine, 1987), as well as for silencing of an auxotrophic marker inserted within heterochromatin (Gottschling, 1990). Using reporter lines carrying w+ insertions, it has been found that Sir2 affects heterochromatic silencing of pericentric markers and markers inserted within repeated DNA arrays (Rosenberg, 2002).
In contrast, Sir2 does not appear to be involved in telomeric position effect. Reduction of Sir2 function suppresses position effect variegation (PEV) at telomere 4, but not at telomeres 2L or 3R. Studies by Cryderman (1999) have shown that subtelomeric and pericentric hsp 70-w+ transposon insertions are suppressed by different mutations, indicating regulation of heterochromatic and telomeric PEV by distinct sets of proteins. This study also showed that telomere 4 is unique among telomere insertions in that it responds to suppressors of heterochromatic silencing, but not to suppressors of telomeric silencing (Cryderman, 1999). That Sir2 is required for silencing of telomere 4 and not other telomeres tested suggests that Sir2 has a role in specific types of heterochromatic silencing which are distinct from telomeric silencing, although a role for Sir2 at telomeres cannot be ruled out. Since Drosophila has four additional sirtuins which all bear the same conserved catalytic core region, the roles of ySIR2 that are not shared by Sir2 may be regulated by these other sirtuins (Rosenberg, 2002).
While ySir2 has generally been described as a dedicated heterochromatic silencing protein, Drosophila Sir2 can also interact with specific euchromatic transcription factors. Sir2 interacts with the euchromatic bHLH repressor Hairy, both genetically and physically. This interaction requires Hairy's basic domain that is highly similar among members of the HES family. However, despite their extensive conservation, Sir2 binds to only a subset of Hairy/E(Spl) family members. Since the four amino acids necessary for mediating Sir2 binding are invariant within this family, there must be additional recognition features within the basic domain or elsewhere within the proteins. Previous reports have shown the basic domain to be an essential domain for DNA binding and dimerization among bHLH proteins. Sir2 binding to this region represents a novel domain for Hairy cofactor binding (Rosenberg, 2002).
The basic domains of bHLH proteins have been shown to undergo a disordered-to-ordered transition upon binding to DNA, making cofactor binding in this region an interesting paradox. Since no supershift has been detected upon addition of Sir2 protein and Sir2 does not appear to affect the ability of Hairy to bind DNA, Sir2 and Hairy may not be in a stable complex with DNA. The simplest explanation for these observations is that the interaction between Sir2 and Hairy in the presence of DNA is weak, requiring other proteins to stabilize the complex in vitro. However, there are several other ways in which Sir2 could affect Hairy function. Upon binding to Hairy, Sir2 may alter chromatin structure, affect distally bound factors on Hairy, or alter Hairy's ability to recruit cofactors required for other Hairy functions. In addition, Sir2 may deacetylate Hairy, altering either its DNA binding or activity, similar to altered p53 activity following deacetylation by human SIRT1 (Vaziri, 2001; Luo, 2001). This would not have been detected by gel shift assays, since bacterially expressed Hairy is not acetylated. Alternatively, other repressors, such as Polycomb Group complexes, can initiate silencing by transient recruitment of repression cofactors during brief interactions of distinct repressor complexes. The ability of Hairy to recruit distinct histone deacetylase containing complexes may represent a mechanism through which Hairy can both initiate and maintain a repressed state of chromatin through distinct and transiently interacting complexes. Such a complex containing Sir2 and DNA-bound Hairy would not have to be very stable, since a short-lived interaction may be sufficient (Rosenberg, 2002).
It is interesting that Hairy has been linked to two distinct histone deacetylases. Hairy and other HES family members recruit Groucho, which in turn has been proposed to recruit the class I HDAC, Rpd3. While Rpd3 mutant embryos exhibit segmentation defects, they involve only minor disruption of the Eve and Engrailed segmentation gene products, leading to the conclusion that Rpd3 is involved in segmentation but cannot represent a major pathway of repression in the early embryo. No dominant interaction has been detected between hairy and Rpd3; however, Sir2 is thought to be required for the processes of segmentation and sex determination in which Groucho is also required by bHLH factors. In contrast to the Rpd3 loss of function phenotypes, the segmentation defects observed in Sir2 loss-of-function embryos are severe (Rosenberg, 2002).
It is proposed that Hairy uses different deacetylases in different contexts. Phenotypic analysis of different hairy mutants suggests that the requirements for Sir2 and Groucho are overlapping but not redundant. Hairy has never been found to activate transcription, in contrast to other factors, such as the bHLHZip protein Myc, which has been shown in different contexts to either activate or repress transcription. Perhaps the requirement for Sir2 in processes that likely involve a separate HDAC complex represents a mechanism of repression by HES proteins that enable them to be dedicated repressors. Alternatively, the ability of Hairy to recruit two distinct histone deacetylases may allow it to independently regulate distinct processes (Rosenberg, 2002).
In light of the requirement for Sir2 throughout embryogenesis, the dynamic subcellular changes in Sir2 expression are intriguing. Dynamic localization of HDACs has been shown to be important for the activity of other bHLH factors, such as myocyte enhancer factor-2 (MEF-2). The class II mammalian HDAC5, which interacts with MEF2, must be removed from the nucleus to permit myocyte differentiation. Phosphorylation of HDAC5 alters its subcellular localization, allowing its export from the nucleus and subsequent progression of myoblast differentiation. Since the early Drosophila embryo is a closed system, it is possible that some developmental programs in the early embryo require removal of Sir2 from the nucleus. It is worth noting that at the times at which Sir2 plays a role in developmental processes (nuclear cycle 9-10 for sex determination and nuclear cycle 14 for segmentation), Sir2 is detectable in the nucleus, while at times in between (nuclear cycle 13), Sir2 is excluded from the nucleus. Future studies that characterize Sir2 localization and its developmental regulation will be informative about the requirements for Sir2 for diverse processes in the early embryo (Rosenberg, 2002).
The results show that Sir2 plays an important role in Drosophila euchromatic gene regulation through its interaction with bHLH repressors. Consistent with this, Sir2 localizes to both distinct euchromatic sites and generally to the centric heterochromatin on polytene chromosomes. van Steensel (2000) has also identified multiple euchromatic targets of Sir2 recruitment using an in vitro chromatin assay. ySIR2 has not been thought to interact with euchromatic repressor complexes. However, a euchromatic role for Sir2 may in fact exist in yeast. Lieb (2001) reported that the ySIR2-interactor RAP1 binds to 5% of yeast genes including intergenic regions that may correspond to promoters. Together, these results suggest that the ability of Sir2 to act as a euchromatic repressor represents a widespread and conserved function for Sir2. The finding that Sir2 is required for PEV also highlights the notion that mechanisms of repression are shared in part by heterochromatin and euchromatin (Rosenberg, 2002).
It is interesting to consider that the role of Sir2 in development may involve additional functions ascribed to the yeast enzyme. ySir2 has been shown by its gene dosage-dependent effects on lifespan (Kaeberlein, 1999) and by its NAD+-dependence (Landry 2000) to be linked to metabolism, perhaps by monitoring redox states in the cell. Sir2 may act during development to coordinate the progression of developmental programs by sensing the metabolic needs and outputs of the embryo and modifying key regulators of development to act accordingly. This may be an important aspect of Drosophila development where much of the early developmental program is executed in a closed system, consisting of maternally contributed factors in the absence of de novo transcription. bHLH factors that are key regulators of circadian rhythms were shown to have altered DNA binding affinity and heterodimerization preferences in response to changing cellular ratios of NAD+:NADH (Rutter, 2001). The bHLH domain mediates responsiveness of these factors to NAD+, which itself can respond both by altered heterodimerization and by altered DNA binding affinity (Rosenberg, 2002).
The finding that Sir2 is required for heterochromatic gene silencing and euchromatic repression represents a common link between the mechanisms of repression utilized by heterochromatin and euchromatin. Future studies on the precise molecular nature of Sir2 activity will likely uncover exciting new roles for it in both euchromatic and heterochromatic silencing.
Sir2 is an evolutionarily conserved NAD+ dependent protein. Although, SIRT1 has been implicated to be a key regulator of fat and glucose metabolism in mammals, the role of Sir2 in regulating organismal physiology, in invertebrates, is unclear. Drosophila has been used to study evolutionarily conserved nutrient sensing mechanisms, however, the molecular and metabolic pathways downstream to Sir2 (dSir2) are poorly understood. This study has knocked down endogenous dSir2 in a tissue specific manner using gene-switch gal4 drivers. Knockdown of dSir2 in the adult fatbody leads to deregulated fat metabolism involving altered expression of key metabolic genes. The results highlight the role of dSir2 in mobilizing fat reserves and demonstrate that its functions in the adult fatbody are crucial for starvation survival. Further, dSir2 knockdown in the fatbody affects dilp5 (insulin-like-peptide) expression, and mediates systemic effects of insulin signaling. This report delineates the functions of dSir2 in the fatbody and muscles with systemic consequences on fat metabolism and insulin signaling. In conclusion, these findings highlight the central role that fatbody dSir2 plays in linking metabolism to organismal physiology and its importance for survival (Banerjee, 2012).
This study reports that dSir2 is a critical factor that regulates metabolic homeostasis and mediates organismal physiology. Using genetic tools (inducible RNAi) that negate background effects, concrete results are provided that highlight the importance of endogenous dSir2 in the whole body, and in metabolically relevant tissues, such as fatbody and muscle. The findings point out the importance of nutrient signaling in eliciting dSir2-dependent molecular changes, which play an important role in tissue specific metabolic functions that affect systemic outputs in flies. By describing a metabolic phenotype in flies that lack dSir2, this study reiterates that Drosophila can be used to study sirtuin biology, but also highlight the evolutionary conservation of dSir2/SIRT1 functions in regulating organismal physiology (Banerjee, 2012)
Until now, the conservation of molecular mechanisms underlying Sir2 biology was poorly addressed in invertebrates. It is only in mammals that a functional interplay between metabolic flux, SIRT1 and its downstream molecular factors has been addressed, thus far (Longo, 2006; Canto, 2009; Finkel, 2009). Results from backcrossed dSir2 mutant and whole body dSir2 knockdown flies indicated that absence or down-regulation of dSir2 expression results in gross metabolic defects. Interestingly, it was observed that the effects on glucose levels were different in these two cases. The differences in glucose levels might reflect the systemic alterations in response to a complete absence of the protein in the case of mutants and down-regulation of expression in the case of knockdowns. It is interesting to note that studies in Sirt1+/-, liver specific Sirt1 knockout and knockdown micehave also yielded seemingly conflicting results. Specifically, with respect to glucose metabolism, these differences indicate that the manifestation of functions of Sir2/SIRT1 might be dependent upon the extent to which its expression is altered. Importantly, this underpins the need to further investigate the molecular interactions that bring about such varied phenotypes, in both mammals and flies (Banerjee, 2012).
It is important to note that consistent phenotypic, metabolic and molecular readouts were obtained with respect to fat metabolism in dSir2-mutant and -RNAi flies. A decrease (or absence) of dSir2 expression was found to result in increased fat storage in the fatbodies, as determined by oil red staining and biochemical analyses. This fat accumulation is due to altered expression of genes involved in fat metabolism. Importantly, it was shown that genes involved in fat breakdown are downregulated in the dSir2 knockdown flies, in addition to an upregulation of genes involved in fat synthesis. These findings are not only in accordance with the results obtained from dSir2 mutant larvae but also implicate dSir2 as a key player in fat metabolism in adult flies (Banerjee, 2012).
A role for dSir2 was uncovered in regulating systemic insulin signaling in flies. To investigate if the ability of dSir2 to mediate insulin signaling emanates from a specific tissue, dilp5 expression was assayed in fatbody and muscle specific dSir2RNAi flies. Interestingly, it was found that knocking down dSir2 only in the fatbody, but not muscles led to increased dilp5 expression, and mimicked dSir2 mutants and whole body dSir2RNAi flies. Specifically, this study addressed the role of dSir2 in the fatbody to mediate systemic effects on insulin signaling. Further investigations should help understand the dSir2-dependent molecular and physiological links between the fatbody and medial secretory neurons (MSNs). Very recently, hepatic SIRT1 was shown to mediate peripheral insulin signaling in mice. Importantly, the current findings underpin the importance of dSir2/SIRT1 in the homologous metabolic tissues, fatbody and liver, on systemic insulin signaling (Banerjee, 2012).
Efforts to link the molecular functions of dSir2 and organismal physiology led to the implication of dSir2 in starvation survival. dSir2 mutants and whole body dSir2RNAi flies succumb to starvation earlier than the controls and interestingly, are phenocopied by fatbody dSir2RNAi flies. Moreover it was shown that this is due to an inability to mobilize fat reserves from the fatbody, and a resultant of decreased expression of lipid breakdown genes, both under fed and starved conditions. The importance of dSir2 in the fatbody and fat mobilization is corroborated by an absence of deregulated fat metabolism in muscle specific dSir2RNAi flies. Further, a lack of starvation phenotype when dSir2 is knocked down from the muscles highlights the physiological relevance of fatbody (Banerjee, 2012).
In summary, this study has elucidated the significance of the functions of dSir2 in the fatbody in mediating central and peripheral effects on metabolic homeostasis and insulin signaling. Therefore, it is concluded that dSir2 is a key component that links dietary inputs with organismal physiology and survival. Most importantly, this study highlights the functions of dSir2 in the fatbody as a deterministic factor in governing fly physiology. This study delineates the functions of dSir2 in two metabolic tissues in affecting organismal survival. Metabolic homeostasis and the ability to utilize stored energy reserves are also crucial for mediating the effects of calorie restriction. These results, which emphasize the importance of dSir2 in maintaining homeostasis, reiterates its role in calorie restriction. Finally, this report highlights the need to further investigate the functions of dSir2, and should motivate future studies to understanding Sir2's interactions with other pathways and importance during aging (Banerjee, 2012).
An alignment of the Sir2 proteins from yeast, Drosophila, and human reveals remarkable conservation within the catalytic core of the enzyme, including the regions that encode the histone deacetylase and ADP-ribosylase functions of the yeast protein (Tanny, 1999; Imai, 2000; Landry, 2000). To test Sir2 for histone deacetylase activity, the ability of recombinant Sir2 to deacetylate labeled histone peptides in vitro was examined (Bedalov, 2001). Sir2 exhibits NAD+-dependent histone deacetylase activity. A small molecule inhibitor of ySir2, Splitomicin, has recently been shown to fully inhibit ySIR2 activity in vivo and recombinant ySIR2 by roughly 20%. Splitomicin also inhibits recombinant Drosophila Sir2 activity, to levels comparable to that of recombinant ySIR2 (20% inhibition; Bedalov, 2001). Together, these data suggest that Sir2 shares considerable functional homology with its yeast and human counterparts within this region (Rosenberg, 2002).
The predicted amino acid sequence of Sir2 is 823 aa in length, making it the longest known Sir2 homolog. The core domain, which encodes the deacetylase activity, is the only region of Sir2 that is conserved with ySir2. Bacterially expressed Sir2 has an intrinsic NAD+-dependent deacetylase activity. Outside the core domain, there is no homology to any known proteins, making it difficult to speculate on the function of these domains. Within the Drosophila genome, five genes contain significant homology to the yeast Sir2. Of the five genes, Sir2 is the most similar to yeast Sir2. This is an important fact when comparing mutant phenotypes across phylogenetic lines (Newman, 2002).
date revised: 7 July 2002
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