HES family repressors are involved in a number of important developmental processes, including sex determination. Deadpan acts as a 'denominator' or negative regulatory element in the process of Drosophila sex determination in which the balance of X-encoded activators, or 'numerator' elements, and autosomally encoded denominator elements regulate the transcription of a master control gene called Sex lethal (Sxl). Sxl activity is required for female development and is expressed in all cells in females, whereas males do not require Sxl activity and do not express active Sxl protein. Loss of Dpn function results in inappropriate activation of Sxl expression, which in turn leads to aberrant dosage compensation and male specific lethality. Since Sir2 interacts physically with Dpn, it is expected that if Dpn requires Sir2 activity to function in sex determination, then lowering Sir2 gene dosage would result in fewer male progeny. Consistent with this hypothesis, a marked decrease is found from the expected number of adult loss-of-function (LOF) Sir2 male progeny and male Sir2 embryos stained for Sxl exhibit ectopic Sxl expression. If Sir2 is involved in assessing the balance of X:A factors in sex determination, it might be expected that overexpression of Sir2 would repress Sxl, resulting in female specific lethality. Indeed, it is found that when Sir2 is overexpressed, female embryos exhibit reduced Sxl expression (Rosenberg, 2002).
New members of the histone deacetylase enzyme family in Drosophila have been identified. dHDAC6 is a class II deacetylase with two active sites, and dSIR2 is an NAD-dependent histone deacetylase. These proteins, together with two class I histone deacetylases, dHDAC1 (Rpd3) and dHDAC3, have been expressed and characterized as epitope-tagged recombinant proteins in Schneider SL2 cells. All these proteins have in vitro deacetylase activity and are able to deacetylate core histone H4 at all four acetylatable lysine residues (5, 8, 12, and 16). Recombinant dHDAC6 and dSIR2 are both insensitive to TSA and HC toxin and resistant, relative to dHDAC1 and dHDAC3, to inhibition by sodium butyrate. Indirect immunofluorescence microscopy of stably transfected SL2 lines reveals that dHDAC1 and dSIR2 are nuclear, dHDAC6 is cytosolic, and dHDAC3 is detectable in both cytosol and nucleus. dHDAC6 and dSIR2 elute from Superose 6 columns with apparent molecular weights of 90 and 200 kDa, respectively. In contrast, dHDAC1 and dHDAC3 elute at 800 and 700 kDa, respectively, suggesting that they are components of multiprotein complexes. Consistent with this, recombinant dHDAC1 coimmunoprecipitates with components of the Drosophila NuRD complex and dHDAC3 with an as yet unknown 45-kDa protein (Barlow, 2001).
Caloric restriction extends lifespan in numerous species. In the budding yeast Saccharomyces cerevisiae this effect requires Sir2, a member of the sirtuin family of NAD1-dependent deacetylases. Sirtuin activating compounds (STACs) can promote the survival of human cells and extend the replicative lifespan of yeast. Resveratrol and other STACs activate sirtuins from Caenorhabditis elegans and Drosophila 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 (Wood, 2004).
Sir2-like proteins (sirtuins) are a family of NAD+-dependent deacetylases conserved from Escherichia coli to humans that play important roles in gene silencing, DNA repair, rDNA recombination and ageing in model organisms. When diet is restricted (caloric restriction), lifespan is extended in diverse species, suggesting that there is a conserved mechanism for nutrient regulation of ageing. In budding yeast, extra copies of SIR2 extend lifespan by 30%, apparently by mimicking caloric restriction. A group of compounds (STACs) have been described that stimulate the catalytic activity of yeast and human sirtuins, and extend the replicative lifespan of yeast cells by up to 60%. To establish whether STACs could activate sirtuins from multicellular animals, a cell-based deacetylation assay was developed for Drosophila S2 cells. Unlike other classes of deacetylases, the sirtuins are insensitive to the inhibitor trichostatin A (TSA). Several classes of polyphenolic STACs (including chalcones, flavones and stilbenes) increase the rate of TSA-insensitive deacetylation. To determine whether this activity is due to direct stimulation of a Sir2 homologue, recombinant SIR-2.1 of C. elegans and Sir2 of D. melanogaster were purified, and the effect of various STACs on enzymatic activity was determined in vitro. In a dose-dependent manner, resveratrol stimulated deacetylation up to 2.5-fold for SIR-2.1 and 2.4-fold for Sir2. As previously observed with the yeast and human Sir2 enzymes, resveratrol lowered the Km of SIR-2.1 for the co-substrate NAD+ (Wood, 2004).
Because resveratrol can significantly extend replicative lifespan in yeast, it was asked whether STACs could also extend lifespan in the metazoans C. elegans and D. melanogaster. Wild-type worms were transferred to plates containing 0 or 100mM of resveratrol shortly after reaching adulthood. Lifespan was extended up to 14%, using either heat-killed or live E. coli as food supply. To test whether the lifespan extension depends on functional SIR-2.1, a sir-2.1 null mutant was constructed. The lifespan of this strain was not appreciably shorter than the wild-type N2 control, and adults treated with resveratrol did not exhibit a significant lifespan extension relative to untreated worms. There was no decrease in fecundity associated with resveratrol treatment. To rule out the possibility that resveratrol was causing the animals to eat less, thereby inducing a caloric restriction effect indirectly, feeding rates of both L4 larval and adult worms with or without resveratrol were measured and no differences were found. Throughout the lifespan trials, the reproductive suppressant FUDR was used to prevent accumulation of progeny on the treatment plates (Wood, 2004).
Whether STACs could extend lifespan in Drosophila was tested using the standard laboratory wild-type strain Canton-S and normal fly culturing conditions (vials), and a yw marked wild-type strain and demographic culturing conditions (cages). Across independent tests in males and females on abundant diet, lifespan was extended up to 23% with fisetin and up to 29% with resveratrol. Increased longevity was associated with reduced mortality before day 40. A restricted diet increased lifespan by 40% in females and by 14% in males (averaged across trials), and under these conditions neither resveratrol nor fisetin further increased longevity. The lack of an additive effect of resveratrol and a low calorie diet suggests that resveratrol extends lifespan through a mechanism related to caloric restriction (Wood, 2004).
Surprisingly, while diet manipulations that extend D. melanogaster longevity typically reduce fecundity, longevity-extending doses of resveratrol modestly increased egg production, particularly in the earliest days of adult life. The increase in egg production suggests that the lifespan extending effect of resveratrol in D. melanogaster is not due to caloric restriction induced by food aversion or lack of appetite. Consistent with this, no decrease in food uptake was seen with resveratrol-fed flies. Furthermore, resveratrol-fed flies maintained normal weight, except during days 3-6 when resveratrol-fed females were laying significantly more eggs than control-fed females (Wood, 2004).
To determine whether resveratrol extends fly lifespan in a Sir2- dependent manner, a Sir2 allelic series with increasing amounts of Sir2 was analysed. Adult offspring from crosses between independently derived alleles of Sir2 were tested. Resveratrol failed to extend lifespan in flies completely lacking functional Sir2 or in flies in which Sir2 is severely decreased. Resveratrol increased longevity a small but statistically significant amount in flies homozygous for a hypomorphic Sir2 allele and increased lifespan up to 17% in flies with one copy of the hypomorphic allele and one copy of awild-type Sir2. These data suggest that the ability of resveratrol to extend fly lifespan requires functional Sir2 (Wood, 2004).
STACs have been reported to extend the replicative lifespan of yeast cells by mimicking caloric restriction. This study shows that STACs can extend lifespan in C. elegans and D. melanogaster, both of which are composed of primarily non-dividing (post-mitotic) cells as adults, and whose somatic and reproductive ageing are independent measures of senescence. In both species, resveratrol increases lifespan in a Sir2-dependent manner and, at least for the fly, this action appears to function through a pathway related to caloric restriction (Wood, 2004).
The observation that resveratrol can increase longevity without an apparent cost of reproduction is counter to prevalent concepts of senescence evolution. However, STACs may still entail trade-offs that involve other traits, or that occur only under some environmental conditions. Plants synthesize STACs such as resveratrol in response to stress and nutrient limitation, possibly to activate their own sirtuin pathways. These molecules may activate animal sirtuins because they serve as plant defence mechanisms against consumers or because they are ancestrally orthologous to endogenous activators within metazoans. Alternatively, animals may use plant stress molecules as a cue to prepare for a decline in their environment or food supply. Understanding the adaptive significance, endogenous function and evolutionary origin of sirtuin activators will lead to further insights into the underlying mechanisms of longevity regulation, and could aid in the development of interventions that provide the health benefits of caloric restriction (Wood, 2004).
To test whether Sir2 interacts with Hairy directly, GST pull-down assays were performed using in vitro translated (IVT-) Sir2. Consistent with the genetic results, IVT-Sir2 does not bind to Groucho or dCtBP or to GST alone. However, it does bind specifically to a full-length GST-Hairy fusion protein. To map the region of Hairy required for this interaction, a series of Hairy protein fragments fused in frame to GST were generated. Sir2 binds to all fragments containing the Hairy basic domain, indicating that this domain is sufficient for binding. A series of small basic domain deletions were generated within the context of full-length Hairy protein to identify the smallest region required for Sir2 binding. One of these deletions, DeltaRRAR, disrupts Sir2 binding, while adjacent four amino acid deletions have no effect. The Hairy DeltaRRAR mutation does not affect Hairy homodimerization or binding to other Hairy-interacting proteins, including dCtBP (Rosenberg, 2002).
The basic domain is highly conserved among HES family proteins, including the invariant RRAR residues, so Sir2 was assayed for binding to other bHLH proteins within this family by GST pull-down. IVT-Sir2 binds to GST-Deadpan (Dpn), but, surprisingly, not efficiently to GST-fusions to the E(Spl)m3 and E(Spl)m8 members of the HES family, suggesting that Sir2 may recognize additional features within the basic domain or in distal regions of HES proteins to permit interaction with a specific subset of these similar proteins in a variety of developmental processes (Rosenberg, 2002).
One possible consequence of cofactor binding to the basic domain of HES proteins could be interference with their DNA binding abilities. Since Sir2 is required for Hairy function but binds to the basic domain, a gel electrophoretic mobility shift assay (EMSA) was used to test whether Sir2 and Hairy could be detected in a stable complex on DNA. Both full-length-Hairy and a fragment containing the bHLH domain of Hairy are able to efficiently shift 32P-labeled N-box probe and are competed by cold wild-type competitor. No complex with altered mobility was detected upon addition of Sir2, although these proteins are able to interact in vitro. Addition of Sir2 to Hairy either before or after incubation of Hairy with DNA does not prevent Hairy from binding to target DNA, suggesting that Sir2 does not interfere with Hairy binding to DNA and that there may be other consequences of Sir2 binding to Hairy within this region (Rosenberg, 2002).
Control of chromosome structure is important in the regulation of gene expression, recombination, DNA repair, and chromosome stability. In a two-hybrid screen for proteins that interact with the Drosophila CREB-binding protein (dCBP), a known histone acetyltransferase and transcriptional coactivator, the Drosophila homolog of a yeast chromatin regulator, Sir2, was identified. In yeast, Sir2 silences genes via an intrinsic NAD+-dependent histone deacetylase activity. In addition, Sir2 promotes longevity in yeast and in Caenorhabditis elegans. In this report, the Drosophila Sir2 gene and its product were characterized and the generation of Sir2 amorphic alleles is described. It was found that Sir2 expression is developmentally regulated and that Sir2 has an intrinsic NAD+-dependent histone deacetylase activity. The Sir2 mutants are viable, fertile, and recessive suppressors of position-effect variegation (PEV), indicating that, as in yeast, Sir2 is not an essential function for viability and is a regulator of heterochromatin formation and/or function. However, mutations in Sir2 do not shorten life span as predicted from studies in yeast and worms (Newman, 2002).
To determine the size, level, and expression pattern of Sir2 transcript, the Sir2 RNAs were analyzed in Northern analyses and in situ hybridization of wild-type and Sir2 mutant embryos. Because many of the important developmental events in Drosophila occur within the first 24 hr of embryogenesis, embryos were collected from different stages within this period. In Northern analyses, the antisense riboprobe hybridized to an ~4-kb transcript in embryos throughout embryogenesis. The Sir2 transcript was first detected in 0-2 hr embryos, which indicates that these transcripts are maternally derived. Expression of Sir2 during cellular blastoderm and gastrulation decreases (2-4 hr) and then increases dramatically during germ-band elongation and morphogenesis (4-8 hr). The dSir25.26/dSir24.5 mutant embryo RNA does not contain Sir2 transcript in Northern blots or whole-mount embryos. The Sir2 deletions remove the 5' end of the Sir2 coding sequence and end ~300 bp before the core domain of dSir2 begins. Thus, it is conceivable that in the mutants a truncated product that contains the core domain is produced. To rule out this possibility, the Northern blot was hybridized with a probe that includes most of the core domain coding sequences (the core domain is from 1085 to 1885 and the C-terminal end of the probe ends at 1514); this probe could not detect an RNA species in the mutant animals (Newman, 2002).
To study the mutant phenotype of Sir2 mutations, mutations were generated on two unrelated chromosomes that deleted a large portion of Sir2 cDNA but left dDnaJ-H sequences intact. The dSir25.26/dSir24.5 trans-heterozygous animals do not express a Sir2 transcript or Sir2 protein at any time during development. The RNA probes and anti-Sir2 antibodies could have detected any truncated product but they did not. dDnaJ-H expression was assessed in whole-mount embryos to ensure that dDnaJ-H coding or regulatory sequences were not interrupted. Flies that were heterozygous for the two mutant chromosomes were used to characterize the Sir2 mutant phenotype so that the second-site lethals on the parental chromosomes would not affect the phenotype. It was found that, as in yeast, loss of Sir2 does not cause lethality. These data contradict a recent report stating that mutations in Sir2 are recessive lethals (Rosenberg, 2002). Both studies used the P{ry+t7.2=PZ} l(2)05327 cn1 chromosome, which was found to carry second-site lethal mutations. The data show that Sir2 mutations are viable and it is suggested that the lethal phenotypes described are due, at least in part, to these additional mutations (Newman, 2002).
In yeast and C. elegans, Sir2 affects life span. In yeast, Sir2 mutants reach senescence more quickly than wild-type cells; in C. elegans, duplication of the Sir2 gene causes a dramatic extension of life span. The data from Drosophila do not support these findings. When the effect of Sir2 mutants on life span was assessed, no significant difference between dSir25.26/dSir24.5 trans-heterozygotes and Sir2-/+ heterozygotes was detected. To sensitize the system and detect a more subtle effect of Sir2 on life span, it was asked whether Sir2 mutations affect life span under more stressful conditions. Surprisingly, the dSir25.26/dSir24.5 flies had a slightly longer median life span than the median life span of the Sir2 heterozygotes and Canton-S controls, although average life spans among the genotypes were not significantly different. The significance of the slight increase in the median life span of the Sir2 mutants is not clear. It may suggest that Sir2 is a negative regulator for stress-related metabolic processes or transcription that in turn may affect vitality. However, it is important to note that this effect does not increase the average life span of the Sir2 mutant flies. Thus, these data argue that the effect of Sir2 on life span is not conserved in flies. While other Sir2-like genes in Drosophila might affect life span, the Sir2 gene described here is most like the Sir2 from yeast and worms that has been shown to affect life span. On the basis of these data, it is likely that dSir2 functions as a transcription and chromatin-remodeling factor that regulates the expression of many genes and thus would not have a specific effect on life span. However, it may be that, in flies, Sir2 acts with other Sir2 family members to elicit an effect on life span. In this case, its effects might be detected only in animals mutant for the other Sir2 proteins (Newman, 2002).
In contrast, it was found that Sir2 mutations are recessive suppressors of PEV, consistent with the model that Sir2 is involved in heterochromatin regulation across phylogenetic lines. Furthermore, it was found that CBP mutations dominantly suppress PEV, suggesting that Sir2 and CBP may act together to control the pattern of heterochromatin histone acetylation. For example, CBP may be inactivated by acetylation and Sir2 may be required to deacetylate CBP for proper heterochromatin function and/or formation. Alternatively, because CBP is known to acetylate proteins other than histones, Drosophila CBP may facilitate heterochromatin formation through modification of other proteins in the complex. The fact that Sir2 mutations are recessive suppressors of PEV while CBP mutations are dominant modifiers of PEV suggests that CBP's role in maintaining heterochromatin formation or function is more dosage sensitive. It is possible that Sir2 helps to stabilize heterochromatin but is not absolutely required for its formation. Sir2 antibodies can co-immunoprecipitate CBP from Drosophila Kc cells, demonstrating that Sir2 and CBP interact in vivo. Dosage studies of Sir2 and CBP on PEV will clarify the nature of the Sir2-CBP interaction. It will also be important to determine whether the deacetylase activity of Sir2 and the acetyltransferase activity of CBP are important for their functions in heterochromatin formation and/or activity (Newman, 2002).
Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. The chromatin profiling method DamID was used to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. DamID profiling was adapted such that tightly gated collections of embryos (2-6 h) could be used, and 20 Hairy targets related to early embryogenesis were found. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. Fifty-nine putative transcriptional targets of Hairy were identified. In addition to finding putative targets for Hairy in segmentation, groups of targets were found suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development (Bianchi-Frias, 2004).
The 59 putative Hairy targets identified correspond to bands of Hairy immunostaining on polytene chromosomes, suggesting that the polytene chromosome staining faithfully represents Hairy binding. Polytene chromosomes are functionally similar in transcriptional activity and display factor/cofactor binding properties similar to chromatin of diploid interphase cells, despite their DNA endoreplication (Bianchi-Frias, 2004).
Since the microarray chips used contained roughly half of Drosophila cDNAs, the actual number of Hairy targets was estimaed to be approximately twice that number (i.e., 118 targets). This predicted number of Hairy targets is close to the approximately 120 strongly staining sites observed on polytene chromosomes. Of the 59 putative Hairy targets identified in both the Kc cell and embryo DamID experiments, 58 correspond to bands of Hairy staining on the polytene chromosomes, suggesting that polytene chromosome staining is representing Hairy binding sites without regard to tissue specificity. It is not yet clear what is limiting Hairy accessibility in different tissues or why Hairy's access does not appear to be limited in salivary glands. It may be that polytene chromosome organization necessitates a looser chromatin structure or that the large number of factors that seem to be endogenously expressed in salivary glands affects accessibility. Ultimately, additional confirmation of the DamID and polytene staining correspondence will require microarray tiling chips containing overlapping genomic DNA fragments; however, such genomic DNA tiling chips are currently unavailable (Bianchi-Frias, 2004).
DNA methylation by tethered Dam has been shown to spread up to a few kilobases from the point where it is brought to the DNA. It was of concern in the beginning that Hairy targets might be missed if the DNA fragments of 2.5 kb or less that were recovered for probes were far away from the start of the transcribed region, especially since the Drosophila microarray chip used was generated using full-length cDNAs. Indeed, Hairy has been described as a long-range repressor; it is likely to bind at a distance from the transcription start site. However, the targets identified by DamID in both Kc cells and in embryos correspond closely to the Hairy staining pattern on polytene chromosomes. As is the case for Hairy, the distribution of DamID-identified loci that recruit the long-range repression-mediating Groucho corepressor corresponds well with the distribution of Groucho binding sites on polytene chromosomes. These results suggest that there is a higher-order structure to the promoter that is allowing factors that bind far upstream of the transcription start site to have physical access to the transcribed region (i.e., DNA looping) or that Hairy does not bind as far away from the transcription start site as it had been proposed to do (Bianchi-Frias, 2004).
Hairy is needed at multiple times during development, where it has primarily been associated with the regulation of cell fate decisions. During embryonic segmentation, ftz has long been thought to be a direct Hairy target. However, the order of appearance of ftz stripes is not inversely correlated with those of Hairy, as would be expected if ftz stripes are generated by Hairy repression. While it was not possible to assess ftz as a direct Hairy target using DamID, no evidence was found for ftz being a direct Hairy target based on the association of Hairy with polytene chromosomes. Indeed, the evidence suggesting that ftz is a direct target of Hairy is based on timing, i.e., that there is not enough time for another factor to be involved. Since the half-life of the pair-rule gene products is very short (less than 5 min), it is possible that additional factors could be acting and that the interaction between Hairy and ftz is indirect (Bianchi-Frias, 2004).
Interestingly, one of the Hairy targets identified in embryos is the homeobox-containing transcriptional regulator, prd. Pair-rule genes have been split into two groups: primary pair-rule genes mediate the transition from nonperiodic to reiterated patterns via positional cues received directly from the gap genes, whereas secondary pair-rule genes take their patterning cues from the primary pair-rule genes and in turn regulate the segment polarity and homeotic gene expression. The transcriptional regulator prd was originally categorized as a secondary pair-rule gene since its expression is affected by mutations in all other known pair-rule genes. However, prd stripes were subsequently shown to require gap gene products for their establishment, and the prd locus has the modular promoter structure associated with primary pair-rule genes. Thus prd has properties of both primary and secondary pair-rule genes and is a good candidate to directly mediate Hairy's effects on segmentation. Hairy can specifically bind to C-box sequences in the prd promoter and interacts genetically with prd. Further experiments will be required to determine if Paired in turn binds to the ftz promoter, such that the order of regulation would be Hairy > prd > ftz (Bianchi-Frias, 2004).
In addition to identifying potential targets for Hairy in segmentation, targets were identified that implicate Hairy in other processes including cell cycle, cell growth, and morphogenesis. The group of targets implicating Hairy in the regulation of morphogenesis includes: concertina, a G-alpha protein involved in regulating cell shape changes during gastrulation; kayak, the Drosophila Fos homolog involved in morphogenetic processes such as follicle cell migration, dorsal closure, and wound healing; pointed and mae, both of which function in the ras signaling pathway to control aspects of epithelial morphogenesis; egh, a novel, putative secreted or transmembrane protein proposed to play a role in epithelial morphogenesis, and Mipp1, a phosphatase required for proper tracheal development (Bianchi-Frias, 2004).
Hairy has been thought to be involved mostly in the regulation of cell fate decisions. However, mosaic experiments in the eye imaginal disc have suggested that Hairy may also play a role in the regulation of cell cycle or cell growth. Consistent with this, another group of Hairy targets implicates Hairy in the regulation of cell cycle or cell growth; this group includes stg, the Drosophila Cdc25 homolog; dacapo, a cyclin-dependent kinase inhibitor related to mammalian p27kip1/p21waf1; IDGF2, a member of a newly identified family of growth-promoting glycoproteins, and ImpL2, a steroid-responsive gene of the secreted immunoglobulin superfamily that functions as a negative regulator of insulin signaling. Consistent with a role for Hairy in growth signaling, mammalian HES family proteins have been linked to insulin signaling (Bianchi-Frias, 2004).
Since cells that are dividing or proliferating cannot simultaneously undergo the cell shape changes and cell migrations required for morphogenetic movements, Hairy may be required to transiently pause the cell cycle in a spatially and temporally defined manner, thereby allowing the cell fate decisions regulated by the transcription cascade to be completed. Since Hairy is itself spatially and temporally expressed, Hairy must be only one of several genes necessary to orchestrate these processes. While much progress has been made in understanding the regulatory networks governing pattern formation, cell proliferation, and morphogenesis, and while it is clear that they must be integrated, the details surrounding their coordination have not yet been elucidated. Thus, the putative Hairy targets identified are consistent with known processes involving Hairy and suggest that in addition to regulating pattern formation, Hairy plays a role in transiently repressing other events, perhaps in order to coordinate cell cycle events with the segmentation cascade. Further experiments will be needed to determine how these different roles for Hairy fit together (Bianchi-Frias, 2004).
The numbers of loci that recruit Groucho, dCtBP, and dSir2 cofactors are consistent with the breadth of interaction they have been shown to exhibit. One hundred and fifth-five loci were identified that recruit Groucho and, as expected, roughly twice as many sites were found on polytene chromosomes. Although Groucho was the first Hairy cofactor identified and its interaction site is often described as Hairy's 'major' repression motif, Groucho is associated with only a minority of Hairy targets in Kc cells. Groucho's dominance as a cofactor during segmentation may reflect a preference for Groucho in the reporter assays used previously to assess corepressor activity, or it may be more heavily recruited to Hairy's targets during segmentation. In the future it will be interesting to determine the loci that recruit Groucho in early embryos and, because Groucho binds a number of other repressors, which, if any, of these factors recruits Groucho as its major cofactor (Bianchi-Frias, 2004).
CtBP was identified as a repressive co-factor, first on the basis of its binding to the C-terminal region of E1A, and in Drosophila by its association with the developmental repressors Hairy and Knirps. CtBP is an integral component in a variety of multiprotein transcriptional complexes. It has been shown to function as a context-dependent cofactor, having both positive and negative effects on transcriptional repression depending upon the repressor to which it is recruited. More than 40 different repressors have been shown to recruit CtBP. Consistent with this wide recruitment of CtBP, 496 loci that recruit dCtBP were found by DamID profiling and roughly twice that many sites on polytene chromosomes. A global protein-protein interaction study has shown that the binding partners for Groucho and dCtBP are largely nonoverlapping. This, along with the near exclusivity of Groucho and dCtBP binding as assayed by DamID and polytene chromosome staining, makes it unlikely that both cofactors work together as a general rule and strengthens the possibility that the binding of each of these factors assembles different protein complexes that are, for the most part, mutually exclusive (Bianchi-Frias, 2004).
dSir2 was only very recently identified as a corepressor for Hairy and other HES family members. 107 loci were identified by DamID profiling that recruit dSir2 and roughly twice that many sites on polytene chromosomes. Surprisingly, the distribution of loci recruiting dSir2 identified by DamID profiling, as well as dSir2′s staining on polytene chromosomes, shows regional binding specificity. This binding specificity may be a reflection of the different nuclear compartments in which these regions of the chromosomes are found. Sir2 has been described mostly as a protein involved in heterochromatic silencing rather than in euchromatic repression. The number of dSir2 euchromatic sites observed is similar to that of Groucho, suggesting that euchromatic repressors (in addition to HES family members) are likely to recruit Sir2. Consistent with this, a recent report has described a role for mammalian Sir2 in repressing the muscle cell differentiation program. The region-specific binding of dSir2 might reflect a difference in the types of factors it can associate with, or the association of dSir2 with particular chromosomal regions or nuclear domains (Bianchi-Frias, 2004).
Interestingly, dCtBP and dSir2 recruitment are largely overlapping, and this association continues outside of those loci where Hairy binds: 90% of dSir2-recruiting loci also recruit dCtBP. dCtBP and dSir2 are unique among transcriptional coregulators in that they both encode NAD+-dependent enzymatic activities. As NAD and NADH levels within the cell exist in closely regulated equilibrium, it is possible that dCtBP and dSir2 function as NAD/NADH redox sensors. In this way, the cell could use coenzyme metabolites to coordinate the transcriptional activity of differentiation-specific genes with the cellular redox state (Bianchi-Frias, 2004).
A polyclonal mouse antibody was generated against a GST fusion to full-length Sir2 that recognizes a single band of ~120 kDa in whole-cell, nuclear, and Kc cell extracts by Western blot, and efficiently detects purified recombinant 6×His epitope-tagged Sir2. This antibody does not recognize other Drosophila sirtuins in these lysates or its yeast homologs even in >5-fold more extract. Wild-type embryos stained with this antibody exhibit dynamic subcellular Sir2 localization in the early embryo. Prior to nuclear cycle 12, Sir2 is detected both in nuclei and in the surrounding cytoplasm. By the syncytial blastoderm stage (nuclear cycle 13), Sir2 is still cytoplasmic but is excluded from nuclei. Since cellularization begins at nuclear cycle 14, Sir2 is present both in nuclei and in the cytoplasm (Rosenberg, 2002).
Since Drosophila Sir2 genetically affects PEV, Sir2 localization on salivary gland polytene chromosomes was examined. By double labeling with antibodies against Sir2 and Histone H3-dimethyl lysine 9 (to label heterochromatin), strong Sir2 localization was observed to discrete euchromatic bands along chromosome arms, as well as at lower levels to centric heterochromatin (Rosenberg, 2002).
Sir2 protein is detected throughout development and, unlike some of the mammalian Sir2 proteins, is primarily nuclear. However, at certain times during development (cellular blastoderm), Sir2 is excluded from the nucleus; at other times, it is both cytoplasmic and nuclear (late in embryogenesis). Sir2 is present during syncitial and cellular blastoderm at high levels in the nuclei surrounding the morphogenic furrows during gastrulation and in the germ band. Later, its expression is primarily detected in the central nervous system. In addition, Sir2 localizes both to heterochromatin and to discrete bands within euchromatic regions of polytene chromosomes. These data suggest that Sir2 is regulated not only in a temporal- and tissue-specific fashion but also at the level of subcellular localization (Newman, 2002).
The Drosophila Sir2 locus is located on the second chromosome at cytological position 34A and encodes a single transcript with one intron. Northern analysis of Sir2 shows that the locus encodes a single mRNA of 3.8 kb expressed at different levels throughout development with a strong maternal component. EP(2)2300 is an EP element insertion ~400 bp upstream of the Sir2 transcription start in the correct orientation to drive overexpression of Sir2. Sir205327, is the result of a PZ element insertion within the Sir2 mRNA at position +14. Sir2ex10 is an excision of Sir205327 that disrupts Sir2 start site (Rosenberg, 2002).
ySIR2 is essential for maintenance of silencing at heterochromatic loci including telomeres (Rine, 1987; Aparicio, 1991); mating-type loci (Rine, 1987), and rDNA arrays (Rine, 1987; Gottlieb, 1989; Cockell, 2000). To determine if Drosophila Sir2 plays a role in heterochromatic silencing (a phenomenon also called position effect variegation [PEV]), the effects of loss of Sir2 function on heterochromatic, centromeric, and telomeric PEV were examined. Drosophila lines containing the white (w+) gene inserted within repeat arrays were used to test heterochromatic silencing. In Sir2/+ heterozygotes, increased expression of w+ is observed. Similarly, in wm4, a line in which w+ is relocated to proximal heterochromatin, there is clear suppression of w+ silencing in a Sir2 heterozygous background, supporting a further role for Sir2 in centromeric heterochromatic silencing. The ability of Sir2 to mediate telomeric silencing was tested using flies that had the w+ gene inserted near telomeres. Silencing of w+ insertions at three telomeres was tested: 2L, 3R, and 4. Silencing at telomere 4 is clearly suppressed in a Sir2 heterozygous background, while silencing of w+ near the telomere on chromosomes 3R and 2L is not affected by reduction of Sir2. Since telomere 4, in contrast to telomeres 2L or 3R, is affected by heterochromatic silencing machinery, these results suggest that Sir2 is involved in heterochromatic, but not telomeric, silencing (Rosenberg, 2002).
Five Drosophila genes belong to the highly conserved sir2 family, which encodes NAD+-dependent protein deacetylases. Of these five, dsir2+ (CG5216) is most similar to the Saccharomyces cerevisiae SIR2 gene, which has profound effects on chromatin structure and life span. Four independent Drosophila strains were found with P-element insertions near the dsir2 transcriptional start site as well as extraneous linked recessive lethal mutations. Imprecise excision of one of these P elements (PlacW 07223) from a chromosome freed of extraneous lethal mutations produced dsir217, a null intragenic deletion allele that generates no Sir2 protein. Homozygosity for dsir217 has no apparent deleterious effects on viability, developmental rate, or sex ratio, and it fully complements sir2ex10. Moreover, through a genetic test, the reported effect of dSir2ex10 on Sex-lethal expression was ruled out. A modest, strictly recessive suppression of whitem4 position-effect variegation and a shortening of life span was attained in dsir2 homozygous mutants, suggesting that dsir2 has some functions in common with yeast SIR2 (Åström, 2003).
Calorie restriction can extend life span in a variety of species including mammals, flies, nematodes, and yeast. Despite the importance of this nearly universal effect, little is understood about the molecular mechanisms that mediate the life-span-extending effect of calorie restriction in metazoans. Sir2 is known to be involved in life span determination and calorie restriction in yeast mother cells. In nematodes increased Sir2 can extend life span, but a direct link to calorie restriction has not been demonstrated. Sir2 is directly involved in the calorie-restriction life-span-extending pathway in Drosophila. An increase in Drosophila Sir2 (dSir2) extends life span, whereas a decrease in dSir2 blocks the life-span-extending effect of calorie reduction or rpd3 mutations. These data led to the proposal of a genetic pathway by which calorie restriction extends life span and provides a framework for genetic and pharmacological studies of life span extension in metazoans (Rogina, 2004).
The Rpd3/Sir2 histone deacetylases have been implicated in both life span determination and calorie restriction in yeast. Rpd3 and Sir2 can effect the activity of a variety of genes and physiological systems by deacetylating histones and other proteins such as p53. A decrease in Rpd3 or an increase in Sir2 extends mother cell life span in yeast, and the effect of Sir2 on yeast life span is linked to calorie restriction. A similar mechanism may operate in metazoans, because an increase in Sir2 extends life span in nematodes, and a decrease in Rpd3 extends life span in flies (Rogina, 2002). The increase in life span associated with decreased Rpd3 in flies is thought to occur through a mechanism related to calorie restriction (Rogina, 2002), The finding of an increase in Drosophila Sir2 (dSir2) transcription in both long-lived rpd3 mutant flies and long-lived calorie-restricted normal flies implicates dSir2 as a potential member of the calorie-restriction life-span-extending pathway (Rogina, 2002). Further evidence of a role for Sir2 in the determination of life span is the finding that the Sir2 agonist resveratrol extends life span in yeast, nematodes, and flies in a Sir2- and calorie-restriction-dependent manner. These data suggest that Sir2 may be one of the primary elements of the calorie-restriction-induced life span extension in flies and other metazoans (Rogina, 2004).
To test whether dSir2 is involved in longevity determination in the fly, the life span was examined of flies in which the level of dSir2 had been increased by using molecular genetic techniques. Flies were constructed that ubiquitously overexpressed dSir2 by combining, in individual flies, the Drosophila tubulin promoter fused to the gene for the yeast GAL4 activator protein (tubulin-GAL4 driver) with a native dSir2 gene that has a P element with GAL4-binding sites (EP-UAS) inserted just upstream. Flies carrying the tubulin-GAL4 driver and each of the different EP-UAS-dSir2 genes, dSir2EP2300, dSir2EP2384, or dSir2EYO3602, had a >4-fold increase in dSir2 mRNA expression over the endogenous level. Consistent with the hypothesis that an increase in dSir2 in flies will increase life span, up to a 57% increase in average life span was seen in the tubulin-GAL4/dSir2EP2300, tubulin-GAL4/dSir22384, and tubulin-GAL4/dSir2EYO3602 flies, with an increase across all lines of 29% for females and 18% for males (Rogina, 2004).
To determine whether a threshold level of dSir2 expression is required for life span extension in the fly, flies were examined in which the armadillo-GAL4 driver was combined with the dSir2EP2300 chromosome. The armadillo-GAL4 driver is a weaker driver than the tubulin-GAL4 driver: compared with control flies, armadillo-GAL4/dSir2EP2300 flies showed only a 10%-20% increase in dSir2 mRNA levels and no life span extension, suggesting that a significant increase in dSir2 mRNA is required to cause an extension in life span (Rogina, 2004).
Knowing that ubiquitous overexpression of dSir2 increases life span, it was of interest to determine which tissues normally express dSir2 in adults and whether an effect in a single tissue could mediate the life span extension caused by dSir2 overexpression. Using anti-dSir2 antibodies, it was found that, similar to embryos and larvae, in adults dSir2 protein is found at high levels in the nuclei of neurons and in the nuclei and cytoplasm of fat body cells. The finding of a prominent expression of dSir2 in the nervous system of normal animals led to an examination of whether an increase in dSir2 in neurons may be one of the primary mediators of the Sir2-related life span extension. Neuronal dSir2 overexpression in flies carrying the pan-neuronal promoter ELAV-GAL4 driver:dSir2EP2300 extended the average life span by 52% in females and 20% in males (Rogina, 2004).
ELAV-GAL4 drives expression in embryos and larvae as well as adults. A different system for overexpressing dSir2 was used to test whether increased expression of dSir2 only in adult neurons might lead to life span extension. The RU-486 Gene-Switch system allows for the comparison of genetically identical animals from the same cohort, one group receiving RU-486, which induces expression of the EP-UAS gene, and the other group receiving only diluent. In two independent trials the maximum life span of adult dSir2EP2300/ELAV-GeneSwitch flies receiving RU-486 was increased by 9% and 16%, respectively, in females and was decreased by 1% and increased by 10%, respectively, in males. In females, respective 5% and 12% increases in median life span were also seen. The smaller increase in life span in flies with the ELAV-GeneSwitch driver, relative to those with the ELAV driver, is consistent with a lower level of dSir2 induction with the ELAV-GeneSwitch driver at the dose of RU-486 used (200 µM). In addition, preliminary studies suggest that RU-486 itself may have some mild deleterious effects on life span in flies, especially on males. The greater life span extension obtained by using the standard ELAV driver could be due partly to increased dSir2 activity before eclosion in adult ELAV-GAL4/dSir2EP2300 flies. Regardless, the results demonstrate that pan-neuronal overexpression of dSir2 during only the adult stage is sufficient to produce a modest extension of maximal life span (Rogina, 2004).
To further explore the possibility that a subset of neurons may be important in dSir2-mediated life span extension, the life span of flies containing the D42-GAL4 motoneuron-specific driver and dSir2EP2300 was examined. No life span extension was seen in the D42/dSir2EP2300 flies (Rogina, 2004).
Decreases in physical activity, reproductive status (especially in females), or calorie intake are known to increase life span in the fly. Although quantitative studies were not performed, visual inspection suggested no obvious decrease in physical activity or fertility in the long-lived dSir2-overexpressing flies compared with their matched controls. Furthermore, the fact that life span was found to be significantly increased in both males and females suggests that a potential decrease in female reproduction is unlikely to be the primary cause of the observed life span extension (Rogina, 2004).
Considered together, the results of experiments driving dSir2 demonstrate that overexpression of dSir2 correlates well with increased life span in flies. In four different driver-GAL4/UAS-dSir2 lines in which dSir2 was substantially overexpressed either ubiquitously or in neurons, the life span of flies was extended significantly. However, when the driver caused only a small ubiquitous increase in dSir2 or an increase only in motor neurons, life span was not extended. Furthermore, an intermediate increase in dSir2 in the adult nervous system caused by the ELAV-GeneSwitch driver caused an intermediate increase in life span (Rogina, 2004).
Flies given low-calorie food, in addition to showing an increase in life span, showed an increase in dSir2 mRNA expression (Rogina, 2002), To determine whether dSir2 is directly in the calorie-restriction life-span-extending pathway in flies, the life span of flies that had reduced or no dSir2 expression on a diet of low-calorie food was compared with that of genetically identical flies from the same cohort on a diet of normal or high-calorie food. Flies with either no dSir2 gene function (e.g., dSir24.5/dSir25.26) or with severely decreased dSir2 gene function (e.g., dSir2KG00871/dSir2KG00871) showed no life span extension on a diet of low-calorie food relative to genetically identical flies on a diet of normal or high-calorie food. The inability of dSir2 mutant flies to increase their life span in response to a low-calorie diet demonstrates that a sufficient level of dSir2 must be available for the activation of life span extension by calorie reduction and, furthermore, that dSir2 is an important element in the calorie-reduction life-span-extending pathway (Rogina, 2004).
If dSir2 mediates the effect of calorie-restriction-induced life span extension, it would also be expected that calorie restriction would not further increase life span in flies in which dSir2 activity is already elevated. The life span of two of the long-lived dSir2-overexpressing lines, tubulin-GAL4/dSir2EP2300 and ELAV-GAL4/dSir2EP2300, was examined under normal and low-calorie food conditions. Under the husbandry conditions used, a decrease in calorie content in the food typically increases the life span of normal flies by 35%-40% or more (Rogina, 2002), However, when the long-lived ELAV-GAL4/dSir2EP2300 and tubulin-GAL4/dSir2EP2300 flies were placed on a diet of low-calorie food, no further increase in life span was seen with the ELAV-GAL4/dSir2EP2300 flies, whereas the tubulin-GAL4/dSir2EP2300 flies showed a reduction in life span toward normal. Control flies for ELAV-GAL4/dSir2EP2300 placed on a diet of low-calorie food demonstrated a 28% and 22% increase in median life span for males and females, respectively. The lack of a cumulative effect of calorie reduction and dSir2 overexpression on life span suggests that life span extensions are mediated through similar or related pathways (Rogina, 2004).
The data presented in this study on dSir2, along with previous work on life span and calorie reduction of rpd3 mutants (Rogina, 2002), indicate that the life-span-extending effects of nutrient reduction in the fly are mediated through dSir2 and Rpd3. The observation of an increase in dSir2 mRNA in long-lived rpd3 mutants (Rogina, 2002) led to the postulate that the effect of Rpd3 may depend partially on an increase in dSir2. Together these data suggest a model for how calorie reduction extends life span in the fly. In this model, the stimulus of calorie reduction triggers a decrease in Rpd3 activity and a subsequent increase in dSir2 activity. The increase in dSir2, either alone or in conjunction with additional changes initiated by the decrease in Rpd3 activity, results in life span extension (Rogina, 2004).
Further confirmation that dSir2 and Rpd3 are in the same life-span-extending pathway was obtained by examining the life span of flies carrying a long-lived rpd3 mutation (rpd3def24) and a dSir2 mutation (dSir217 or dSir2EP2300). dSir2 mutations do not reduce life span in otherwise normal flies (Newman, 2002). Therefore, if dSir2 were not in the same pathway as Rpd3, the life span would not be altered from the extended life span of rpd3 mutant flies. As predicted by the model, flies with both a dSir2 mutation and rpd3def24 mutation were not long-lived, whereas their counterparts, flies mutant for only rpd3def24, remained long-lived. dSir2 and Rpd3, therefore, seem to be in the calorie-reduction life-span-extending pathway in flies. The finding that long-lived rpd3 mutations increase dSir2 levels (Rogina, 2002) suggests that dSir2 is downstream of Rpd3 (Rogina, 2004).
The search for elements that extend life span in metazoans has identified the involvement of the insulin-signaling, nutrient-sensing, and Sir2 pathways. Although the Sir2 pathway has been linked to calorie availability in yeast, it has not been shown to function in the calorie restriction pathway in metazoans. The data presented in this study demonstrate a direct link between the life-span-extending effects of dSir2 and calorie restriction in the fly. Five different GAL-4 drivers (tubulin, ELAV, armadillo, ELAV-Gene-Switch, and D42-motoneuron) were used to drive expression of endogenous dSir2 genes with three separate nearby insertions of UAS elements. In four strains in which dSir2 expression was increased substantially, either ubiquitously or in neuronal cells, the life span of the flies was extended substantially, up to 57% when dSir2 mRNA expression was increased 4-fold. Conversely, in two other similarly constructed strains in which dSir2 expression was not elevated or was only marginally elevated, life span was not altered relative to that of control flies. Thus, in six fly strains constructed by using different combinations of drivers and dSir2 responders, increased longevity correlated very well with elevation of dSir2. Furthermore, life span cannot be extended by calorie restriction in flies that lack dSir2 activity, nor can life span be further increased by calorie restriction in flies in which dSir2 activity is already raised. The recent findings that a Sir2 agonist, resveratrol (shown to increase the activity of yeast, nematode, fly, and human Sir2) extends life span in yeast, nematodes, and flies in a manner that is Sir2-dependent and associated with calorie restriction provide additional evidence for a primary role of Sir2 activity in determining life span in metazoans. Together, these observations make a strong case that calorie restriction extends life span in flies by increasing dSir2 activity (Rogina, 2004).
The data presented here, in conjunction with previous work on Rpd3 (Rogina, 2002), show that dSir2 and Rpd3 are important components in the calorie-restriction life-span-extending pathway of flies. A decrease in dSir2 prevents the life-span-extending effect of calorie restriction, and the life-span-extending effect of calorie restriction is not cumulative with the life-span-extending effect of increased dSir2. Similarly, the life-span-extending effect of Rpd3 mutations is not cumulative with the effect of calorie restriction (Rogina, 2002). Long-lived flies with reduced Rpd3 activity have elevated dSir2 mRNA (Rogina, 2002). This study shows that, in flies with decreases in both Rpd3 and dSir2 activity, life span is not extended, indicating that an increase in dSir2 activity in response to a decrease in Rpd3 activity is necessary for life span extension. Together these data suggest that dSir2 is downstream of Rpd3 in the calorie-restriction life-span-extending pathway in flies. This model provides a useful framework and testable model for examining the relationship of Sir2, calorie reduction, and longevity by using genetic, molecular, and pharmaceutical approaches. The documentation of a molecular genetic pathway responsible for effecting calorie-restriction-related life span extension will be useful for identifying biochemical mediators and drug interventions that can mimic calorie restriction. Given the conservation of elements of the calorie restriction/Rpd3/Sir2 pathway in extending life span in yeast and now flies, agents that stimulate the activity of Sir2 are potential tools for extending life span in metazoans (Rogina, 2004).
Afshar, G. and Murnane, J. P. (1999). Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2. Gene 234(1): 161-8. 10393250
Anderson, R. M., et al. (2002). Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J. Biol. Chem. 277(21): 18881-90. 11884393
Aparicio, O. M., Billington, B. L. and Gottschling, D. E. (1991). Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell 66: 1279-1287. 11950950
Åström, S. U., Cline, T. W. and Rine, J. (2003). The Drosophila melanogaster sir2+ gene is nonessential and has only minor effects on position-effect variegation. Genetics 163: 931-937. 12663533
Barlow, A. L., van Drunen, C. M., Johnson, C. A., Tweedie, S., Bird, A. and Turner, B.M. (2001). dSIR2 and dHDAC6: two novel, inhibitor-resistant deacetylases in Drosophila melanogaster. Exp. Cell Res. 265(1): 90-103. 11281647
Bedalov, A., Gatbonton, T., Irvine, W. P., Gottschling, D. E. and Simon, J. A. (2001). Chemical genetics of SIR2: continuous deacetylase activity is required for silencing. Proc. Natl. Acad. Sci. USA, 98:15113-15118. 11935028
Bernstein, B. E., Tong, J. K. and Schreiber, S. L. (2000). Genomewide studies of histone deacetylase function in yeast. Proc. Natl. Acad. Sci. 97: 13708-13713. 11095743
Bianchi-Frias, D., et al. (2004). Hairy transcriptional repression targets and cofactor recruitment in Drosophila. PLoS Biol. 2(7): E178. 15252443
Borra, M. T., O'Neill, F. J., Jackson, M. D., Marshall, B., Verdin, E., Foltz, K. R. and Denu, J. M. (2002). Conserved enzymatic production and biological effect of O-Acetyl-ADP-ribose by Silent information regulator 2-like NAD+-dependent deacetylases. J. Biol. Chem. 277: 12632-12641. 11812793
Brachmann, C. B., et al. (1995). The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 9: 2888-2902. 11714726
Cockell, M. M., Perrod, S. and Gasser, S. M. (2000) Analysis of sir2p domains required for rDNA and telomeric silencing in saccharomyces cerevisiae Genetics, 154:1069-1083. 10393187
Fulco, M., et al. (2003). Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state. Molec. Cell 12: 51-62. 12887892
Gottlieb, S. and Esposito, R. E. (1989). A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA. Cell 56: 771-776. 2225075
Gottschling, D. E. (2000). Gene silencing: two faces of SIR2. Curr. Biol. 10: R708-R711. 11483616
Guarente L. (2000). Sir2 links chromatin silencing, metabolism, and aging. Genes Dev. 14:1021-1026. 10809662
Hoppe, G. J., Tanny, J. C., Rudner, A. D., Gerber, S. A., Danaie, S., Gygi, S. P. and Moazed, D. (2002). Steps in assembly of silent chromatin in yeast: Sir3-independent binding of a Sir2/Sir4 complex to silencers and role for Sir2-dependent deacetylation. Mol. Cell. Biol. 22: 4167-4180. 12024030
Imai, S., Armstrong, C. M., Kaeberlein, M. and Guarente, L. (2000). Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403:795-800. 10521401
Landry, J., et al. (2000). The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases. Proc. Natl. Acad. Sci. 97:5807-5811. 10811920
Langley, E., Pearson, M., Faretta, M., Bauer, U.-M., Frye, R. A., Minucci, S., Pelicci, P. G. and Kouzarides, T. (2002). Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. EMBO J. 21: 2383-2396. 12006491
Lieb, J. D., Liu, X., Botstein, D. and Brown, P. O. (2001). Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat. Genet. 28: 327-334. 11455386
Luo, J., et al. (2001). Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 107:137-148. 12080091
Moretti, P., Freeman, K., Coodly, L. and Shore D. (1994). Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 8: 2257-2269. 11689698
Newman, B. L., et al. (2002). A Drosophila homologue of Sir2 modifies position-effect variegation but does not affect life span. Genetics 162: 1675-1685. 12524341
Rine J. and Herskowitz I. (1987). Four genes responsible for a position effect on expression from HML and HMR in S. cerevisiae Genetics 116: 9-22. 3297920
Rogina, B. Helfand, S. L. and Frankel, S. (2002). Longevity regulation by Drosophila Rpd3 deacetylase and caloric restriction, Science 298: 1745. 12459580
Rogina, B. and Helfand, S. L. (2004). Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc. Natl. Acad. Sci. 101: 15998-16003. 15520384
Rosenberg, M. I. and Parkhurst, S. M. (2002). Drosophila Sir2 is required for heterochromatic silencing and by euchromatic Hairy/E(Spl) bHLH repressors in segmentation and sex determination. Cell 109: 447-458. 12086602
Rutter, J., Reick, M., Wu, L. C. and McKnight, S. L. (2001). Regulation of Clock and NPAS2 DNA binding by the redox state of NAD cofactors. Science 293: 510-514. 11441146
Tanny, J. C., Dowdk G. J., Huangk J., Hilzk H. and Moazedk D. (1999). An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing. Cell 99: 735-745. 1061942
Tissenbaum, H. A. and Guarente, L. (2001). Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410(6825): 227-30. 11242085
van Steensel, B. and Henikoff, S. (2000). Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat. Biotechnol. 18:424-428. 10748524
Vaziri, H., et al. (2001). hSIR2 SIRT1 functions as an NAD-dependent p53 deacetylase. Cell 107: 149-159. 15254550
date revised: 15 January 2006
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