Sir2


REGULATION

Targets of Activity

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).

Sir2 acts through Hepatocyte Nuclear Factor 4 to maintain insulin signaling and metabolic homeostasis in Drosophila

SIRT1 is a member of the sirtuin family of NAD+-dependent deacetylases, which couple cellular metabolism to systemic physiology. This study shows that loss of the Drosophila SIRT1 homolog sir2 leads to the age-progressive onset of hyperglycemia, obesity, glucose intolerance, and insulin resistance. Tissue-specific functional studies show that Sir2 is both necessary and sufficient in the fat body to maintain glucose homeostasis and peripheral insulin sensitivity. This study reveals a major overlap with genes regulated by the nuclear receptor Hepatocyte Nuclear Factor 4 (HNF4). Drosophila HNF4 mutants display diabetic phenotypes similar to those of sir2 mutants, and protein levels for dHNF4 are reduced in sir2 mutant animals. Sir2 exerts these effects by deacetylating and stabilizing dHNF4 through protein interactions. Increasing dHNF4 expression in sir2 mutants is sufficient to rescue their insulin signaling defects, defining this nuclear receptor as an important downstream effector of Sir2 signaling. This study provides a genetic model for functional studies of phenotypes related to type 2 diabetes and establishes HNF4 as a critical downstream target by which Sir2 maintains metabolic health (Palu, 2016).

This study shows that sir2 mutants display a range of metabolic defects that parallel those seen in mouse Sirt1 mutants, including hyperglycemia, lipid accumulation, insulin resistance, and glucose intolerance. These results suggest that the fundamental metabolic functions of Sirt1 have been conserved through evolution and that further studies in Drosophila can be used to provide insights into its mammalian counterpart. An additional parallel with Sirt1 is seen in tissue-specific studies, where sir2 function is shown to be necessary and sufficient in the fat body to maintain insulin signaling and suppress hyperglycemia and obesity, analogous to the role of Sirt1 in the liver and white adipose. These results are also consistent with published studies of insulin sensitivity in Drosophila, which have shown that the fat body is the critical tissue that maintains glucose and lipid homeostasis through its ability to respond properly to insulin signaling (Palu, 2016).

These studies also define the dHNF4 nuclear receptor as a major target for Sir2 regulation. Consistent with this, dHNF4 mutants display a range of phenotypes that resemble those of sir2 mutants, including hyperglycemia, obesity, and glucose intolerance. As expected, these defects are more severe in dHNF4 loss-of-function mutants, consistent with sir2 mutants only resulting in a partial loss of dHNF4 protein. Sir2 interacts with dHNF4 and appears to stabilize this protein through deacetylation. This is an established mechanism for regulating protein stability, either through changes in target protein conformation that allow ubiquitin ligases to bind prior to proteasomal degradation, or through alternate pathways. Further studies, however, are required to determine if this is a direct protein-protein interaction or part of a higher order complex (Palu, 2016).

Although two papers have shown that mammalian Sirt1 can control HNF4A transcriptional activity through a protein complex, only one gene was identified as a downstream target of this regulation, PEPCK, leaving it unclear if this activity is of functional significance. The current study suggests that this regulatory connection is far more extensive. The observation that one third of the genes down-regulated in sir2 mutants are also down-regulated in dHNF4 mutants (including pepck), and most of the genes up-regulated in sir2 mutants are up-regulated in dHNF4 mutants, establishes this nuclear receptor as a major downstream target for Sir2 regulation. It will be interesting to determine if the extent of this regulatory connection has been conserved through evolution (Palu, 2016).

Despite this regulatory control, the over-expression of an HNF4 transgene was only able to partially restore the insulin signaling response and not the defects in carbohydrate homeostasis in sir2 mutants. This lack of complete rescue is not surprising, given that the Sirt1 family targets a large number of transcription factors, histones, and enzymes, providing multiple additional pathways for metabolic regulation. Moreover, the activity or target recognition of dHNF4 may be altered when it is hyperacetylated, in which case merely over-expressing this factor would not fully restore normal function. Future studies can examine more direct targets, both previously characterized and uncharacterized, for their functions in suppressing diabetes downstream of Sir2-dependent regulation (Palu, 2016).

Finally, sir2 mutants represent a new genetic model for studying the age-dependent onset of phenotypes related to type 2 diabetes. Newly-eclosed sir2 mutant adults are relatively healthy, with elevated levels of free glucose and glycogen but otherwise normal metabolic functions. Their health, however, progressively worsens with age, with two-week-old sir2 mutants displaying lipid accumulation, fasting hyperglycemia, and reduced insulin signaling accompanied by insulin resistance. This is followed by the onset of glucose intolerance by three weeks of age. Previous studies of type 2 diabetes in Drosophila have relied on dietary models using wild-type animals that are subjected to a high sugar diet. Although this is a valuable approach to better define the critical role of diet in diabetes onset, it is also clear that the likelihood of developing type 2 diabetes increases with age. The discovery that sir2 mutants display this pathophysiology provides an opportunity to exploit the power of Drosophila genetics to better define the mechanisms that lead to the stepwise onset of metabolic dysfunction associated with diabetes (Palu, 2016).

Characterization of Sir2 enzymatic activity

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).

Sirtuin activators mimic caloric restriction and delay ageing in metazoans

Caloric restriction extends lifespan in numerous species (see Drosophila as a Model for Human Diseases: Aging and Lifespan). 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).

Protein Interactions

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).

Hairy transcriptional repression targets and cofactor recruitment in Drosophila

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).

Drosophila poly(ADP-ribose) glycohydrolase mediates chromatin structure and SIR2-dependent silencing

Protein ADP ribosylation catalyzed by cellular poly(ADP-ribose) polymerases [PARPs; see Poly-(ADP-ribose) polymerase] and tankyrase [an enzyme that modulates the activity of target proteins through poly(ADP-ribosyl)ation], modulates chromatin structure, telomere elongation, DNA repair, and the transcription of genes involved in stress resistance, hormone responses, and immunity. Using Drosophila genetic tools, the expression and function of poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme responsible for degrading protein-bound ADP-ribose moieties, was characterize. Strongly increasing or decreasing PARG levels mimics the effects of Parp mutation, supporting PARG's postulated roles in vivo both in removing ADP-ribose adducts and in facilitating multiple activity cycles by individual PARP molecules. PARP is largely absent from euchromatin in PARG mutants, but accumulates in large nuclear bodies that may be involved in protein recycling. Reducing the level of either PARG or the silencing protein SIR2 weakens copia transcriptional repression. In the absence of PARG, SIR2 is mislocalized and hypermodified. It is proposed that PARP and PARG promote chromatin silencing at least in part by regulating the localization and function of SIR2 and possibly other nuclear proteins (Tulin, 2006).

ADP-ribose modification of nuclear proteins mediates DNA repair, gene transcription, telomere elongation, and chromatin structure. Protein ADP-ribosylation levels are ultimately determined by the location and activity of poly(ADP-ribose) polymerase (PARP) and tankyrase enzymes that utilize NAD to add such residues, as well as poly(ADP-ribose) glycohyrolase (PARG) enzymes that remove them. Although a great deal has been learned about the biochemical properties of these enzymes in vitro, exactly how they function in vivo remains poorly known. Most of the time, the vast majority of PARP molecules are enzymatically inactive, unmodified, and thought to act only during brief bursts of activity. Damaged or altered DNA conformation, along with other uncharacterized signals, can cause nearby PARP molecules within small chromosome regions to dimerize and become transiently active before they are dissociated and shut off by automodification with long poly(ADP-ribose) chains (pADPr). Histones and other chromosomal proteins in the affected chromatin domain adopt a looser configuration, either by binding avidly to PARP-linked poly(ADP-ribose) polymers or by direct modification, thereby facilitating repair or gene activation. When PARG eventually removes all the ADP-ribosyl groups from a PARP monomer, the cycle can repeat until the inducing conditions are no longer present. Other mechanisms of PARP action have been proposed as well, including some that do not require PARP enzymatic function (Tulin, 2006 and references therein).

Genetic analysis of this system is greatly facilitated in Drosophila, which contains a single Parp gene located in 3R heterochromatin that encodes an enzyme with the same domain structure as that of the major mammalian PARP1 protein . Drosophila also contains a single tankyrase gene (tankyrase) and a single gene (Parg) predicted to encode a PARG. Parp mutations are lethal and drastically alter many aspects of developmental physiology. These include the ability to activate and maintain nucleoli, to form polytene chromosome puffs, and to activate genes located therein that respond to stress, infection, or steroid hormones (Tulin, 2006).

Heterochromatin forms in early embryonic cells and additional chromatin domains are silenced as individual cell types differentiate. The ability to compact heterochromatin and to silence specific gene regions also requires Parp. For example, the 30-50 normally quiescent genomic copies of the copia retrotransposon are overexpressed >50-fold in Parp mutants. Normally, copia transcription is suppressed by a chromatin-based mechanism related to gene silencing in other regions. Thus, in addition to its role as an activator, PARP contributes to the repression of at least some chromatin domains (Tulin, 2006).

The evolutionarily conserved silent information repressor protein 2 (SIR2; see Drosophila Sir2) protein contributes to heterochromatin formation through the action of its NAD-dependent histone deacetylase activity. NAD is cleaved in conjunction with removal of acetyl groups from the target, forming nicotinamide and O-acetyl-ADP-ribose. In addition, many SIR2 protein family members catalyze protein ADP ribosylation. Drosophila contains five genes related to yeast SIR2, but the Sir2 gene residing at 34A7 shows the highest level of conservation and exhibits NAD-dependent histone deacetylase activity. While nonessential, Sir2 participates in chromatin silencing (Tulin, 2006).

To better understand how poly(ADP)-ribose metabolism regulates chromatin activity, this study characterized the Drosophila Parg gene. The findings reinforce previous evidence that PARP-catalyzed ADP ribosylation plays widespread roles in the nucleus, which are not limited to DNA repair. They support the view that PARP acts in vivo by undergoing bursts of activation limited by automodification and reversed by PARG action. In addition, PARG was found to control the localization of other nuclear proteins. In Parg mutants, SIR2 protein is mislocalized and hypermodified; endogenous copia retrotransposon expression is elevated, suggesting that chromatin silencing is compromised. These experiments further document important roles played by ADP-ribose modification in controlling chromatin structure and activity and suggest that some of these effects are mediated through SIR2 (Tulin, 2006).

A previous study found that Parg mutations are lethal or semilethal, and an effect of mutations on pADPr levels in neural tissue and on organismal life span was reported. However, the relationship between PARG and the roles played by PARP remained unclear. This study has shown that reducing or increasing PARG causes phenotypic effects very similar to disrupting Parp on nucleolar function, chromatin structure, immunity, and heat-shock sensitivity. This shows that PARG acts in many common pathways with PARP, presumably by virtue of its enzymatic action on ADP-ribose groups (Tulin, 2006).

The phenotypic similarity observed among Parg disruption, PARG overproduction, and Parp mutation argues that protein ADP ribosylation, rather than a direct structural function, underlies many of the reported actions of PARP. Molecular studies of Parg mutants directly verified the predicted increase in ADP-ribose modification levels. In the absence of PARG, newly synthesized PARP molecules would still be expected to function until they became automodified. Thus, the strong phenotypic effects of Parg mutation imply that recycling of automodified PARP molecules is quantitatively important, at least locally. For example, the developmental delays that were observed prior to each molt are probably caused by the extra time needed to synthesize de novo enough new PARP to support molting gene expression. PARG overproduction would also be expected to interfere with PARP action. Poly(ADP-ribose) chains on automodified PARP might remain too short to function, and protein recycling that depends on the kinetics of poly(ADP-ribose) modification might be disrupted. In support of these interpretations, it was found that the phenotypic effects of PARG overproduction are completely suppressed by simultaneously producing extra PARP (Tulin, 2006).

Consequently, these studies strongly support the view that localized episodes of poly(ADP)-ribose modification under the control of PARP, and recycling of ADP-ribose-modified proteins under the control of PARG, play a major role in controlling chromatin structure, gene activity and nuclear function in vivo. However, it still remains unclear how PARP is incorporated into chromatin in an inactive state, how it becomes locally activated (except in the case of DNA damage), and to what extent other chromatin proteins in addition to PARP itself are important substrates for PARP and PARG enzymatic activities in vivo. Moreover, this work does not rule out the possibility that PARP also acts via other mechanisms, including some that do not require enzymatic function. Drosophila oocytes and early embryos contain an essential isoform, PARP-e, that lacks a catalytic domain. In vitro, human PARP-1 represses chromatin by binding to nucleosomes, displacing histone H1, and compacting its local architecture independently of PARP enzymatic activity. In addition, PARP may form a stable component of repressive chromatin complexes on target genes (Tulin, 2006 and references therein).

The phenotype of Parg disruption is not identical to Parp mutation, suggesting that PARG carries out some functions independently of PARP. In particular, PARP protein and Fibrillarin accumulate in large nucleoplasmic and peri-centromeric bodies in Parg mutant cells, in contrast to the Fibrillarin-rich cytoplasmic body seen in Parp mutants. It is suggested that these structures correspond to intermediates in a process that normally recycles ADP-ribose-modified proteins within the cell. In this respect, they are reminiscent of Cajal bodies, which have been postulated to serve as staging, storage, or assembly sites of factors involved in transcript production and processing. The normal rate of this recycling may be greater in regions of high gene activity. When ADP-ribose groups cannot be removed, the recycling process backs up, causing the observed breakdown of the nucleolus and loss of PARP from eukaryotic chromosome regions. The nucleolus might be particularly sensitive if ongoing ADP ribosylation is needed to maintain rDNA genes, which do not exhibit a normal nucleosomal organization, in an active state. The phenotypic differences between Parp and Parg mutants may result from different arrest points within a common recycling pathway or because PARG also reverses the action of other poly(ADP-ribose) polymerases in addition to PARP (Tulin, 2006).

PARP activation or PARG reduction might block SIR2 action simply by depleting cellular NAD pools. However, PARG overexpression should not reduce NAD pools, and yet nucleolar structure was disrupted in animals with elevated PARG. Instead of acting via NAD, the observation that, in the absence of PARG, a higher-molecular-weight form of SIR2 accumulates in the cell cytoplasm suggests that SIR2 is modified by an ADP-ribose addition as part of its function. Many SIR2 protein family members themselves exhibit protein ADP-ribosylation activity (Frye, 1999; Furuyama, 2004), and mouse SIRT6, a predominantly nuclear protein, can direct its own mono-(ADP) ribosylation (Liszt, 2005). PARG may be needed to remove ADP-ribose groups from SIR2 that are added by these or other mechanisms that are independent of PARP (Tulin, 2006).

Taken together, these experiments suggest a model in which PARP, PARG, and SIR2 cooperate to silence specific chromosomal domains. It is proposed that activation and ADP ribosylation of PARP molecules (and possibly other local chromatin proteins) loosen chromatin early in the silencing process and that this facilitates SIR2 access to acetylated histone tails. In some cases this process would transiently strip the target chromatin proteins off the affected region and transfer them in an organized fashion to the branched ADP-ribose polymers on auto-inactivated PARP molecules within the immediately adjacent nucleoplasm. Here the ADPr/chromatin complex would encounter PARG and SIR2, possibly in conjunction with other proteins involved in chromatin remodeling (Furuyama, 2004). SIR2 molecules would undergo autoADP-ribosylation and deacetylate histones such as H4 within the complex, while PARG begins to cleave their ADP-ribose moieties. Since the ADPr tails shorten, the chromatin proteins would be driven to reassemble onto their former chromosome region. PARG action on SIR2 might also help coordinate these events (Tulin, 2006).

When Parp is mutated or when PARG activity becomes too high or too low, chromatin activation and silencing would be drastically disrupted. Without PARP or in the presence of excess PARG, chromatin proteins would fail to loosen and become accessible to modification. The state of chromatin would become 'frozen' at whatever state it had reached when the deficiency became acute (i.e., when maternal PARP is depleted in the case of a zygotic Parp mutant, or when expressed PARG reaches a critical level). When PARG levels are too low, in contrast, excess levels of ADP-ribose-modified SIR2 would build up, driving it into the cytoplasm. Following a single activation, PARP molecules would be trapped in the inactive automodified state. Large amounts of pADPr would accumulate, shunting chromatin proteins into remodeling complexes that cannot break down. It is now possible to look forward to obtaining a more detailed understanding of these events using the genetic tools available for the study of chromatin organization in Drosophila (Tulin, 2006).

Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals

Feeding behavior, one of the most essential activities in animals, is tightly regulated by neuroendocrine factors. Drosophila short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. This study found that minibrain (mnb) and the mammalian ortholog Dyrk1a, target genes of sNPF and NPY signaling, regulate food intake in Drosophila and mice. In Drosophila neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, and increased food intake. These findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals (Hong, 2012).

The production of sNPF and NPY in sNPFnergic and hypothalamic neurons of flies and mammals respectively, is increased during fasting. These neuropeptides are secreted to produce paracrine and endocrine effects but also feedback upon their synthesizing neurons where they respectively induce mnb and Dyrk1a gene expression through the PKA-CREB pathway. This Mnb/Dyrk1a kinase phosphorylates and activates the Sir2/Sirt1 deacetylase, which in turn deacetylates and activates the FOXO transcription factor. Among its many potential targets, FOXO then increases sNPF/NPY mRNA expression. Negative controls modulate the positive feedback of sNPF/NPY. Feeding activates the insulin receptor-PI3K-AKT pathway. FOXO becomes phosphorylated and transcriptionally inactivated by translocation to the cytoplasm. In this state the induction of sNPF/NPY by FOXO is decreased. Because sNPF and NPY are orexogenic, their positive feedback during fasting should reinforce the propensity for food intake whereas the negative regulation of sNPF and NPY mRNA during feeding condition would then contribute to satiety (Hong, 2012).

FOXO family transcriptional factors are involved in metabolism, longevity, and cell proliferation. FOXO is in part regulated in these processes by post-transcriptional modifications including phosphorylation and acetylation. In many model systems, the ligand activated Insulin-PI3K-AKT pathway phosphorylates FOXO to inactivate this transcription factor by moving it to the cytoplasm. The cytoplasmic localization of FOXO is mediated by 14-3-3 chaperone proteins in Drosophila and mammals. FOXO may also be acetylated, as is FoxO1 of mice, by the CREB-binding protein (CBP)/p300 acetylase and this inhibits FOXO transcriptional function by suppressing its DNA-binding affinity. Such FoxO1 acetylation can be reversed by SirT1 to help activate the FoxO1 transcription factor. This study describes for Drosophila how dFOXO in sNPFR1 neurons regulates the expression of sNPF and food intake. This mechanism parallels how hypothalamic FoxO1 regulates food intake through its control of orexigenic NPY and Agrp in rodents. Post-transcriptional modification of FOXO is central to these controls in both animals. sNPF and NPY expression is increased when FOXO is deacetylated by Sir2/Sirt1, while sNPF and NPY are decreased when FOXO is phosphorylated via the Insulin-PI3K-AKT pathway. Post-transcriptional modifications of FOXO proteins play a critical role for controlling food intake through the sNPF and NPY expression in flies and rodents (Hong, 2012).

Mnb/Dyrk1a participate in olfactory learning, circadian rhythm, and the development of the nervous system and brain. Mnb and Dyrk1a proteins contain a nuclear targeting signal sequence, a protein kinase domain, a PEST domain, and a serine/threonine rich domain. The kinase domains are evolutionary well-conserved from flies to humans. In Down syndrome (DS), chromosome 21 trisomy gives patients three copies of a critical region that includes the Mnb/Dyrk1a; trisomy of this region is associated with anomalies of both the nervous and endocrine systems. DS patients often show high Body Mass Index due to the increased fat mass. Children with DS have elevated serum leptin coupled with leptin resistance, both of which contribute to the obesity risk common to DS patients. This study found a novel function of Mnb/Dyrk1a that may underlay this metabolic condition of DS patients. Mnb/Dyrk1a regulates food intake in flies and mice. This is controlled by sNPF/NPY-PKA-CREB upstream signaling and thus produces downstream affects upon Sir2/Sirt1-FOXO-sNPF/NPY. Fasting not only increases the expression of mnb, but also of sNPF, suggesting that Mnb kinase activates a positive feedback loop where Sir2-dFOXO induces sNPF gene expression. Notably, fasting increases Sirt1 deacetylase activity and localizes FoxO1 to the nucleus in the orexogenic AgRP neurons of the mouse hypothalamus. Increased dosage of Dyrk1a in DS patients may reinforce the positive feedback by NPY and disrupt the balance between hunger and satiety required to maintain a healthy body mass (Hong, 2012).

Insulin produced in the pancreas affects the hypothalamus to regulate feeding in mammals. Insulin injected into the intracerebroventrical of the hypothalamus reduces food intake while inhibiting insulin receptors of the hypothalamic ARC nucleus causes hyperphasia and obesity in rodent models. This study showed a similar pattern for Drosophila where overexpression of insulin-like peptide (Dilp2) at insulin producing neurons decreased food intake while food intake was increased by inhibiting the insulin receptor in sNPFR1 expressing neurons. Likewise, during fasting, serum insulin and leptin levels are decreased in mammals, as is mRNA for insulin-like peptides of Drosophila. Thus, the mechanism by which insulin and insulin receptor signaling suppresses food intake is conserved from fly to mammals in at least some important ways (Hong, 2012).

Previous work has shown how sNPF signaling regulates Dilp expression through ERK in IPCs and controls growth in Drosophila (Lee, 2008). This study shows that sNPF signaling regulates mnb expression through the PKA-CREB pathway in non-IPC neurons and controls food intake. Since sNPF works through the sNPFR1 receptor, sNPFR1 in IPCs and non-IPCs neurons might transduce different signals and thereby modulate different phenotypes. Four Dilps (Dilp1, 2, 3, and 5) are expressed in the IPCs of the brain. Interestingly, levels of Dilp1 and 2 mRNA are reduced in the sNPF mutant, which has small body size, but this study finds only Dilp3 and 5 mRNA levels are reduced upon 24 h fasting. Likewise, only Dilp5 is reduced when adult flies are maintained on yeast-limited diets. In addition, Dilp1 and 2 null mutants show slight reduced body weights but Dilp3 and Dilp5 null mutants do not. These results suggest that Dilp1 and 2 behave like a mammalian insulin growth factor for size regulation while Dilp3 and 5 act like a mammalian insulin for the regulation of metabolism. However, in the long term starvation, Dilp2 and Dilp5 mRNA levels are reduced and Dilp3 mRNA expression is increased (Hong, 2012).

During fasting, sNPF but not sNPFR1 mRNA expression was increased in samples prepared from fly heads increasing food intake. In contrast, in feeding, the high level of insulin signaling reduced sNPF but not sNPFR1 mRNA expression and suppresses food intake. Interestingly, in the antenna of starved flies, sNPFR1 but not sNPF mRNA expression is increased and induces presynaptic facilitation, which results in effective odor-driven food search. However, high insulin signaling suppresses sNPFR1 mRNA expression and prevents presynaptic facilitation in DM1 glomerulus. These results indicate that starvation-mediated or insulin signaling-mediated sNPF-sNPFR1 signaling plays a critical role in Drosophila feeding behavior including food intake and food search even though the fine tuning is different (Hong, 2012).

This study presents a molecular mechanism for how sNPF and NPY regulate food intake in Drosophila and mice. A system of positive feedback regulation for sNPF and NPY signaling is described that increases food intake and a mode of negative regulation for sNPF and NPY by the insulin signaling that suppresses food intake. Modifications of the FOXO protein play a critical role for regulating sNPF and NPY expression, resulting in the control of food intake (Hong, 2012).


DEVELOPMENTAL BIOLOGY

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).

Effects of Mutation or Deletion

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).

Sir2 mediates longevity in the fly through a pathway related to calorie restriction

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).

Sir2 mediates apoptosis through JNK-dependent pathways in Drosophila

Increased expression of the histone deacetylase sir2 has been reported to extend the life span of diverse organisms including yeast, Caenorhabditis elegans, and Drosophila melanogaster. A small molecule activator of Sir2, resveratrol, has also been suggested to extend the fitness and survival of these simple model organisms as well as mice fed high calorie diets. However, other studies in yeast have shown that Sir2 itself may prevent life extension, and high expression levels of Sir2 can be toxic to yeast and mouse cells. This conflicting evidence highlights the importance of understanding the mechanisms by which Sir2 expression or activation affects survival of organisms. To investigate the downstream signaling pathways affected by Sir2 in Drosophila, transgenic flies were generated expressing sir2. Overexpression of sir2 in Drosophila promotes caspase-dependent but p53-independent apoptosis that is mediated by the JNK and FOXO signaling pathways. Furthermore, a loss-of-function sir2 mutant partially prevents apoptosis induced by UV irradiation in the eye. Together, these results suggest that Sir2 normally participates in the regulation of cell survival and death in Drosophila (Griswold, 2008).

Drosophila has five sir2-like genes, with sir2 being most homologous to the yeast sir2, C. elegans sir2.1, and human sirt1. Ubiquitous overexpression of sir2 in Drosophila by using EP lines has been reported to extend life span. To understand the consequences of Drosophila sir2 overexpression and to identify its downstream signaling pathways, transgenic flies were generated that can express sir2 in the Drosophila eye, a well established system for characterizing signaling pathways. The use of the gmr-Gal4 driver line to overexpress this gene in developing eyes causes a phenotype, specifically a lack of pigmentation and a rough, bristled appearance. The severity of this phenotype correlates well with dosage of sir2. Consistent with the known pattern of gmr-Gal4 expression, Sir2 expression was seen in the developing eye imaginal disc in cells posterior to the morphogenic furrow. The endogenous Sir2 is also found at a low level in whole heads of Drosophila as well as in photoreceptor cells posterior to the morphogenic furrow and regions of the antennal disc. Ubiquitous sir2 overexpression using the actin-5C-Gal4 or the pan-neuronal driver elav-gal4 resulted in premature death during development, suggesting that Sir2 affects survival in other cell types as well. Additionally, it was found that the overexpressed Sir2 was enzymatically functional because it increased NAD+-dependent deacetylase activity in both larval eye imaginal discs and adult fly heads (Griswold, 2008).

To verify whether the observed phenotype is Sir2-specific, transgenic flies were generated to express a Sir2 paralog, CG5085, which shares 43% identity and 63% similarity in amino acid sequence in the deacetylase domain. CG5085 is indeed a functional Sir2 deacetylase family member because recombinant CG5085 demonstrates NAD+-dependent deacetylase activity. However, although structurally and enzymatically similar, when overexpressed by using the gmr-Gal4 driver, CG5085 did not alter the phenotype of the eye. Furthermore, overexpression of lacZ or the Drosophila G protein-coupled receptor methuselah had no effect on eye morphology. This indicates that the eye phenotype in the transgenic flies overexpressing sir2 is indeed Sir2-specific and not due to either general overexpression of proteins or the deacetylase activity itself (Griswold, 2008).

The finding that overexpression of sir2 results in a deleterious effect on various tissues of the fly contrasts with a previously reported effect of sir2 overexpression on longevity. To clarify this contradictory result, a sir2 overexpression line (EP2300) used in the previous report was crossed with the gmr-Gal4 driver but no defective eye phenotype was found, although it highly expressed sir2. It is plausible that the insertion of EP2300 could affect genes neighboring sir2, thus leading to modification of the eye phenotype. EP2300 is inserted in a 500-bp region upstream of a chaperone gene dnaJ-H, and an increase was observed in the transcription level of dnaJ-H in adult heads when EP2300 was crossed with the gmr-Gal4 driver. Because overexpression of dnaJ-H can suppress the effects of toxic proteins in the eye, transgenic flies were generated overexpressing UAS-sir2 and UAS-dnaJ-H together in the eye, and up-regulation of dnaJ-H was found to ameliorate the defective phenotype caused by sir2 overexpression. This result suggests an explanation for the lack of an eye phenotype in EP2300 line despite an increase in sir2 expression. Coexpression of sir2 and dnaJ-H by EP2300 raises the possibility that the reported effects of this line may give a misleading picture of the role of sir2 in Drosophila aging (Griswold, 2008).

Based on the defective eye phenotype, it was hypothesized that sir2 expression may cause cell death. Thus, the developing eye in third-instar larval imaginal discs was examined by using acridine orange staining, a vital dye that detects dying cells, and the TUNEL assay, which identifies cells undergoing programmed cell death. Staining with acridine orange showed an increase in dying cells in the posterior part of eye discs overexpressing sir2, suggesting that sir2 overexpression causes cell death. Numerous TUNEL-positive cells in the imaginal discs with sir2 overexpression were also found, whereas the control showed few positive cells, indicating that the phenotype is mediated by apoptotic cell death in the developing eye. In addition, in vitro caspase-3 activity in the eye imaginal disc overexpressing sir2 increased 1.5-fold when compared with the control. This increase in caspase-3 activity was also verified by immunostaining for active caspase-3 in the imaginal discs; overexpression of sir2 showed an increase in the number of more intensely stained cells (Griswold, 2008).

A previous report has characterized mammalian Sir2 as an apoptosis inhibitor through its deacetylation of p53. In Drosophila, the role of p53 is still under investigation, although it has been shown to regulate cell death in response to stress, similar to its mammalian homolog. However, the relationship of p53 and Sir2 in Drosophila has not been explored. To determine whether p53 and Sir2 are in the same genetic pathway to cause apoptosis in the eye, p53 and sir2 were coexpressed. The eyes of the flies overexpressing both p53 and sir2 were more severely affected than either p53 or sir2 overexpressed alone. However, overexpression of dominant negative p53 constructs did not rescue the sir2 phenotype. This suggests that the sir2 overexpression effect is p53-independent and that p53 and sir2 overexpression work in parallel to induce cell death in Drosophila (Griswold, 2008).

Sir2 can alter the activity of mammalian FOXO3a by deacetylation. Additionally, to increase life span in C. elegans, overexpression of sir2.1 requires DAF-16, the FOXO3a transcription factor homolog. No such direct link between Sir2 and FOXO has been established in Drosophila; however, overexpression of foxo in the Drosophila eye exhibits a defective eye phenotype. Because the results show that sir2 overexpression induces apoptotic cell death in the Drosophila eye, whether FOXO activity might be involved in the induction of apoptosis as a result of sir2 overexpression in Drosophila was investigated. sir2 was overexpressed in a foxo null mutant background, and a less severe eye phenotype was found, suggesting a genetic interaction between Sir2 and FOXO in cell death pathways in Drosophila (Griswold, 2008).

Recently, it was shown that the foxo-induced defective eye phenotype can be modulated through the JNK signaling pathway. Furthermore, increased activation of JNK is associated with an apoptotic eye phenotype in Drosophila. Because JNK signaling interacts with FOXO and influences apoptotic pathways, whether the JNK pathway is also involved in the Sir2-induced eye phenotype was examined. The transcription level of the JNK phosphatase puc, a downstream target of the JNK signaling pathway, is increased in the heads of flies overexpressing sir2, suggesting an increase in JNK-dependent transcription. Inhibition of this signaling pathway by overexpression of bskDN, a dominant negative form of Drosophila JNK, resulted in a major improvement of the eye phenotype caused by sir2 overexpression. Additionally, inhibition of JNK signaling by coexpression of puc with sir2 demonstrated a significant rescue in the eye, consistent with a report that constitutive overexpression of puc can rescue the eye phenotype caused by increased JNK activity. These rescue flies do not reduce the level of Sir2 below that expressed by coexpression of lacZ. Together, these results suggest that sir2 overexpression requires JNK signaling to induce cell death in the eye (Griswold, 2008).

In summary, the evidence that endogenous Sir2 in the Drosophila eye plays a role in apoptosis is consistent with the finding that sir2 overexpression induces apoptotic cell death in the eye imaginal discs. An important issue is thus the identification of signaling pathways that mediate these effects. JNK signaling is implicated by phenotypic alleviation via the expression of a dominant negative JNK or an inhibitor JNK signaling. Also, loss of function of foxo can ameliorate the eye phenotype induced by sir2 overexpression, suggesting that sir2 can activate a proapoptotic function of FOXO. These pathways may intersect at JNK to induce the proapoptotic function of FOXO. The result of these pathways is the observed increase in proapoptotic gene expression of reaper, grim, and hid. This in turn leads to increased caspase activity and ultimately cell death (Griswold, 2008).

The results show that sir2 overexpression in Drosophila does not necessarily promote longevity, and endogenous Sir2 plays a critical role in regulating cell survival and death in the animal. Hence, it will be of future interest to study the signaling pathways induced by sir2 expression that lead to JNK activation as well as the relationship between Sir2 and FOXO in modulating apoptotic and survival pathways in Drosophila. Together, these will determine pathways affected by sir2 expression and give insights as to how it can mediate both cell survival and cell death (Griswold, 2008).

Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant

PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In a fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin (Koh, 2012).

From these findings, the following model is proposed for PINK1-mediated mitochondrial protection. To protect mitochondria, PINK1 translocates Parkin to mitochondria and activates its E3 ubiquitin ligase activity. In mitochondria, Parkin ubiquitinates mitochondrial proteins such as voltage-dependent anion channel 1 (VDAC1) and mitofusin (Mfn) to regulate mitochondrial remodeling process. In addition to the direct action in mitochondria, PINK1 transduces signals to the cytosol and activates Sir2. Sir2 deacetylates FOXO and induces the FOXO-dependent transcription of mitochondrial protective genes including SOD2 and Thor in the nucleus. The expressed proteins locate to the cytosol or mitochondria and play their roles such as scavenging harmful reactive oxygen species (ROS) and enhancing production of mitochondrial proteins. Through the direct regulation of mitochondrial protein turnover and the induction of mitochondrial protective gene expression, PINK1 can efficiently protect cells from mitochondrial damages (Koh, 2012).


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Sir2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 October 2014

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