InteractiveFly: GeneBrief

charybde and scylla: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Overexpression | Evolutionary Homologs | References

Gene name - charybde and scylla

Synonym - charybdis (char)

Cytological map positions - 68C

Function - unknown

Keywords - negative regulation of growth, insulin pathway, response to hypoxia

Symbol - chrb and scyl

FlyBase IDs: FBgn0036165 and FBgn0041094

Genetic map position - 3L

Classification - conserved protein

Cellular location - cytoplasmic and nuclear

NCBI links for Charybde: Entrez Gene
NCBI links for Scylla: Entrez Gene

chrb orthologs: Biolitmine
scyl orthologs: Biolitmine

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters said to have lived in the Strait of Messina between Sicily and Italy, that posed a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) as is its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).

The evolutionarily conserved Insulin/IGF receptor (Inr)/Target of Rapamycin (TOR) signaling network plays an important role in modulating growth, metabolism, reproduction, and life span in response to intracellular and extracellular signals in species ranging from invertebrates to humans. In Drosopohila, viable mutant combinations of positive components of the Drosopohila Inr cascade such as Inr, chico (the homolog of vertebrate IRS1-4), PKB (Protein kinase B, also known as Akt) and PDK1 (3-phosphoinositide-dependent protein kinase-1) lead to developmentally delayed and proportionally reduced small flies, displaying a reduction in cell size and number. In contrast, loss of PTEN (phosphatase and tensin homolog on chromosome ten), which antagonizes PI3K activity by dephosphorylating the 3'-position of phosphoinositides, leads to hypertrophy and hyperplasia. In humans, loss of the tumor suppressor PTEN is observed frequently in glioblastomas, prostate cancers, and endometrial cancers, and PTEN germline mutations are linked to dominant hamartoma syndromes like Cowden syndrome, Lhermitte-Duclose disease, Proteus syndrome, and Bannayan-Zonana syndrome. Genetic studies in Drosophila indicate that PKB has a crucial role in signaling downstream of PTEN since flies completely lacking PTEN function can be rescued to viability by lowering PKB activity (Reiling, 2004 and references therein).

The TOR/S6K (S6 kinase) branch of the growth modulatory network is negatively regulated by the tumor suppressor Tsc1 (hamartin)/Tsc2 (tuberin: Drosophila homolog is Gigas) complex. Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by the formation of hamartomas, benign tumors that arise in various tissues. In Drosophila, cells devoid of Tsc1/Tsc2 function are increased in size. Tsc2 and, more weakly, Tsc1 were found to physically associate with dTOR, thereby inhibiting dTOR kinase activity. Other studies reported an inhibitory role of PKB on Tsc1-Tsc2 by PKB-mediated phosphorylation of Tsc2. Recently, the small GTPase Rheb (Ras homolog enriched in brain) has been identified as a new positive growth effector acting downstream of Tsc1-Tsc2 and upstream of TOR. Mechanistically, Tsc2 acts as GTPase-activating protein (GAP) toward Rheb. The molecular mechanism to explain how Rheb relays the signal to TOR is currently unknown. dTOR mutants show a growth deficit that is more pronounced in endoreplicative tissues than in mitotic tissues. An effector of mTOR is S6K, which upon activation by mTOR phosphorylates ribosomal protein S6. S6K-mediated S6 phosphorylation has been thought to lead to a preferential translation of mRNAs encoding ribosomal proteins and proteins of the translational apparatus although the significance of this S6K function has been questioned. Inr/TOR signaling activity culminates in the regulation of translation rate by controlling S6K and the translational repressor 4E-BP1. S6K mutant flies are small but in contrast to mutants of the Inr pathway, only cell size is reduced without a change in cell number. Therefore, loss of S6K function reduces growth and body size to a lesser extent than loss of other positive components acting further upstream in the cascade (Reiling, 2004).

Growth is modulated by extrinsic factors such as nutrients, temperature, and hypoxia. However, their link to the Inr/TOR signaling network is not well defined. Although it is known that starvation results in a reduction in the levels of insulin-like peptides and a reduction in S6K activity in Drosopohila, little is known about whether temperature or hypoxia regulates the activity of this pathway. It is conceivable that mutations in genes coding for factors mediating the modulation of growth in response to external stimuli have escaped detection because they do not exhibit a very strong phenotype under standard culture conditions. For example, overexpression of the Forkhead transcription factor FOXO3a, the human homolog of Drosopohila dFOXO, produces a very subtle small eye phenotype under normal nutrient conditions. Under starvation, however, this phenotype is massively exacerbated, inducing a further eye size reduction and cell death. Furthermore, dFOXO mutant flies are viable and do not show an (over)growth phenotype in an otherwise wild-type background under normal conditions. Genes like dFOXO were missed in genetic loss-of-function screens aimed at identifying growth-regulatory genes (1) because they have only mild or missing mutant phenotypes under normal conditions and/or (2) because their function is masked by redundancy (Reiling, 2004).

Genes acting in a redundant manner can be identified in a complementary gain-of-function approach using EP (enhancer/promoter) elements. Screening >4000 novel EP lines, two insertions were found in the scylla locus as suppressors of a PKB/PDK1-dependent eye overgrowth phenotype. A second gene was identified in the Drosopohila genome with homology to scylla, and named charybdis. Homologs of these genes also exist in mammals, and they have been implicated in the response of a cell to hypoxia or more generally as stress-induced genes having either pro- or anti-apoptotic functions. Evidence is presented that scylla and charybdis, like some of their mammalian homologs, are induced by hypoxia and that Scylla and Charybdis act as partially redundant negative regulators of growth by controlling S6K but not PKB activity (Reiling, 2004).

Therefore the two related proteins, Scylla and Charybdis, are negative modulators of Inr/TOR signaling in response to different external stress situations including hypoxia and starvation. scylla and charybdis single mutants do not show obvious growth phenotypes. scylla charybdis double-mutant flies are also viable and fertile and exhibit a slight increase in body size. Viability of the double mutants is strongly reduced, however, when they are reared under hypoxic conditions. Thus, although Scylla and Charybdis are largely dispensable for normal development, they have a critical role for the endurance of prolonged hypoxia. scylla is transcriptionally induced as a target of Drosopohila HIF-1 (the Tango-Similar dimer) and that scylla and charybdis are up-regulated under hypoxic conditions like their mammalian homolog RTP801/REDD1. Furthermore, Scylla negatively regulates Inr/TOR signaling by reducing S6K but not PKB activity (Reiling, 2004).

RTP801 was described as a hypoxia/HIF-1-inducible factor with a role in apoptosis (Shoshani, 2002). scylla/charybdis, like their mammalian homolog, are induced by hypoxia, and scylla is a direct target of Drosopohila HIF-1. However, there is no indication that scylla/charybdis overexpression promotes cell death. Coexpression of the caspase inhibitors p35 or DIAP1 did not rescue the small eye phenotype induced by expression of either scylla or charybdis in the eye. Moreover, acridine orange staining revealed no increased cell death upon scylla/charybdis coexpression. Accordingly, signs of necrotic eye tissue were never observed upon scylla/charybdis overexpression (Reiling, 2004).

Dig2, a murine Scylla/Charybdis homolog, is also a stress-responsive protein induced by a variety of treatments including dexamethasone, thapsigargin, tunicamycin, and heat shock (Wang, 2003). Together with the finding by Zinke (2002) that scylla/charybdis expression is increased during starvation conditions and the current analysis, showing that Scylla and Charybdis act as growth inhibitors, these data support a model wherein Scylla and Charybdis, induced by stresses like hypoxia and starvation, act to dampen growth under certain stress conditions (Reiling, 2004).

Overexpressing either scylla or charybdis on their own is sufficient to reduce growth. Coexpression of both proteins seemed to have a slight cooperative effect on the PKB/PDK1-dependent eye phenotype with respect to ommatidial structure. Thus, an obvious question is whether Scylla and Charybdis bind to each other and exert their effect only in the presence of the other. Notably, eye-specific charybdis overexpression in a scylla-/- background results in the same phenotype as in a wild-type situation. This indicates that Charybdis can act independently of Scylla. This is further supported by their mostly nonoverlapping mRNA expression patterns. Indeed, charybdis but not scylla is expressed in neuromuscular junctions. Moreover, under hypoxia only scylla is induced in the fat body but not charybdis, indicating that they may be differentially regulated (Reiling, 2004).

Several lines of evidence indicate that Scylla and Charybdis feed into the Inr pathway downstream of PKB: (1) Scylla antagonizes PKB/PDK1-induced overgrowth in the eye, but in vitro kinase assays demonstrate that Scylla and Charybdis do not reduce PKB kinase activity, nor does the loss of Scylla enhance PKB kinase activity. (2) scylla or charybdis coexpression can rescue PKB-induced developmental lethality, and ubiquitous scylla expression suppresses the lethality associated with hypomorphic PTEN mutants. (3) The weight reduction of hypomorphic PKB3 flies is partially rescued by the simultaneous absence of Scylla function. (4) Eye-specific PKB/PDK1 expression in a scylla-/- background leads to a mild enhancement of the eye phenotype. (5) Overexpression of PTEN or Dp110DN does not suppress the big eye phenotype of the tester system, suggesting that the screening system identifies components that act downstream of PKB (Reiling, 2004).

S6K assay has demonstrated that Scylla is capable of reducing S6K activity. Thus, Scylla acts upstream of S6K. This result is consistent with in vivo results. (1) scylla overexpression in the wing reduces wing size by decreasing cell size but not cell number (in fact, cell number is slightly increased), and (2) a S6K scylla mutant combination has the same weight as S6K single mutants. S6K mutants are smaller because of a reduction in cell size but not cell number, making it distinct from other Inr signaling pathway mutants. Scylla and Charybdis do not control S6K activity directly but require its upstream regulator TSC. Tsc1/2 mutants cannot be rescued by overexpression of scylla, and the big head phenotype caused by loss of TSC function is not enhanced by the absence of Scylla and Charybdis. Coexpression of scylla or charybdis and Tsc1/2 does not further decrease eye size compared to Tsc1 and Tsc2 co-overexpression on their own. Moreover, a Rheb-dependent big eye phenotype or lethality induced by ubiquitous Rheb expression cannot be suppressed by scylla expression. These results indicate that Scylla regulates S6K activity by acting upstream of Tsc1/2 and Rheb. This function appears to be conserved between mammals and flies because RTP801/REDD1 (Brugarolas, 2004) can reduce S6 phosphorylation only in the presence of TSC (Reiling, 2004).

The findings indicate that scylla/charybdis overexpression mainly affects the TSC/TOR/S6K branch of the pathway downstream of PKB. The PKB-FOXO axis appears not to be influenced by Scylla and Charybdis. Eye-specific overexpression of scylla/charybdis in conjunction with FOXO is unable to induce 4EBP. In contrast, simultaneous expression of FOXO and PTEN or a dominant-negative form of PI3K leads to a strong induction of the reporter gene (Reiling, 2004).

Consistent with an interplay between the Inr and TOR/S6K pathways, Inr lethality is suppressed by heterozygosity of Tsc1. Furthermore, overexpressed PKB phosphorylates and inactivates Tsc2 and thereby activates S6K. The finding that scylla overexpression is sufficient to rescue the lethality associated with PKB overexpression indicates that the lethality caused by PKB overexpression is due to the hyperactivation of the TOR/S6K pathway. Thus, oncogenic activation of PI3K/PKB signaling seems to be mainly mediated by TOR/S6K signaling. This may explain the beneficial effect of Rapamycin treatment (or its derivatives CCl-779 and RAD001) on PTEN-deficient tumors or cells overexpressing PKB (Reiling, 2004 and references therein).

TSC and TOR receive multiple inputs reflecting the metabolic state of the cell. AMP-activated kinase (AMPK) is a heterotrimeric kinase that is activated by high AMP/ATP ratios in the cell. ATP depletion induces Tsc2-phosphorylation, and it was found that AMPK could interact with and phosphorylate Tsc2. Interestingly, loss of Tsc2 in MEFs and U2OS osteosarcoma cells under low serum and prolonged hypoxia conditions results in HIF-1alpha accumulation and concomitantly increased expression of HIF-1 targets in a Rapamycin-dependent manner. It has been shown that mTOR is regulated by decreased oxygen concentration resulting in a dephosphorylation of mTOR at Ser 2481, an mTOR autophosphorylation site. This effect is accompanied by reduced S6K phosphorylation but does not correlate with changes in adenine nucleotide levels and AMPK phosphorylation. Hence, these findings suggest a role for AMPK/Tsc2/mTOR in the integration of oxygen sensing/energy metabolism and growth (Reiling, 2004).

Frei (2004) found mutations in the gene encoding Drosopohila HIF-1 prolyl hydroxylase (Hph), the enzyme rendering HIF-1alpha a substrate for proteasomal destruction under normoxic conditions, function as dominant suppressors of a Cyclin D/Cdk4-induced bulging eye phenotype. Cells defective for hph show a growth deficit, and its overexpression stimulated growth. This study suggested that the growth-promoting function of Hph is independent of HIF-1alpha/Sima. The results raise the possibility that the Sima target scylla is important under hypoxia for growth inhibition (Reiling, 2004).

Directed expression of Tgo-Sima in the fat body induces scylla expression. That this regulation is physiologically relevant can be inferred from three findings: (1) scylla is also induced under hypoxic conditions; (2) directed expression of other bHLH-PAS proteins like Tgo-Trh or Sim alone did not induce scylla expression; (3) survival of flies lacking scylla and charybdis function is severely compromised under hypoxic conditions. It is suggested that scylla and charybdis are induced in response to external stress stimuli (e.g., hypoxia and starvation) to inhibit growth downstream of PKB but upstream of Tsc1/2. Scylla suppresses growth by reducing S6K activity. This could be achieved by relieving the inhibitory effect of PKB on Tsc2. Alternatively, Scylla/Charybdis could be negatively regulated targets of PKB. This is unlikely, however, since Scylla and Charybdis lack PKB consensus phosphorylation sites. AMPK, activated by drops in energy levels, may also contribute to the induction process of scylla and charybdis for growth inhibition, presumably under prolonged stress exposure. However, it is also possible that AMPK is controlled by Scylla and/or Charybdis. AMPK decreases protein synthesis by inhibition of S6K in a Rapamycin-sensitive manner, suggesting that mTOR is involved in mediating AMPK signaling (Kimura, 2003). AMPK also phosphorylates Tsc2, an event important for the cellular energy response pathway (Reiling, 2004 and references therein).

In tumors, hypoxic microenvironments are often encountered. Tumor hypoxia is associated with poor prognosis and resistance to radiation-induced cell death. Mutations in the tumor suppressor von Hippel-Lindau (VHL), the subunit of an E3 ubiquitin ligase complex that recognizes proline-hydroyxlated residues in HIF-1alpha, led to the formation of a variety of tumors including clear cell carcinomas of the kidney, pheochromocytomas, and hemangioblastomas. VHL-defective tumors exhibit increased HIF-1alpha expression. The induction of RTP801/REDD1 in cells exposed to hypoxia in tumors raises the possibility that these genes may play a role in tumor development. RTP801/REDD1 may act as a tumor suppressor. Cells having lost RTP801/REDD1 function may not stop growing under hypoxic conditions and hence risk accumulating further mutations that promote their tumorigenic state. The analysis of RTP801/REDD1 expression or mutations in a variety of tumor cell lines should help to test this hypothesis (Reiling, 2004).


Enhancer loops appear stable during development and are associated with paused polymerase

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. This study generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. These results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase (Ghavi-Helm, 2014).

Drosophila embryogenesis proceeds very rapidly, taking 18 h from egg lay to completion. Underlying this dynamic developmental program are marked changes in transcription, which are in turn regulated by characterized changes in enhancer activity. However, the role and extent of dynamic enhancer looping during this process remains unknown. To address this, 4C-seq (chromosome conformation capture sequencing) experiments were performed, anchored on 103 distal or promoter-proximal developmental enhancers (referred to as 'viewpoints'), and absolute and differential interaction maps were constructed for each, varying two important parameters: (1) developmental time, using embryos at two different stages, early in development when cells are multipotent (3-4 h after egg lay; stages 6-7), and mid-embryogenesis during cell-fate specification (6-8 h; stages 10-11); and (2) tissue context, comparing enhancer interactions in mesodermal cells versus whole embryo. To perform cell-type-specific 4C-seq in embryos, a modified version of BiTS-ChIP (batch isolation of tissue-specific chromatin for immunoprecipitation) was established. Nuclei from covalently crosslinked transgenic embryos, expressing a nuclear-tagged protein only in mesodermal cells, were isolated by fluorescence-activated cell sorting (FACS; (>98% purity) and used for 4C-seq on 92 enhancers at 6-8 h and a subset of 14 enhancers at 3-4 h. The same 92 enhancers, and 11 additional regions, were also used as viewpoints in whole embryos at both time points. The enhancers were selected based on dynamic changes in mesodermal transcription factor occupancy between these developmental stages and the expression of the closest gene. This study was thereby primed to detect dynamic three-dimensional (3D) interactions, focusing on developmental stages during which the embryo undergoes marked morphological and transcriptional changes (Ghavi-Helm, 2014).

All 4C-seq experiments had the expected signal distribution, with high concordance between replicates. To assess data quality further, ten known enhancer-promoter pairs (of the ap, Abd-b, E2f, pdm2, Con, eya, stumps, Mef2, sli and slp1 genes) were compared, and in all cases the expected interactions were recovered. For example, using an enhancer of the apterous (ap) gene, the expected interaction was detected with the ap promoter, 17 kilobases (kb) away, illustrating the high quality and resolution of the data (Ghavi-Helm, 2014).

In chromosome conformation capture assays, interaction frequencies decrease with genomic distance between regions. To adjust for this, the 4C signal decay was modelled as a function of distance using a monotonously decreasing smooth function. Subtracting this trend, the residual interaction signal was converted to z-scores and interacting regions defined by merging neighbouring high-scoring fragments within 1 kb. Using this stringent approach, 4,247 high-confidence interactions were identified across all viewpoints and conditions, representing 1,036 unique interacting regions (Ghavi-Helm, 2014).

Each enhancer (viewpoint) interacted with, on average, ten distinct genomic regions, less than half (41%) of which were annotated enhancers or promoters. Distal enhancers had a higher than expected interaction frequency with other enhancers. Similarly, promoter-proximal elements had extensive interactions with distal active promoters, 98% of which are >10 kb away. Enhancer-promoter interactions, although not significantly enriched, involve active promoters, with high enrichment for H3K27ac and H3K4me3, and active enhancers, defined by H3K27ac, RNA Pol II and H3K79me3. These results are similar to recent findings in human cells and the mouse β-globin locus, indicating similarities in 3D regulatory principles from flies to human (Ghavi-Helm, 2014).

The extent of 3D connectivity is surprising given the relative simplicity of the Drosophila genome. On average, each promoter-proximal element interacted with four distal promoters and two annotated enhancers, whereas each distal enhancer interacted with two promoters and three other enhancers. These numbers are probably underestimates, as 60% of interactions involved intragenic or intergenic fragments containing no annotated cis-regulatory elements. Despite this, the level of connectivity is similar to that recently observed in humans, where active promoters contacted on average 4.75 enhancers and 25% of enhancers interacted with two or more promoters. The multi-component contacts that were observed for Drosophila enhancers indicate topologically complex structures and suggest that, despite its non-coding genome being an order of magnitude smaller than humans, Drosophila may require a similar 3D spatial organization to ensure functionality (Ghavi-Helm, 2014).

Insulators, and associated proteins, are thought to have a major role in shaping nuclear architecture by anchoring enhancer-promoter interactions or by acting as boundary elements between topologically associated domains (TADs). Occupancy data from 0 to 12 h Drosophila embryos revealed a 50% overlap of interacting regions with occupancy of one or more insulator protein. Insulator-bound interactions are enriched in enhancer elements, suggesting that insulators may have a role in promoting enhancer-enhancer interactions. In contrast to mammalian cells, this study observed no association between insulator occupancy and the genomic distance spanned by chromatin loops, although there was a modest increase in average interaction strength. Conversely, 50% of interacting regions are not bound by any of the six Drosophila insulator proteins, suggesting that these 3D contacts are formed in an insulator-independent manner, or are being facilitated by neighbouring interacting regions (Ghavi-Helm, 2014).

If enhancer 3D contacts are involved in transcriptional regulation, then genes linked by interactions with a common enhancer should share spatio-temporal expression. For the four loci examined-pdm2, engrailed, unc-5 and charybde-this is indeed the case. For example, the pdm2 CE8012 enhancer interacts with both the pdm2 and nubbin (nub, also known as pdm1) promoters, located 2.5 and 47 kb away, respectively. Both genes, producing structurally related proteins, are co-expressed in the ectoderm, overlapping the activity of the pdm2 enhancer. Although there are examples of long-range interactions in Drosophila, often involving Polycomb response elements (PREs) and insulator elements, the vast majority of characterized enhancers are within 10 kb of their target gene, with few known to act over 50 kb. However, as investigators historically tested regions close to the gene of interest, characterized Drosophila enhancers are generally close to the gene they regulate. In contrast, although 4C cannot assess the full extent of short-range interactions, it provides an unbiased systematic measurement of the distance of enhancer interactions, far beyond 10 kb (Ghavi-Helm, 2014).

The distance distribution of all significant interactions reveals extensive long-range interactions within the ~180 megabase (Mb) Drosophila genome; 73% span >50 kb, with the median interaction-viewpoint distance being 110 kb. Two striking examples of long-range interactions are the unc-5 and charybde loci. The unc-5 promoter interacts with multiple regions, including a weak but significant interaction with the promoter of slit (sli), at a distance of >500 kb. These genes produce structurally unrelated proteins that are co-expressed in the heart, and are essential for heart formation (Ghavi-Helm, 2014).

A promoter-proximal element near the charybde (chrb) promoter has a strong interaction with the promoter of the scylla (scyl) gene, almost 250 kb away. Both genes are closely related in sequence and co-expressed throughout embryogenesis. These long-range interactions were confirmed by reciprocal 4C, using either the promoter of chrb or scyl, or an interacting putative enhancer as viewpoint. This interaction was further verified using DNA fluorescence in situ hybridization (FISH) in embryos. As a control, the distance was assessed between the chrb promoter (probe A) and an overlapping probe A' or a region on another chromosome (probe D), to determine the distances between regions very close or far away, respectively. Comparing the distance between the chrb and scyl promoters (probes A and B) showed a high, statistically significant co-localization, in contrast to the distance between the chrb promoter and a non-interacting region with equal genomic distance (probes A and C) (Ghavi-Helm, 2014).

The reciprocal 4C revealed several intervening interactions that are consistently associated with loops to both the scyl and chrb promoter. The activity was examined of two of these in transgenic embryos. Both interacting regions can function as enhancers in vivo, recapitulating chrb expression in the visceral mesoderm and nervous system (Ghavi-Helm, 2014).

When considering a 1-Mb scale around this region, the 4C interaction signal drops to almost zero just after the promoters of both genes. This 'contained block' of interactions is reminiscent of TADs, although the boundaries don't exactly match TADs defined at late stages of embryogenesis, which may reflect differences in the developmental stages used. However, the boundaries do overlap a block of conserved microsynteny between drosophilids spanning ~50 million years of evolution, suggesting a functional explanation underlying the maintained synteny. Expanding this analysis across all viewpoints, ~60% of interactions are located within the same TAD and the same microsyntenic domain as the viewpoint. In the case of the chrb and scyl genes, this constraint may act to maintain a regulatory association between a large array of enhancers, facilitating their interaction with both genes' promoters (Ghavi-Helm, 2014).

These examples, and the other 555 unique interactions >100 kb, provide strong evidence that long-range interactions are widely used within the Drosophila genome, potentially markedly increasing the regulatory repertoire of each gene. As enhancer-promoter looping can trigger gene expression, it follows that enhancer contacts should reflect the dynamics of transcriptional changes during development and therefore be temporally associated with gene expression. To assess this, looping interactions were directly compared between the two different time points and tissue contexts. Given the non-discrete nature of chromatin contacts, the quantitative 4C-seq signal was used to identify differential interactions based on a Gamma-Poisson model, and they were defined as having >2-fold change and false discovery rate <10% (Ghavi-Helm, 2014).

Despite the marked differences in development and enhancer activity between these conditions, surprisingly few changes were found in chromatin interaction frequencies, with ~6% of interacting fragments showing significant changes between conditions. Of these, 87 interactions were significantly reduced during mid-embryogenesis (6-8 h) compared to the early time point (3-4 h), and 90 interactions significantly increased. Similarly, 105 interactions had a higher frequency in mesodermal cells, compared to the whole embryo, and For example, a promoter-proximal viewpoint in the vicinity of the Antp promoter identified many interactions, two of which are significantly decreased at 6-8 h, although the expression of the Antp gene itself increases. For one region, the reduction in 4C interaction at 6-8 h corresponds to a loss in a H3K4me3 peak from 3-4 h to 6-8 h, suggesting that this 3D contact is associated with the transient expression of an unannotated transcript. The activity of the other interacting peak was examined in transgenic embryos, and it was shown to act as an enhancer, driving specific expression in the nervous system overlapping the Antp gene at 6-8 h. Along with the two enhancers discovered at the chrb locus, this demonstrates the value of 3D interactions to identify new enhancer elements, even for well-characterized loci like Antp (Ghavi-Helm, 2014).

A viewpoint in the vicinity of the Abd-B promoter interacted with a number of regions spanning the bithorax locus, three of which correspond to previously characterized Abd-B enhancers; iab-5, iab-7 and iab-8. The iab-7 and iab-8 enhancers are active in early embryogenesis, and have much reduced or no activity at the later time point. Notably, although the loop to those two enhancers is strong at the early time point, it becomes significantly reduced later in development, when both enhancers' activities are reduced. Conversely, the iab-5 enhancer contacts the promoter at a much higher frequency later in development, at the stage when the enhancer is most active. This locus therefore exhibits dynamic 3D promoter-enhancer contacts that reflect the transient activity of three developmental enhancers. It is interesting to note that in all loci examined, the dynamic contacts of specific elements are neighboured by stable contacts, as seen in the Antp and Abd-B loci. Dynamic changes, therefore, appear to operate in the context of larger, more-stable 3D landscapes (Ghavi-Helm, 2014).

Ninety-four per cent of enhancer interactions showed no evidence of dynamic changes across time and tissue context, which is remarkable given the marked developmental transitions during these stages. To investigate this further, enhancer-promoter interactions were examined of genes switching their expression state between time points or tissue contexts. The ap gene, for example, is not expressed at 2-4 h but is highly expressed during mid-embryogenesis (6-8 h). Despite the absence of expression, the interaction between the apME680 enhancer and the ap promoter is already present at 3-4 h, several hours before the gene's activation. To examine this more globally, differentially expressed genes, going either from on-to-off or off-to-on, were selected. Even for these dynamically expressed genes, there was no correlation with changes in their promoter-enhancer contacts. Similar 'stable' interactions were observed between tissue contexts. Genes predominantly expressed in the neuroectoderm at 6-8 h, for example, have interactions at the same locations in whole embryos and purified mesodermal nuclei at 6-8 h, despite the fact that they are not expressed in the mesoderm at this stage (Ghavi-Helm, 2014).

Pre-existing loops were recently observed in human and mouse cells, and suggested to prime a locus for transcriptional activation. However, why they are formed and how transcription is eventually triggered remains unclear. To investigate this, this study focused on the subset of genes that have both off-to-on expression and no evidence for differential interactions (20 genes; differentially expressed with stable loops (DS) genes). Despite changes in their overall expression, DS genes have similar levels of RNA polymerase II (Pol II) promoter occupancy at both time points. The presence of promoter-bound Pol II in the absence of full-length transcription is indicative of Pol II pausing. Using global run-on sequencing (GRO-seq) data to define a stringent set of paused genes, it was observed that most (75%) DS genes are paused (15 of 20 DS genes), and have a significantly higher pausing index. This percentage is significantly higher than expected by chance when sampling over all off-to-on genes, and is robust to using a strict or more relaxed) definition of Pol II pausing. This association is very evident when examining specific loci, showing Pol II occupancy, short abortive transcripts, and loop formation before the gene's expression. Taken together, these results indicate that 'stable' chromatin loops are associated with the presence of paused Pol II at the promoter (Ghavi-Helm, 2014).

To understand how transcription is ultimately activated, changes were examined in DNase I hypersensitivity at the promoter of DS genes. DNase I hypersensitivity is significantly increased at interacting promoters at the stages when the gene is expressed, suggesting that the recruitment of additional transcription factor(s) later in development might act as the trigger for transcriptional activation (Ghavi-Helm, 2014).

In summary, these data reveal extensive long-range interactions in an organism with a relatively compact genome, including pairs of co-regulated genes contacting common enhancers often at distances greater than 200 kb. Comparing enhancer contacts in different contexts revealed that chromatin interactions are very similar across developmental time points and tissue contexts. Enhancers therefore do not appear to undergo long-range looping de novo at the time of gene expression, but are rather already in close proximity to the promoter they will regulate. Within this 3D topology, highly dynamic and transient contacts would not be visible when averaging over millions of nuclei. As transcription factor binding is sufficient to force loop formation, these results suggest a model where through transcription factor-enhancer occupancy, an enhancer loops towards the promoter and polymerase is recruited, but paused in the majority of cases. The subsequent recruitment of transcription factor(s) or additional enhancers at preformed 3D hubs most likely triggers activation by releasing Pol II pausing. Such preformed topologies could thereby promote rapid activation of transcription. At the same time, as paused promoters can exert enhancer-blocking activity, the presence of paused polymerase within these 3D landscapes could safeguard against premature transcriptional activation, but yet keep the system poised for activation (Ghavi-Helm, 2014).

Transcriptional Regulation

RTP801/REDD1 was shown to be induced by hypoxia (Shoshani, 2002). This prompted an investigation of the effects of hypoxia on the scylla charybdis double mutants. scylla charybdis mutant larvae were raised on normal food at room temperature in a hypoxia chamber containing 9% oxygen during their entire development. In general, control flies as well as scylla charybdis homozygotes were 3-4 d delayed in development under these hypoxic conditions. However, whereas adult homozygous scylla charybdis double mutants could readily be recovered under standard culture conditions in normoxia, homozygous mutants of three independent scylla charybdis allelic combinations (char180 in combination with scy31, scy113, or scyEP9.85) were strongly underrepresented compared to normoxic conditions. The appearance of an increased number of dead pupae in the vials was observed. The scylla113 char180 double-mutant combination had the strongest effect, and only two escapers (out of 326 scored flies) hatched, whereas under normoxia flies with this genotype were recovered with nearly the expected Mendelian ratio. The eclosed homozygotes raised under hypoxia did not show obvious morphological defects. Thus, whereas simultaneous loss of Scylla and Charybdis under normoxic conditions results in a slight increase in growth, their absence under reduced oxygen concentrations severely compromises larval development. This indicates that Scylla and Charybdis have a critical function for survival under hypoxic conditions (Reiling, 2004).

The transcription factor HIF-1 is the key regulator of changes in gene expression in response to hypoxia. It consists of two bHLH-PAS domain protein subunits (HIF-1alpha and HIF-1beta). Under conditions of low oxygen, the HIF-1 protein complex is stabilized and binds to Hypoxia Response Elements (HRE), short regulatory DNA sequences (core recognition sequence 5'-TACGTG-3') located in the genomic region of target genes. Both the scylla and charybdis loci possess several HREs. Since RTP801/REDD1, a mammalian homolog of scylla and charybdis, is a direct target gene of HIF-1 and is induced under hypoxic conditions, it was enquired whether this function is evolutionarily conserved. Wild-type larvae were subjected to hypoxia (between 2% and 5% O2) and checked for the induction of scylla and charybdis expression. It is mainly the endoreplicative tissue such as fat body, gut, salivary glands, and tracheae that respond to changes in oxygen concentrations. scylla mRNA expression was up-regulated in the larval fat body and in the gut after hypoxia. charybdis, in contrast, is mildly induced in the midgut but not in the fat body. Whether hypoxia had an effect on Scylla protein levels and distribution was also tested. To detect the endogenous Scylla protein, advantage was taken of a transgenic Scylla-reporter line (a so-called protein trap line). This protein trap line bears a promoter-less green fluorescent protein (GFP)-reporter transgene in the scylla locus generating a Scylla-GFP fusion protein. Scylla-GFP is expressed in most larval tissues. Under normoxic conditions, nuclear accumulation of Scylla protein is observed in some cells of the endoreplicative tissue. Consistent with the mRNA data, upon exposure of third instar larvae to various hypoxia conditions, an up-regulation and nuclear localization of Scylla protein was observed in the fat body and in the gut (Reiling, 2004).

In Drosopohila, the bHLH-PAS family comprises Period (Per), Trachealess (Trh), Single-minded (Sim), Spineless (Ss), Dysfusion (Dys), Tango (Tgo), and Similar (Sima). Tgo is the ubiquitously expressed HIF-1beta ortholog, which dimerizes with any of the alpha-subunits. Sima has been shown to fulfill analogous functions to its mammalian homolog HIF-1 (Reiling, 2004).

To test whether scylla/charybdis transcription is regulated by bHLH-PAS proteins that recognize the same DNA stretch, sim, trh or sima were overexpressed together with tgo using the Lsp2-Gal4-driver that is active specifically in the fat body during the third larval stage. For Sima, a form lacking the oxygen-dependent degradation domain (ODD) was used, rendering it refractory to proteolytic destruction under normoxic conditions. Only the coexpression of tgo and sima induced scylla but not charybdis expression as assessed by mRNA in situ hybridization. This does not preclude, however, the possibility that charybdis is a target of Tgo-Sima, since its endogenous induction was observed upon hypoxia, but only in the gut and not in the fat body. Since neither expression of trh with tgo nor sim induced scylla or charybdis expression, the regulation of scylla by the Tgo-Sima heterodimer is specific. Thus, scylla and charybdis, like their mammalian homolog RTP801/REDD1, are induced by hypoxia, and at least scylla appears to be a direct target gene of the HIF-1 homolog Tgo-Sima (Reiling, 2004).

Scylla acts downstream of PKB but upstream of TSC

Although loss of Scylla function does not produce a mutant phenotype on its own, whether it would alter the PKB/PDK1 overexpression eye phenotype was tested. Indeed, loss of Scylla function enhances the PKB/PDK1 overgrowth phenotype. Thus, Scylla is essential for attenuating the increased growth in response to hyperactivation of the Inr pathway. Furthermore, loss of Scylla partially suppresses the growth reduction associated with reduced PKB function as assessed by comparing weights of PKB3 single mutants to scy31 PKB3 double mutants. In contrast, complete loss of Scylla in a heteroallelic S6K combination does not rescue the S6K single mutant phenotype indicating that S6K is epistatic over scylla (Reiling, 2004).

Moreover, overexpression of scylla and charybdis not only suppresses the growth phenotype caused by over-activation of the Inr pathway in the eye but to a certain extent also rescues the lethality associated with the ubiquitous increase in Inr pathway activity due to either overexpression of PKB or loss of PTEN. scylla rescues the male-specific lethality caused by ubiquitous expression of PKB and organismal lethality associated with the partial but not complete loss of PTEN function. Similarly, PKB-associated male lethality is also rescued by charybdis overexpression. This indicates that scylla and charybdis have the capacity to act as potent negative regulators of insulin signaling downstream of PKB and PDK1 (Reiling, 2004).

Several lines of evidence suggest that Scylla and Charybdis act upstream of TSC and Rheb. Tsc1/2 mutant flies can be rescued to adulthood by reducing S6K signaling, and a mere reduction of one TOR copy in a Tsc1 mutant context results in a rescue to the pupal stage. Whether ubiquitous scylla overexpression could rescue the larval lethality of heteroallelic Tsc1/2 mutant combinations (Tsc12G3/Tsc1Q87X and Tsc256/Tsc2192) was examined using the da-Gal4 or Act5C-Gal4 drivers in combination with a UAS-scy transgene or EPscy at 18°C, 25°C, and 29°C. Ubiquitous overexpression of scylla/charybdis in a Tsc1/2 mutant background did in no case extend larval development beyond first/second instar, and these larvae died at the same time as Tsc1/2 mutants. Moreover, the big head phenotype of Tsc2192 (and Tsc256) induced by the eyflp/FRT system was not further enhanced in scyEP9.85 char180 Tsc2 triple-mutant heads. It has been shown that heads composed almost entirely of scylla charybdis double-mutant cells are enlarged. Conversely, GMRGal4-driven co-overexpression of Tsc1, Tsc2, and scylla or charybdis in the eye does not further reduce the small eye phenotype induced by coexpression of Tsc1 and Tsc2 on their own. The absence of an additive growth effect upon loss of Tsc2, scylla, and charybdis or overexpression of Tsc1/2 and scylla or charybdis suggests that they function in the same pathway. These results are consistent with the idea that Scylla and Charybdis act upstream of the TSC complex. This conclusion is further supported by the fact that neither a Rheb-dependent bulging eye phenotype nor organismal lethality could be suppressed by scylla/charybdis coexpression (Reiling, 2004).

scylla and charybde are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation

Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).

The foundation for the current study was a survey of RNA expression patterns by automated whole-mount RNA hybridization in situ. This screening protocol led to the identification of scyl as a dorsally restricted transcript in blastoderm stage embryos. Based on its spatial and temporal expression properties, which represent a subset of the dpp expression pattern, it was postulated that scyl is a transcriptionally regulated target of the Dpp signaling cascade that specifies early embryonic dorsal fates. To test this hypothesis, scyl transcript distributions were compared in wild-type and dorsoventral patterning mutant embryos. Fate determining genes functioning downstream of the ventral morphogen Dorsal (Dl) and/or the dorsal morphogen Dpp are expected to exhibit altered expression patterns in Dl-deficient/Dpp-constitutive and Dl-constitutive/Dpp-deficient mutant embryos. Indeed, this was found to be the case for the scyl transcript. Transcription of scyl in wild-type and mutant embryos was similar to that of zen, the best characterized target of Dpp, indicating that Dpp is both necessary and sufficient for scyl transcription. In blastoderm stage embryos, zen is expressed at the posterior pole and in the dorsal-most 40% of the developing embryo. In like fashion, scyl is expressed at the posterior pole and is dorsally restricted. scyl transcripts, however, are confined to the subset of zen-expressing cells that correspond to the dorsal-most 10% of the developing embryo. Both scyl and zen are ubiquitously expressed in dorsalized embryos derived from dl mutant females and in which the dorsal morphogen Dpp is ubiquitously expressed. Neither scyl nor zen is expressed in the abdominal regions of ventralized embryos, which are derived from cactus (cact) mutant females and which lack zygotic Dpp (Scuderi, 2006).

Two observations that led to an examination of the regulation of scyl and chrb by downstream components of the Dpp signaling cascade are: (1) scyl, like zen, is a transcriptionally regulated target of Dpp-mediated signaling and (2) scyl, chrb and zen are expressed in overlapping dorsal fields. The gene encoding the divergent homeobox transcription factor Zen is itself activated by Dpp-mediated signaling in dorsal domains of the blastoderm stage embryo. In zen mutant embryos, dorsally restricted scyl and chrb transcripts are lost, placing both genes downstream of the Zen transcriptional effector of Dpp-mediated signal transduction (Scuderi, 2006).

In addition to the dorsal field of scyl and chrb expression in early embryos, segmental expression along the anteroposterior axis suggests that scyl and chrb may also be sensitive to regulation by homeobox genes other than zen. Both scyl and chrb transcripts are localized in anteroposteriorly segmented patterns in ventral regions of the blastoderm and in the three thoracic segments of stage 13 embryos. In stage 13 embryos, genes of the bithorax complex (BX-C) repress expression of target genes in abdominal segments, restricting their expression to the thorax. The expression of scyl and chrb was examined in BX-C mutant embryos: expansion was observed of thoracic expression into abdominal segments of mutant embryos, thereby placing scyl and chrb downstream of the BX-C homeobox transcription factors, Ubx, Abd-A and/or Abd-B. Consistent with this observation is the finding (Chauvet, 2000) that Ubx binds to regulatory regions of both scyl and chrb (Scuderi, 2006).

Finally, as an extension of the observation that scyl and chrb are downstream targets of homeobox genes acting in distinct signaling pathways, bioinformatic tools were used to identify conserved elements of the scyl and chrb promoters in D. melanogaster and D. pseudoobscura. In a computational cross-genome comparison utilizing algorithms based on both Gibbs sampling and Artificial Neural Networks, one 24-nucleotide motif and three 16-nucleotide motifs were identified that are conserved in the promoter regions of both genes in both species. Motif 1 was found much more frequently than expected for a random sequence, suggestive of its role as a generic transcription factor binding site or regulatory element. Motifs 2-4 were observed much less frequently, and this statistic is interpreted to be indicative of more specific roles for motifs 2-4 in the co-regulation of scyl and chrb. With respect to the identification of defined binding sites, neither motif 1 nor motif 2 corresponds to any canonical transcription factor binding sites listed in the TRANSFAC transcription factor database. Motif 3, however, is GC-rich and contains canonical binding sites for two widely used transcriptional activators: SP1 (GCCCGCCCCCC) and AP2 (GCCCGCGGC). More notable, however, is the characterization of motif 4, which was found only twice in each of the Drosophila genomes (in the promoter regions of scyl and chrb). In scans of the TRANSFAC database, it was found that motif 4 harbors a 10-bp canonical binding site for Zen (ATTTAAATGA) (Scuderi, 2006).

Scylla decreases S6K but not PKB activity

To test whether the placement of Scylla between PKB and TSC can be corroborated biochemically, the effect of scylla overexpression on PKB and S6K activity was examined. PKB activity of adult female heads overexpressing scylla or charybdis was tested in conjunction with PKB and PDK1 under control of the GMR-Gal4 enhancer. The same experimental setup has previously been used to demonstrate that PDK1 increases PKB activity. Total fly head protein was extracted and PKB activity was assayed by incorporation of 32P-labeled phosphate into a synthetic PKB substrate (Crosstide, CT). Although scylla/charybdis overexpression substantially suppresses the PKB/PDK1-induced bulging eye phenotype, PKB activity is not reduced in these eyes. Moreover, PKB activity is also unaffected in a scylla-/- background (Reiling, 2004).

These results are consistent with the placement of Scylla downstream of PKB. To test the effect of Scylla on S6K activity, second instar larvae expressing scylla under the control of Act5C-Gal4 were collected, and larval extracts were assayed for S6K activity. On average, S6K activity was down-regulated by >50%. Although there may also be a slight reduction in total S6K protein levels, this effect cannot account for the much stronger reduction in S6K activity. Taken together with the genetic evidence, these results strongly support the argument that Scylla acts between PKB and TSC to regulate S6K activity. Furthermore, Brugarolas (2004) provide direct biochemical evidence that a functional TSC complex is required for RTP801/REDD1 to affect S6 phosphorylation. Altogether, these data indicate that Scylla functions upstream of TSC (Reiling, 2004).

FOXO-regulated transcription restricts overgrowth of Tsc mutant organs

FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. This study further characterizes this phenomenon. Tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species (Harvey, 2008).

To investigate mechanisms by which the TOR pathway controls tissue growth, transcriptional profiles were analyzed of tissue lacking Tsc1, which leads to hyperactivation of the TOR pathway and excessive growth. Eye-antennal imaginal discs from third instar Drosophila larvae were generated that were composed almost entirely of tissue derived from one of two different genotypes: Tsc1 or wild-type isogenic control. Three biologically independent first strand cDNA samples from each genotype were hybridized to microarray chips. Expression levels of 157 genes were elevated 1.5-fold or more, whereas 211 genes were repressed 1.5-fold or more when compared with control tissue. These genes have been implicated in diverse cellular functions including metabolism, membrane transport, stress response, cell growth, and cell structure (Harvey, 2008).

Observed transcriptional changes were validated for several genes using Drosophila gene-enhancer trap lines. The UAS-Gal4 system was used to activate the TOR pathway in a specific tissue domain by driving expression of Rheb under the control of the glass multiple reporter (GMR) promoter. Induction of astray (aay) and 4E-BP (both of which were found to be elevated in Tsc1 tissue by microarray analysis) were observed in the GMR expression domain (posterior to the morphogenetic furrow) when Rheb was misexpressed but were not induced when the negative control Gal4 gene was misexpressed. QPCR was also used to confirm expression changes observed in Tsc1 tissue for charybdis (chrb), scylla (scy), phosphoenolpyruvate carboxy kinase, 4E-BP, and aay (Harvey, 2008).

Intriguingly, several gene products whose expression was elevated in Tsc1 tissue have been implicated in tissue growth controlled by the insulin and TOR pathways, including 4E-BP, Chrb, and Scy. 4E-BP is a repressor of cap-dependent translation. Upon phosphorylation by TOR, 4E-BP dissociates from eIF4E, allowing assembly of the initiation complex at the mRNA cap structure, ribosome recruitment, and subsequent translation. Scy and Chrb, and their mammalian orthologues REDD1 and REDD2, inhibit insulin and TOR signaling in response to hypoxia and energy stress and restrict growth during Drosophila development. The finding that inhibitors of growth are highly expressed in Tsc1 tissue led to the hypothesis that such genes are transcriptionally induced as part of a feedback loop that restricts tissue growth under conditions of excessive TOR activity. Feedback loops are an important activity-modulating feature of many signaling pathways, including the TOR and insulin pathways (Harvey, 2008).

To examine the mechanism whereby transcription of growth inhibitors is induced in response to TOR hyperactivation, attempts were made to determine which transcription factors were responsible for their expression. One obvious candidate was FOXO, a member of the forkhead transcription factor family, which has a well-established role as an effector of insulin signaling. If FOXO has a role in inducing expression of negative regulators of growth in Tsc tissue, then expression of some of those genes should be elevated under conditions of increased FOXO activity. To investigate this hypothesis, the expression profiles were examined of Tsc1 LOF tissue and Drosophila S2 cells expressing FOXOA3, a mutant version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt. This analysis revealed that 25 genes were up-regulated 1.5-fold or greater in both Tsc1 LOF and FOXO GOF expression profiles, which represents a highly significant degree of overlap as determined by calculation of the hypergeometric distribution. A highly statistically significant P value strongly suggests that there is a functional overlap between these two datasets that cannot be explained by random variation (Harvey, 2008).

Interestingly, two genes previously implicated in tissue growth regulated by the insulin and TOR pathways 4E-BP and scy were elevated in both microarray experiments, whereas the chrb growth-inhibiting gene was not. Thus, a subset of genes elevated in Tsc1 tissue appears to respond to FOXO activity and was investigated further (Harvey, 2008).

4E-BP is a well-characterized FOXO target gene. To determine whether FOXO could directly activate transcription of genes that were elevated in Tsc1 tissue other than 4E-BP, focus was placed on scy and the phosphoserine phosphatase aay (one of the most highly elevated transcripts in each microarray experiment). scy and aay both possess consensus FOXO recognition elements (FREs) in their promoters comparable to those found in dInR and 4E-BP promoters. Therefore, whether these genes are bona fide FOXO targets was examined by measuring their expression in Drosophila S2 cells misexpressing FOXOA3 in the presence of insulin. aay and scy mRNAs were up-regulated 19.4- and 4.3-fold, respectively, relative to a control gene, actin, as determined by QPCR (Harvey, 2008).

Luciferase reporter assays in S2 cells were used to determine whether the aay promoter region containing putative FREs was sensitive to FOXO activity. Luciferase activity dependent on the aay promoter was strongly induced by FOXOA3. In addition, using in vitro band shift assays, it was demonstrated that FOXO directly binds to the aay promoter, indicating that FOXO likely activates expression of aay by directly binding to the FRE. Surprisingly, in parallel luciferase reporter assays, activation of the scy promoter by FOXO could not be demonstrated, despite the fact that strong binding of FOXO to the putative scy FRE was observed using in vitro band-shift assays. A possible explanation is that the scy-promoter construct lacked the minimal promoter elements required for transcription of luciferase (Harvey, 2008).

TOR pathway hyperactivation caused by Tsc deficiency has been shown to strongly repress activity of Akt. FOXO is normally inactivated by Akt-dependent phosphorylation, which restricts nuclear entry of FOXO and leads to its ubiquitin-dependent destruction. Therefore, in response to TOR pathway hyperactivation, it was predicted that reduced Akt activity would cause FOXO protein to accumulate. To examine this hypothesis, expression of FOXO protein was analyzed in mosaic Tsc1 imaginal discs. It was found that FOXO protein was markedly increased in Tsc1 clones when compared with neighboring wild-type tissue (Harvey, 2008).

In addition, FOXO protein appeared to be mostly nuclear in Tsc1 tissue and cytoplasmic in wild-type tissue. Consistent with this observation, nuclear localization of the mouse FOXO orthologue FOXO1 is observed in endothelial cells of Tsc2 mutant hemangiomas, whereas FOXO1 is mostly cytoplasmic in normal cells. FOXO mRNA levels are unchanged in Tsc1 tissue as determined by microarray analysis, which suggests that changes in translation or stability of FOXO protein account for its accumulation in Tsc1 tissue. The presence of increased FOXO protein in the nuclei of Tsc1 cells is consistent with the hypothesis that FOXO is responsible for increased expression of some of the growth inhibitors that are up-regulated in Tsc1 cells (Harvey, 2008).

To determine whether FOXO was necessary for transcriptional induction of genes that were elevated in Tsc1 tissue, QPCR analysis was used to measure 4E-BP, aay, and scy expression in Tsc1 and Tsc1-FOXO double mutant eye-antennal imaginal discs. Consistent with microarray analysis, increased expression of 4E-BP, aay, and scy was observed in Tsc1 tissue. In Tsc1-FOXO tissue, however, 4E-BP was expressed at approximately equivalent amounts as in wild-type tissue, whereas aay and scy expression was only partially reduced. This demonstrates that elevated expression of 4E-BP in Tsc1 tissue is dependent on the FOXO transcription factor and provides evidence that FOXO activity increases when the TOR pathway is hyperactivated. Expression of aay and scy appear to be partially dependent on FOXO but are likely stimulated by additional transcription factors in Tsc1 tissue (Harvey, 2008).

Next, attempts were made to determine whether FOXO is required to limit growth of tissues with increased TOR pathway activity. In addition, a potential role was examined for another transcription factor, HIF-1, for retardation of TOR-driven growth. HIF-1 is a dual-subunit transcription factor consisting of α and β subunits that functions in response to insulin/TOR signaling and drives transcription of the growth-inhibiting genes scy and chrb, both of which are elevated in Tsc1 tissue (Harvey, 2008).

Drosophila possesses several HIF-1α subunits and a sole HIF-1β subunit, tango (tgo), which partners with each HIF-1α subunit. If FOXO and/or HIF-1 are required to induce expression of genes that limit tissue growth when the TOR pathway is hyperactivated, one might predict that Tsc1-FOXO and/or Tsc1-tgo double mutant tissue would possess a greater capacity to grow than Tsc1 tissue alone. To test this hypothesis, the size was examined of Drosophila eyes comprised almost entirely of the following genotypes: control, tgo, FOXO, Tsc1, Tsc1-tgo, and Tsc1-FOXO. Mutant eyes were created by driving mitotic recombination of chromosomes bearing flipase recognition target (FRT) sites and the appropriate gene mutations, specifically in developing Drosophila eye-antennal imaginal discs. Eyes lacking either tgo or FOXO were approximately the same size as control eyes, whereas Tsc1 eyes were considerably larger. Tsc1-tgo double mutant eyes did not exhibit a further increase in size, which suggests that HIF-1 is not required to inhibit tissue growth in response to Tsc1 loss. In contrast, Tsc1-FOXO double mutant eyes were substantially larger than Tsc1 eyes. This finding is particularly significant in light of the finding that eyes lacking FOXO were indistinguishable in size from wild-type eyes. Thus, it appears that FOXO is normally dispensable for control of eye size, but when growth control is altered by virtue of increased TOR activity, FOXO partially offsets the increased tissue growth. These findings are consistent with observations that FOXO protein accumulates in Tsc1 tissue and that transcriptional profiles of FOXO GOF and Tsc1 LOF cells overlap significantly (Harvey, 2008).

Because individual components of the insulin and TOR pathways are highly conserved among eukaryotes, important regulatory mechanisms that control tissue growth via these pathways are also likely to be conserved. To investigate this idea, transcriptional control was analyzed of mouse orthologues of genes that were elevated in D. melanogaster Tsc1 tissue. Initially, Northern blotting analysis was performed on Tsc2 primary mouse embryonic fibroblasts (MEFs; derived on a p53 background to overcome premature senescence induced by Tsc2 loss). It is reasonable to predict that transcriptional changes that occur because of loss of either Tsc1 or Tsc2 should be very similar because TSC1 and TSC2 function together in an obligate fashion, and mutation of either gene leads to almost indistinguishable phenotypes. It was found that several gene expression changes observed in Drosophila Tsc1 tissue are conserved in Tsc2 MEFs (Harvey, 2008).

The homologues of aay, heat shock protein (hsp) 23, scy, and chrb (PSPH, hsp 27, REDD1, and REDD2, respectively) were all significantly up-regulated in Tsc2 MEFs when compared with control MEFs and expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. To demonstrate that these expression changes were a specific consequence of Tsc2 loss, Tsc2 expression was reconstituted in Tsc2 null cells, which substantially suppressed mammalian TOR activity and expression of these genes. Interestingly, expression of phosphoenolpyruvate carboxy kinase and 4E-BP1/2 was not altered between wild-type and Tsc2 cells, which might reflect tissue- or species-specific differences in the transcriptome of Drosophila epithelial cells and MEFs (Harvey, 2008).

To determine whether the mode of transcription of these genes was also conserved in mammals, expression was analyzed of the scy homologue REDD1. Like scy, mammalian REDD1 orthologues possess a putative consensus FRE within their proximal promoters. Cotransfection of a version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt (TM-FKHRL-1) induced robust activation of a mouse REDD1 reporter construct in primary MEFs. To determine whether induction was mediated through the identified FRE, a mutant reporter was created lacking this sequence. Deletion of the REDD1 FRE consistently reduced FOXO-mediated induction of the REDD1 promoter. Finally, to directly assess whether FOXO-dependent transcription was activated in mammalian cells lacking Tsc2, activity of the REDD1 promoter reporter or the corresponding mutant FRE reporter was examined in wild-type and Tsc2 MEFs. As predicted, the wild-type REDD1 promoter exhibited robust activation in Tsc2 cells compared with wild-type cells, and this activation was substantially reduced by deletion of the FRE. Together, these findings provide evidence that transcriptional changes resulting from Tsc1/Tsc2 deficiency are conserved in diverse species (Harvey, 2008).

This study has identified of an evolutionary conserved transcriptional program important for restricting tissue overgrowth driven by excessive activation of the TOR pathway. The FOXO transcription factor plays a key role in this transcriptional response, likely by stimulating expression of several growth inhibitory genes. Thus, although the requirement for FOXO in restricting growth under normal development conditions appears dispensable, this is no longer the case under conditions of excessive TOR activation. These findings have important implications for cancer syndromes that arise because of inappropriate TOR pathway activation, such as the human hamartomatous syndrome, tuberous sclerosis. TOR-dependent feedback inhibition is thought to contribute to the benign nature of Tsc1 and Tsc2 tumors (Ma, 2005; Manning, 2005). Conceivably, inactivating mutations in FOXO family transcription factors and/or FOXO target genes that possess growth-inhibiting properties could promote further growth in normally benign Tsc1 and Tsc2 tumors (Harvey, 2008).



scylla and charybdis are both expressed during embryogenesis in dynamic, partially overlapping patterns. In contrast to the broadly expressed scylla mRNA, charybdis transcripts are predominantly restricted to neurons of the CNS and PNS as assessed by mRNA in situ hybridization. During late larval stages scylla mRNA is uniformly expressed without apparent tissue-specific distribution, whereas charybdis mRNA expression could not be detected in third instar imaginal discs (Reiling, 2004).


In order to identify novel genes involved in growth regulation by the Inr/TOR pathway, an EP overexpression screen was performed using a double-headed EP element. A genetically sensitized system involving coexpression of PKB and PDK1 (achieved by using EP837 that drives endogenous PDK1) was used in the eye; this leads to a big eye phenotype. Pilot experiments demonstrated that overexpression of PTEN or a dominant-negative version of the catalytic subunit of Drosopohila PI3K, Dp110, were not able to suppress the PKB/PDK1-dependent phenotype. Thus, the screening system is likely to identify components acting downstream of or in parallel to PKB/PDK1. For example, coexpression of Tsc1/Tsc2 strongly suppresses the phenotype of the tester flies (Reiling, 2004).

Two EP insertions (EP22.1, hereafter named EPscy, and EP9.85) were identified in the scylla locus as suppressors of the PKB/PDK1 bulging eye phenotype. BLASTP search with the Scylla amino acid sequence revealed another homologous protein encoded in the Drosopohila genome termed Charybdis. scylla (scy, CG7590) and charybdis (char, CG7533) are separated by ~232 kb of genomic DNA. Their gene products share a high degree of homology (38% identity, 49% similarity), suggestive of a gene duplication event. Whether charybdis overexpression would behave similarly to scylla was examined in the PKB/PDK1 overexpression assay using EP1035 (hereafter named EPchar). Indeed, the big eye phenotype of the tester system is also suppressed by EPchar. UAS transgenes with either the scylla or charybdis cDNA recapitulate the suppression phenotype of the corresponding EP element. Coexpression of scylla and charybdis further ameliorates the suppression phenotype to a nearly wild-type situation. Notably, scylla or charybdis overexpression on their own using a panel of different eye/wing Gal4 drivers reduces adult organ size. Coexpression of the caspase inhibitors p35 or DIAP1 does not rescue the small eye phenotype induced by expression of either scylla or charybdis in the eye. Moreover, no elevated cell death in eye imaginal discs overexpressing scylla/ charybdis under control of the GMR-Gal4 driver was observed by acridine orange staining. This suggests that apoptosis is not the cause for the eye size reduction. Thus, scylla and charybdis overexpression antagonizes the growth-promoting effects of PKB/PDK1 and is sufficient to negatively regulate growth (Reiling, 2004).

To investigate the function of Scylla and Charybdis in more detail, loss-of-function mutations were generated in both genes and the analysis was complemented with overexpression studies. Partial scylla deletions were obtained by imprecise excisions of EP9.85, which is integrated in the scylla open reading frame (ORF) and therefore already represents a scylla allele (hereafter named scyEP9.85). For charybdis, a local hop strategy of EPchar was used to obtain char180, constituting a new EP insertion (EP1035*) in the charybdis 5'-untranslated region (UTR) plus the original EPchar. Quantitative real time-PCR showed that in char180 homozygotes, charybdis mRNA expression is decreased to ~25% of wild-type levels. It is assumed that this charybdis allele is a strong hypomorph, since mRNA levels were only slightly more reduced (to 23% or 15%, respectively) when RNA was extracted from flies heterozygous for char180 over either one of two independent deficiencies uncovering charybdis and scylla (Reiling, 2004).

All scylla mutant combinations and the char180 homozygotes are viable and fertile without apparent mutant phenotype. scylla and char180 mutant animals have the same weight as control flies. Measurement of wing size and hair density in the adult wing of scylla mutants revealed no differences in cell size and cell number as compared to control animals. scylla loss-of-function clones were created in imaginal disks using FLP/FRT-mediated mitotic recombination to test the effect on growth properties of the mutant tissue. One would expect a growth advantage of cells in clones lacking a bona fide negative growth regulator, as is the case for PTEN. However, larval scylla mutant clones were the same size as their wild-type sister clones. Likewise, clones obtained in adult eyes revealed no increase in cell size of scylla or char180 mutant ommatidia. Thus, loss of Scylla or Charybdis function is dispensable for growth under normal conditions. It is conceivable that Scylla and Charybdis act in a redundant manner. Therefore, it was important to create scylla charybdis double mutants (Reiling, 2004).

char180 were combined with three of scylla alleles by meiotic recombination. All double-mutant scylla charybdis combinations produced viable adult flies. Weight analysis of heteroallelic scylla charybdis flies demonstrated that simultaneous loss of Scylla and Charybdis significantly increases body weight. Consistent results were obtained by combining one copy of scyEP9.85/31/113 char180 and the deficiency Df(3L)vin4 uncovering scylla and charybdis. Mutant females were on average 6%-23% and males 9%-17% heavier than control flies. Conversely, ubiquitous scylla/charybdis overexpression using the Act5CGal4 driver generated flies that are decreased in size and weight (15.1% using EPscy and 14.3% using UAS-char#53). The majority of scylla/charybdis-overexpressing flies eclosed with a minor delay (0.5-1 d) (Reiling, 2004).

To assess whether charybdis/scylla overexpression affects cell size, scylla and charybdis gain-of-function flip-out clones in the eye, marked by the absence of the red pigment, were generated. A moderate reduction in cell size was observed in cells overexpressing scylla or charybdis. Ommatidia overexpressing scylla or charybdis exhibit no patterning defects. In contrast, simultaneous removal of Scylla and Charybdis in clones of photoreceptor cells resulted in slightly enlarged cells. Consistently, when most of the head capsule and the eyes were made homozygous by means of the eyflp/FRT system in an otherwise heterozygous mutant background, a mild big head phenotype was generated in the double mutant but not in either single mutant (Reiling, 2004).

The gain-of-function clonal analysis in the eye showed that cell size is reduced upon forced scylla/charybdis expression, but it did not address the question whether cell number is affected. To clarify this issue, the wing size and cell number of flies that ubiquitously overexpress scylla were measured. The insect wing is a double-layered epithelial structure, and each cell in the wing secretes a single hair (trichome). Therefore, by counting the number of trichomes per defined area, hair density can be taken as a measure for cell number. Overall wing size was reduced by ~25% in males or ~15% in females overexpressing scylla. By extrapolating the number of cells per measured area, it was found that cell size is decreased by ~29% in males and ~21% in females. It is concluded that the size reduction brought about by overexpressing scylla is caused by a reduction in cell size. Cell density is even slightly increased (5.8% in males, 7.6% in females). The data show that Scylla and Charybdis have a growth-inhibitory role and that they share some functional redundancy (Reiling, 2004).

Zinke (2002) performed a whole-genome DNA microarray analysis of 2-d-old larvae (48 h AEL) grown on normal food that were subsequently subjected to a starvation regime for different time periods. Under these conditions, upon 12 h of starvation, scylla and charybdis expression were found to be on average 6.3 times and 4.6 times up-regulated, respectively. Therefore, the effects of starvation were tested on the viability of scylla and charybdis mutants, as well as on flies overexpressing both genes by exposing adult flies to a water-only diet. Various scylla heteroallelic combinations did not show elevated susceptibility to starvation. However, char180 mutants lived significantly shorter lives than control flies, suggesting that Charybdis has a protective effect for the animal under nutrient-deprived conditions. Strikingly, forced expression of scylla/charybdis extends mean life span by up to twofold. Lipid and glycogen content of these flies were analyzed to see whether energy stores were altered. Indeed, flies overexpressing scylla and/or charybdis showed significantly elevated lipid levels. Glycogen levels were also measured but no statistically significant changes could be detected, although there was a tendency toward increased glycogen content (Reiling, 2004).

scylla and charybde are required for head involution in Drosophila

The similarities in the expression profiles of scyl and chrb (both spatially and temporally) led to the hypothesis that the two closely related genes share overlapping functions in the developing embryo. No mutant alleles of chrb exist to test this hypothesis. With respect to scyl, the EY03729 fly line harbors a P-element insertion 253 bp from the scyl transcription start site; flies homozygous for the EY03729 insertion are, however, fully viable and exhibit no detectable phenotypes. Thus, to directly assess the functional contributions of scyl and chrb to embryonic development, RNA interference (RNAi) techniques were used to disrupt expression of one or both genes and the effects of interference were monitored throughout embryogenesis (Scuderi, 2006).

Injection of either scyl or chrb dsRNA resulted in no observable phenotype. Injected embryos were monitored for alterations in their developmental program throughout gastrulation, as well as by cuticle assay. Absence of an observable phenotype was reproducible using up to 5 μM dsRNA, 10-fold more than the amount required to produce a phenotype in engrailed RNAi control experiments. In contrast, coinjection of both scyl and chrb dsRNAs (either dorsally or posteriorly) led to defects in head involution that were clearly visible at the level of the larval cuticle. The scyl chrb co-RNAi embryonic lethal phenotype is reminiscent of that exhibited by homozygotes harboring hypomorphic mutations in the Zen transcription factor that is required to pattern the embryonic head ectoderm. Even more notably, the scyl chrb co-RNAi embryonic lethal phenotype is identical to that exhibited by embryos harboring amorphic mutations in the Hid (Head involution defective) apoptotic activator that functions morphogenetically to sculpt the embryonic head (Scuderi, 2006).

The scyl chrb co-RNAi phenotype was next reproduced in genetic studies exploiting deficiencies that removed one or both genes and thereby confirmed the specificity of the RNAi-induced phenotype. Three deficiency lines that mapped cytologically to the region of 68B–68C were PCR-genotyped with respect to scyl and chrb. Whereas Df(3L)lxd6 deletes scyl only, Df(3L)vin4 and Df(3L)vin2 delete both genes. Although these deficiencies span multiple genes, embryonic lethal phenotypes will correspond to the loss-of-function phenotype of the earliest acting gene(s) uncovered by the deficiency -- in this case, presumably scyl and/or chrb (Scuderi, 2006).

The cuticle phenotypes observed in the deficiency lines are entirely consistent with results from RNAi assays. Cuticles derived from Df(3L)lxd6 homozygotes develop normally and exhibit no visible defects, consistent with the determination that scyl alone is deleted in this deficiency. In contrast, cuticles derived from Df(3L)vin4 and Df(3L)vin2 homozygotes, in which both scyl and chrb are missing exhibit clear defects in head involution, with the head structures compressed anteriorly. Structures that comprise the head skeleton include the mouth hooks, which are the most anterior structures, as well as the chitinous labrum and the episomal sclerites, which connect the mouth hooks to the pigmented structures of the cephalopharyngeal skeleton (CPS). In scyl chrb double mutants, mouth hooks are present, but the labrum and epistomal sclerites are missing; errors in head involution result in at least part of the CPS remaining at the surface of the embryo (Scuderi, 2006).

Finally, because deficiency and RNAi analyses revealed a requirement for both scyl and chrb in normal head development, whether these genes, like Hox genes, compensate for one another in a dose-dependent manner was assayed. In this regard, the effect of removing a total of three copies of the genes was examined. Cuticles derived from Df(3L)lxd6/Df(3L)vin4 transheterozygotes (scyl+/scyl chrb) revealed defects in head involution, indistinguishable from those observed in cuticles derived from embryos lacking all four copies of the sister genes. Notably, only 10 genes, none of which has a previously characterized embryonic lethal phenotype, map to the Df(3L)lxd6/Df(3L)vin4 region of overlap. These data demonstrate that quantitative changes in gene pair activity are of greater consequence than qualitative differences between the two gene products (Scuderi, 2006).

Based upon the observations that: (1) simultaneous loss of scyl and chrb function leads to a hid-analogous, cell death defective phenotype and (2) scyl and chrb are homologous to the mammalian apoptotic gene RTP801, it was postulated that the scyl and chrb gene products have pro-apoptotic functions in the embryonic Drosophila head. Two lines of experimentation were employed to test this hypothesis. (1) hid expression was examined in scyl chrb double mutant embryos in situ. The scyl and chrb gene products do not function as transcriptional modulators of hid since hid transcription is unaffected in scyl chrb double mutant embryos. (2) A Caspase-3 activity assay was employed to monitor apoptosis in wild-type and scyl chrb double mutant embryos. Activated Caspase-3 has been used previously to specifically label apoptotic cells in Drosophila. Anti-Caspase-3 staining mirrors cell death patterns previously defined by acridine orange and TUNNEL assays in the Drosophila embryo and pupal retina. In this study, dying cells expressing activated Caspase-3 were evident in the head and the nervous system of 95% of embryos derived from matings of Df(3L)vin4/twi:GFP heterozygotes 0-8 h AEL (n = 278). When GFP screening was used to enrich for similarly staged mutant embryos, it was noted that Caspase-3 activity was greatly diminished in mid-stage scyl chrb double mutants. By 8 AEL, 75% of the mutant-enriched population was caspase-negative, in contrast to the unselected population in which only 8% of the embryos were found to be caspase-negative. No gross differences in Caspase-3 activity were found prior to the onset of germ band retraction and head involution. Since cleaved Caspase-3 is a key executioner (and hence marker) of apoptosis, these data support the hypothesis that Scylla and Charybde have pro-apoptotic roles in Drosophila head involution. More generally, Scylla and Charybde likely function as essential death activators in Drosophila since Caspase-3 activation in scyl chrb double mutants is disrupted in the nervous system as well as in the head. The scylla and charybde gene products are not, however, sufficient for cell death since (1) immunostains reveal wild-type patterns of Caspase-3 activation in embryos derived from dl mutant mothers and in which expression of scylla and charybde is greatly expanded and (2) neither scyl nor chrb (alone or in combination) can mimic hid-induced apoptosis in cultured Cos or Hela cells (Scuderi, 2006).

Several lines of evidence indicate that Scylla and Charybde function in the Hid-mediated cell death pathway. (1) A previous phenotypic analysis of scyl chrb mutants revealed their essential roles in regulating cell death in the developing Drosophila eye (Reiling, 2004). Loss-of-function studies have similarly revealed a requirement for Hid in modulating cell death events in early and late stages of Drosophila eye development. (2) In this study, which relied upon deficiencies and RNAi methodologies to generate scyl chrb null double mutants, an earlier developmental requirement for the scyl and chrb gene products was documented. scyl chrb double mutants suffer an embryonic lethality that is associated with defects in the morphogenetic process of head involution. Drosophila homozygous for loss-of-function hid alleles similarly suffer an embryonic lethality and exhibit signature defects in head involution. (3) Molecular characterization of the embryonic lethality in scyl chrb double mutants revealed that Caspase-3 activation is disrupted not only in the morphogenetically aberrant head, but in the CNS as well. In Drosophila, Hid induces apoptosis in midline glia cells failing to activate the EGFR signaling cascade. Together, the significant homologies of scyl and chrb to the mammalian RTP801 gene product that functions as an apoptotic factor in mammalian cell culture systems, as well as the scyl chrb embryonic and eye phenotype studies establish redundant roles for scyl and chrb in Hid-mediated cell death in both embryonic and post-embryonic stages of the Drosophila life cycle (Scuderi, 2006).

Each of the three cell death proteins, hid, rpr and grim, has been implicated in apoptotic events defining segmental boundaries and/or neuronal fates in the CNS, albeit in different paradigms. In the CNS, specificity in neuronal apoptosis is achieved via differential expression of the BX-C Hox gene abd-A, which prevents neuronal apoptosis in posterior segments. Viewed from this perspective, the finding that the Zen and BX-C Drosophila Hox gene products regulate transcription of the scyl and chrb pro-apoptotic genes (and thereby potentially sculpt head and segment boundaries during development) is reminiscent of the Deformed Drosophila Hox protein functioning as a transcriptional activator of the rpr cell death gene. Together, these studies strengthen the idea that Hox-gene-dependent induction of cell death is a general phenomenon in Drosophila (Scuderi, 2006).

Intriguingly, the pro- and anti-apoptotic roles of the Zen and BX-C Homeobox transcription factors in Drosophila embryogenesis correspond to their activation and repression effects on scyl and chrb gene expression. In this regard, scyl, chrb and cell death are activated by Zen in dorsal domains of the developing embryo, whereas ventrally scyl, chrb and cell death are repressed by one or more of BX-C gene products. Hence, in addition to the pro-apoptotic role of Zen, there is evidence for an anti-apoptotic role for the BX-C gene product(s) and in flies as in mouse related transcription factors function in context-specific fashion (Scuderi, 2006).

As a final point, both TGF-β and BMP mammalian members of the TGF-β cytokine superfamily have been documented to induce cell death in numerous developmental contexts. Along these same lines, previous reports in Drosophila have suggested a link between Dpp and cell death but have stopped short of designating this link as direct. Based on molecular and genetic evidence, it is suggested that the Drosophila pro-apoptotic scyl and chrb gene products serve as direct links between Dpp/Zen-mediated patterning and differentiation, in this case, cell death. Thus, in Drosophila as in vertebrates, cytokines of the TGF-β superfamily control both cell death and cell proliferation within the contexts of their cellular environments (Scuderi, 2006).

Given the importance of cell death regulation in development and disease, it is likely that there are several mechanisms by which cell death can be regulated, and, in like fashion, several nodes where independent regulatory pathways may in specific contexts converge. With respect to members of the RTP801 family of apoptotic factors, evidence points to at least two triggers of regulation: cell death can be a pathologic response to stresses such as hypoxia (as is the case for mammalian RTP801) or cell death can be a developmental response to a spatially and temporally restricted cell signaling pathway, such as the Dpp/TGF-β cytokine-mediated signaling pathway (as is the case for Drosophila Scylla and Charybde). Within the context of pathway convergence nodes, it is particularly notable that several reports document cross-talk between the HIF-1 and TGF-β pathways in regulating gene expression and cell death, and thus it is possible that the RTP801/Scylla/Charybde death effectors represent a point of convergence between these two death activating pathways. Consistent with this model is the demonstration that scyl and chrb are hypoxia-inducible in Drosophila (Reiling, 2004). Viewed from this perspective, the genetically defined roles of Scylla and Charybde as pro-apoptotic effectors establish a clear basis for future genetic and biochemical characterization of the mechanism by which activation of cell death programs might occur via Dpp/TGF-β-mediated signaling (Scuderi, 2006).


Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). A novel HIF-1-responsive gene, designated RTP801, has been identified and cloned. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H2O2-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases (Shoshani, 2002).

REDD1 has been identified as a novel transcriptional target of p53 induced following DNA damage. During embryogenesis, REDD1 expression mirrors the tissue-specific pattern of the p53 family member p63, the most ancient family member most closely related to the single gene present in Drosophila, and TP63 null embryos show virtually no expression of REDD1, which is restored in mouse embryo fibroblasts following p63 expression. In differentiating primary keratinocytes, TP63 and REDD1 expression are coordinately downregulated, and ectopic expression of either gene inhibits in vitro differentiation. REDD1 appears to function in the regulation of reactive oxygen species (ROS): TP63 null fibroblasts have decreased ROS levels and reduced sensitivity to oxidative stress, which are both increased following ectopic expression of either TP63 or REDD1. Thus, REDD1 encodes a shared transcriptional target that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation (Ellisen, 2002).

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. This study shows that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia (Brugarolas, 2004).

Glucocorticoid hormones induce apoptosis in lymphoid cells. This process requires de novo RNA/protein synthesis. A novel dexamethasone-induced gene designated dig2 has been identified and cloned. Using Affymetrix oligonucleotide microarray analysis of approximately 10,000 genes and expressed sequence tags, it was found that the expression of dig2 mRNA is significantly induced not only in the murine T cell lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes following dexamethasone treatment. This result was confirmed by Northern blot analysis. The induction of dig2 mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor since it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Furthermore, it is demonstrated that dig2 is a novel stress response gene, since its mRNA is induced in response to a variety of cellular stressors including thapsigargin, tunicamycin, and heat shock. In addition, the levels of dig2 mRNA are up-regulated after treatment with the apoptosis-inducing chemotherapeutic drug etoposide. Though the function of dig2 is unknown, dig2 appears to have a pro-survival function, because overexpression of dig2 reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced apoptosis (Wang, 2003).

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. The stress response REDD1 gene has been identified as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, because regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway (Sofer, 2005).

Cancer cells frequently evade apoptosis during tumorigenesis by acquiring mutations in apoptotic regulators. Chronic activation of the PI 3-kinase-Akt pathway through loss of the tumor suppressor PTEN is one mechanism by which these cells can gain increased protection against apoptosis. REDD1 (RTP801) can act as a transcriptional downstream target of PI 3-kinase signaling in human prostate cancer cells (PC-3). REDD1 expression is markedly reduced in PC-3 cells treated with LY294002 (LY) or Rapamycin and strongly induced under hypoxic conditions in a hypoxia-inducible factor-1 (HIF-1)-dependent manner. Loss of function studies employing antisense molecules or RNA interference indicate that REDD1 is essential for invasive growth of prostate cancer cells in vitro and in vivo. Reduced REDD1 levels can sensitize cells towards apoptosis, whereas elevated levels of REDD1 induced by hypoxia or overexpression desensitize cells to apoptotic stimuli. Taken together these data designate REDD1 as a novel target for therapeutic intervention in prostate cancer (Schwarzer, 2005).

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an essential role in cell growth control. mTOR stimulates cell growth by phosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by a wide variety of cellular signals, including mitogenic growth factors, nutrients, cellular energy levels, and stress conditions. Recent studies have proposed several mechanisms to explain how mTOR is regulated by growth factors and cellular energy levels. However, little is known as to how mTOR is regulated by stress conditions. Two stress-induced proteins, RTP801/Redd1 and RTP801L/Redd2, potently inhibit signaling through mTOR. The data support that RTP801 and RTP801L work downstream of AKT and upstream of TSC2 to inhibit mTOR functions. These results add a new dimension to mTOR pathway regulation and provide a possible molecular mechanism of how cellular stress conditions may regulate mTOR function (Corradetti, 2005).

Hypoxia induces rapid and dramatic changes in cellular metabolism, in part through inhibition of target of rapamycin (TOR) kinase complex 1 (TORC1) activity. Genetic studies have shown the tuberous sclerosis tumor suppressors TSC1/2 and the REDD1 protein to be essential for hypoxia regulation of TORC1 activity in Drosophila and in mammalian cells. The molecular mechanism and physiologic significance of this effect of hypoxia remain unknown. This study demonstrates that hypoxia and REDD1 [also know as RTP801/Dig1/DDIT4, a member of a gene family that includes its paralog REDD2 (RTP801L, DDIT4L) and the Drosophila orthologs Scylla and Charybdis] suppress mammalian TORC1 (mTORC1) activity by releasing TSC2 from its growth factor-induced association with inhibitory 14-3-3 proteins. Endogenous REDD1 is required for both dissociation of endogenous TSC2/14-3-3 and inhibition of mTORC1 in response to hypoxia. REDD1 mutants that fail to bind 14-3-3 are defective in eliciting TSC2/14-3-3 dissociation and mTORC1 inhibition, while TSC2 mutants that do not bind 14-3-3 are inactive in hypoxia signaling to mTORC1. In vitro, loss of REDD1 signaling promotes proliferation and anchorage-independent growth under hypoxia through mTORC1 dysregulation. In vivo, REDD1 loss elicits tumorigenesis in a mouse model, and down-regulation of REDD1 is observed in a subset of human cancers. Together, these findings define a molecular mechanism of signal integration by TSC1/2 that provides insight into the ability of REDD1 to function in a hypoxia-dependent tumor suppressor pathway (DeYoung, 2008).


Search PubMed for articles about Drosophila charybde and scylla

Brugarolas, J., et al. (2004). Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004 18(23): 2893-904. 15545625

Chauvet, S., Maurel-Zaffran, C., Miassod, R., Jullien, N., Pradel, J., Aragnol, D. (2000). Characterization of charybde and scylla, two paralogous target genes of Hox and cofactor proteins in Drosophila. GenBank, Direct Submission

Corradetti, M. N., Inoki, K. and Guan, K. L. (2005). The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. J. Biol. Chem. 280(11): 9769-72. 15632201

DeYoung, M. P., et al. (2008). Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling. Genes Dev. 22: 239-251. PubMed Citation: 18198340

Ellisen, L. W., et al. (2002). REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Mol. Cell 10: 995-1005. 12453409

Frei, C. and Edgar, B.A. (2004). Drosophila cyclin D/Cdk4 requires Hif-1 prolyl hydroxylase to drive cell growth. Dev. Cell 6: 241-251. 14960278

Ghavi-Helm, Y., Klein, F. A., Pakozdi, T., Ciglar, L., Noordermeer, D., Huber, W., Furlong, E. E. (2014) Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512(7512): 96-100. PubMed ID: 25043061

Harvey, K. F., et al. (2008). FOXO-regulated transcription restricts overgrowth of Tsc mutant organs. J. Cell Biol. 180(4): 691-6. PubMed Citation: 18299344

Kimura, N., Tokunaga, C., Dalal, S., Richardson, C., Yoshino, K., Hara, K., Kemp, B. E., Witters, L. A., Mimura, O. and Yonezawa, K. (2003). A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8: 65-79. 12558800

Ma, L., et al. (2005). Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression. Genes Dev. 19: 1779-1786. PubMed Citation: 16027168

Manning, B. D., et al. (2005). Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev. 19: 1773-1778. PubMed Citation: 16027169

Reiling, J. H. and Hafen, E. (2004). The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila. Genes Dev. 18(23): 2879-92. 15545626

Schwarzer R, Tondera, D., Arnold, W., Giese, K., Klippel, A. and Kaufmann, J. (2005). REDD1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase. Oncogene 24(7): 1138-49. 15592522

Scuderi, A., Simin, K., Kazuko, S. G., Metherall, J. E. and Letsou, A. (2006). scylla and charybde, homologues of the human apoptotic gene RTP801, are required for head involution in Drosophila. Dev. Biol. 291(1): 110-22. 1642334

Shoshani, T., Faerman, A., Mett, I., Zelin, E., Tenne, T., Gorodin, S., Moshel, Y., Elbaz, S., Budanov, A., Chajut, A., et al. (2002). Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol. Cell. Biol. 22: 2283-2293. 11884613

Sofer, A., Lei, K., Johannessen, C. M. and Ellisen, L. W. (2005). Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol. Cell. Biol. 25(14): 5834-45. 15988001

Wang, Z., Malone, M. H., Thomenius, M. J., Zhong, F., Xu, F. and Distelhorst, C. W. (2003). Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals. J. Biol. Chem. 278: 27053-27058. 12736248

Zinke, I., Schutz, C. S., Katzenberger, J. D., Bauer, M. and Pankratz, M. J. (2002). Nutrient control of gene expression in Drosophila: Microarray analysis of starvation and sugar-dependent response. EMBO J. 21: 6162-6173. 12426388

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date revised: 12 January 2018

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