RESUMEN
Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAA and HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.
Asunto(s)
Alelos , Proteínas de Drosophila , Drosophila melanogaster , Alas de Animales , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/genéticaRESUMEN
Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.
Asunto(s)
Proteínas de Drosophila , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Células Sanguíneas/metabolismo , Homeostasis , Serina/metabolismo , Treonina/metabolismoRESUMEN
Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel HΔCT allele by genome engineering. Phenotypically, HΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, we define the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the HΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Proteínas Represoras/genética , Proteínas de Drosophila/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Alelos , Receptores Notch/genética , Receptores Notch/metabolismo , Drosophila/genética , Cromatina/metabolismoRESUMEN
The primary role of Notch is to specify cellular identities, whereby the cells respond to amazingly small changes in Notch signalling activity. Hence, dosage of Notch components is crucial to regulation. Central to Notch signal transduction are CSL proteins: together with respective cofactors, they mediate the activation or the silencing of Notch target genes. CSL proteins are extremely similar amongst species regarding sequence and structure. We noticed that the fly homologue suppressor of hairless (Su(H)) is stabilised in transcription complexes. Using specific transgenic fly lines and HeLa RBPJKO cells we provide evidence that Su(H) is subjected to proteasomal degradation with a half-life of about two hours if not protected by binding to co-repressor hairless or co-activator Notch. Moreover, Su(H) stability is controlled by MAPK-dependent phosphorylation, matching earlier data for RBPJ in human cells. The homologous murine and human RBPJ proteins, however, are largely resistant to degradation in our system. Mutating presumptive protein contact sites, however, sensitised RBPJ for proteolysis. Overall, our data highlight the similarities in the regulation of CSL protein stability across species and imply that turnover of CSL proteins may be a conserved means of regulating Notch signalling output directly at the level of transcription.
Asunto(s)
Proteínas de Drosophila , Humanos , Animales , Ratones , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Co-Represoras/metabolismo , Receptores Notch/metabolismo , Fosforilación , Proteínas Represoras/metabolismo , Unión ProteicaRESUMEN
The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand-receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand-receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.
Asunto(s)
Drosophila melanogaster , Receptores Notch , Secretasas de la Proteína Precursora del Amiloide , Animales , Transducción de SeñalRESUMEN
Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor Suppressor of Hairless (Su(H)) bound to the Notch antagonist Hairless (H) and the general co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). The latter two are shared by different repressors from numerous pathways, raising the possibility that they are rate-limiting. We noted that the overexpression during wing development of H mutants HdNT and HLD compromised in Su(H)-binding induced ectopic veins. On the basis of the role of H as Notch antagonist, overexpression of Su(H)-binding defective H isoforms should be without consequence, implying different mechanisms but repression of Notch signaling activity. Perhaps excess H protein curbs general co-repressor availability. Supporting this model, nearly normal wings developed upon overexpression of H mutant isoforms that bound neither Su(H) nor co-repressor Gro and CtBP. Excessive H protein appeared to sequester general co-repressors, resulting in specific vein defects, indicating their limited availability during wing vein development. In conclusion, interpretation of overexpression phenotypes requires careful consideration of possible dominant negative effects from interception of limiting factors.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Alas de Animales/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Proteínas Co-Represoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Fenotipo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alas de Animales/anatomía & histología , Alas de Animales/metabolismoRESUMEN
Activation and repression of Notch target genes is mediated by transcription factor CSL, known as Suppressor of Hairless (Su(H)) in Drosophila and CBF1 or RBPJ in human. CSL associates either with co-activator Notch or with co-repressors such as Drosophila Hairless. The nuclear translocation of transcription factor CSL relies on co-factor association, both in mammals and in Drosophila. The Drosophila CSL orthologue Su(H) requires Hairless for repressor complex formation. Based on its role in transcriptional silencing, H protein would be expected to be strictly nuclear. However, H protein is also cytosolic, which may relate to its role in the stabilization and nuclear translocation of Su(H) protein. Here, we investigate the function of the predicted nuclear localization signals (NLS 1-3) and single nuclear export signal (NES) of co-repressor Hairless using GFP-fusion proteins, reporter assays and in vivo analyses using Hairless wild type and shuttling-defective Hairless mutants. We identify NLS3 and NES to be critical for Hairless function. In fact, HâNLS3 mutant flies match H null mutants, whereas HâNLS3âNES double mutants display weaker phenotypes in agreement with a crucial role for NES in H export. As expected for a transcriptional repressor, Notch target genes are deregulated in HâNLS3 mutant cells, demonstrating nuclear requirement for its activity. Importantly, we reveal that Su(H) protein strictly follows Hairless protein localization. Together, we propose that shuttling between the nucleo-cytoplasmic compartments provides the possibility to fine tune the regulation of Notch target gene expression by balancing of Su(H) protein availability for Notch activation.
Asunto(s)
Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Drosophila/genética , Femenino , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/genética , Fenotipo , Receptores Notch/genética , Factores de Transcripción/genética , Alas de Animales/crecimiento & desarrolloRESUMEN
Members of the Protein Kinase D (PKD) family are involved in numerous cellular processes in mammals, including cell survival after oxidative stress, polarized transport of Golgi vesicles, as well as cell migration and invasion. PKD proteins belong to the PKC/CAMK class of serine/threonine kinases, and transmit diacylglycerol-regulated signals. Whereas three PKD isoforms are known in mammals, Drosophila melanogaster contains a single PKD homolog. Previous analyses using overexpression and RNAi studies indicated likewise multi-facetted roles for Drosophila PKD, including the regulation of secretory transport and actin-cytoskeletal dynamics. Recently, involvement in growth regulation has been proposed based on the hypomorphic dPKDH allele. We have generated PKD null alleles that are homozygous viable without apparent phenotype. They largely match control flies regarding fertility, developmental timing and weight. Males, but not females, are slightly shorter lived and starvation sensitive. Furthermore, migration of pole cells in embryos and border cells in oocytes appears normal. PKD mutants tolerate heat, cold and osmotic stress like the control but are sensitive to oxidative stress, conforming to the described role for mammalian PKDs. A candidate screen to identify functionally redundant kinases uncovered genetic interactions of PKD with Pkcδ, sqa and Drak mutants, further supporting the role of PKD in oxidative stress response, and suggesting its involvement in starvation induced autophagy and regulation of cytoskeletal dynamics. Overall, PKD appears dispensable for fly development and survival presumably due to redundancy, but influences environmental responses.
Asunto(s)
Drosophila melanogaster/fisiología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Alelos , Animales , Femenino , Genotipo , Humanos , Masculino , Mutación , Estrés Oxidativo , Fenotipo , Recombinación Genética , Estrés Fisiológico , Transcripción GenéticaRESUMEN
During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins HFA, HLLAA and HWA. For in vivo analysis, UAS-lines HFA, HLLAA and HWA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the HLD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.
Asunto(s)
Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Mutación , Receptores Notch/metabolismo , Factores de Transcripción/genética , Animales , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
BACKGROUND: In Drosophila, the development of the fly eye involves the activity of several, interconnected pathways that first define the presumptive eye field within the eye anlagen, followed by establishment of the dorso-ventral boundary, and the regulation of growth and apoptosis. In Lobe (L) mutant flies, parts of the eye or even the complete eye are absent because the eye field has not been properly defined. Manifold genetic interactions indicate that L influences the activity of several signalling pathways, resulting in a conversion of eye tissue into epidermis, and in the induction of apoptosis. As information on the molecular nature of the L mutation is lacking, the underlying molecular mechanisms are still an enigma. RESULTS: We have identified Protein Kinase D (PKD) as a strong modifier of the L mutant phenotype. PKD belongs to the PKC/CAMK class of Ser/Thr kinases that have been involved in diverse cellular processes including stress resistance and growth. Despite the many roles of PKD, Drosophila PKD null mutants are without apparent phenotype apart from sensitivity to oxidative stress. Here we report an involvement of PKD in eye development in the sensitized genetic background of Lobe. Absence of PKD strongly enhanced the dominant eye defects of heterozygous L 2 flies, and decreased their viability. Moreover, eye-specific overexpression of an activated isoform of PKD considerably ameliorated the dominant L 2 phenotype. This genetic interaction was not allele specific but similarly seen with three additional, weaker L alleles (L 1 , L 5 , L G ), demonstrating its specificity. CONCLUSIONS: We propose that PKD-mediated phosphorylation is involved in underlying processes causing the L phenotype, i.e. in the regulation of growth, the epidermal transformation of eye tissue and apoptosis, respectively.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Epistasis Genética , Proteínas del Ojo/genética , Ojo/embriología , Ojo/metabolismo , Mutación , Proteína Quinasa C/genética , Animales , Ojo/ultraestructura , Estudios de Asociación Genética , Organogénesis/genética , FenotipoRESUMEN
BACKGROUND: DNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, we have generated transgenic flies allowing temporal and tissue-specific induction of mei-41. RESULTS: Overexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, we show that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, we further show that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo. CONCLUSION: We conclude that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.
Asunto(s)
Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Daño del ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Animales Modificados Genéticamente , División Celular , Drosophila melanogaster/efectos de la radiación , Fase G2RESUMEN
Throughout the animal kingdom, the Notch signalling pathway allows cells to acquire diversified cell fates. Notch signals are translated into activation of Notch target genes by CSL transcription factors. In the absence of Notch signals, CSL together with co-repressors functions as a transcriptional repressor. In Drosophila, repression of Notch target genes involves the CSL homologue Suppressor of Hairless (Su(H)) and the Notch (N) antagonist Hairless (H) that together form a repressor complex. Guided by crystal structure, three mutations Su(H)LL, Su(H)LLF and Su(H)LLL were generated that specifically affect interactions with the repressor H, and were introduced into the endogenous Su(H) locus by gene engineering. In contrast to the wild type isoform, these Su(H) mutants are incapable of repressor complex formation. Accordingly, Notch signalling activity is dramatically elevated in the homozygotes, resembling complete absence of H activity. It was noted, however, that heterozygotes do not display a dominant H loss of function phenotype. In this work we addressed genetic interactions the three H-binding deficient Su(H) mutants display in combination with H and N null alleles. We included a null mutant of Delta (Dl), encoding the ligand of the Notch receptor, as well as of Su(H) itself in our genetic analyses. H, N or Dl mutations cause dominant wing phenotypes that are sensitive to gene dose of the others. Moreover, H heterozygotes lack bristle organs and develop bristle sockets instead of shafts. The latter phenotype is suppressed by Su(H) null alleles but not by H-binding deficient Su(H) alleles which we attribute to the socket cell specific activity of Su(H). Modification of the dominant wing phenotypes of either H, N or Dl, however, suggested some lack of repressor activity in the Su(H) null allele and likewise in the H-binding deficient Su(H) alleles. Overall, Su(H) mutants are recessive perhaps reflecting self-adjusting availability of Su(H) protein.
Asunto(s)
Alelos , Proteínas de Drosophila/genética , Proteínas Represoras/genética , Animales , Proteínas Co-Represoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epistasis Genética , Homocigoto , Fenotipo , Receptores Notch/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
One of the key players in genome surveillance is the tumour suppressor p53 mediating the adaptive response to a multitude of stress signals. Here we identify Cyclin G (CycG) as co-factor of p53-mediated genome stability. CycG has been shown before to be involved in double-strand break repair during meiosis. Moreover, it is also important for mediating DNA damage response in somatic tissue. Here we find it in protein complexes together with p53, and show that the two proteins interact physically in vitro and in vivo in response to ionizing irradiation. In contrast to mammals, Drosophila Cyclin G is no transcriptional target of p53. Genetic interaction data reveal that p53 activity during DNA damage response requires the presence of CycG. Morphological defects caused by overexpression of p53 are ameliorated in cycG null mutants. Moreover, using a p53 biosensor we show that p53 activity is impeded in cycG mutants. As both p53 and CycG are likewise required for DNA damage repair and longevity we propose that CycG plays a positive role in mediating p53 function in genome surveillance of Drosophila.
Asunto(s)
Ciclina G/genética , Drosophila/genética , Inestabilidad Genómica/genética , Proteína p53 Supresora de Tumor/genética , Animales , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Drosophila/genética , Meiosis/genéticaRESUMEN
The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in Drosophila melanogaster. It binds to the Drosophila CSL homologue Suppressor of Hairless [Su(H)] and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the Hairless gene from Daphnia pulex and, hence, for the first time from a crustacean. Daphnia and Drosophila Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the Daphnia Hairless protein and the respective Drosophila interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between Daphnia Hairless and Drosophila Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during Drosophila development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from Daphnia and Drosophila are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Daphnia/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Daphnia/genética , Daphnia/crecimiento & desarrollo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Notch/antagonistas & inhibidores , Proteínas Represoras/química , Proteínas Represoras/genética , Homología de Secuencia , Transducción de Señal , Homología Estructural de Proteína , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Notch signalling activity governs cellular differentiation in higher metazoa, where Notch signals are transduced by the transcription factor CSL, called Suppressor of Hairless [Su(H)] in Drosophila. Su(H) operates as molecular switch on Notch target genes: within activator complexes, including intracellular Notch, or within repressor complexes, including the antagonist Hairless. Mass spectrometry identified phosphorylation on Serine 269 in Su(H), potentially serving as a point of cross-regulation by other signalling pathways. To address the biological significance, we generated phospho-deficient [Su(H)S269A] and phospho-mimetic [Su(H)S269D] variants: the latter displayed reduced transcriptional activity despite unaltered protein interactions with co-activators and -repressors. Based on the Su(H) structure, Ser269 phosphorylation may interfere with DNA-binding, which we confirmed by electro-mobility shift assay and isothermal titration calorimetry. Overexpression of Su(H)S269D during fly development demonstrated reduced transcriptional regulatory activity, similar to the previously reported DNA-binding defective mutant Su(H)R266H. As both are able to bind Hairless and Notch proteins, Su(H)S269D and Su(H)R266H provoked dominant negative effects upon overexpression. Our data imply that Ser269 phosphorylation impacts Notch signalling activity by inhibiting DNA-binding of Su(H), potentially affecting both activation and repression. Ser269 is highly conserved in vertebrate CSL homologues, opening the possibility of a general and novel mechanism of modulating Notch signalling activity.
Asunto(s)
ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Animales , Línea Celular , ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Espectrometría de Masas , Fosforilación/fisiología , Unión Proteica , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Represoras/genéticaRESUMEN
Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutación , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Sitios de Unión , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Unión Proteica , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Cell communication in metazoans requires the highly conserved Notch signaling pathway, which is subjected to strict regulation of both activation and silencing. In Drosophila melanogaster, silencing involves the assembly of a repressor complex by Hairless (H) on Notch target gene promoters. We previously found an in-frame internal ribosome entry site in the full length H transcript resulting in two H protein isoforms (Hp120 and Hp150). Hence, H may repress Notch signalling activity in situations where cap-dependent translation is inhibited. Here we demonstrate the in vivo importance of both H isoforms for proper fly development. To this end, we replaced the endogenous H locus by constructs specifically affecting translation of either Hp150 or Hp120 isoforms using genome engineering. Our findings indicate the functional relevance of both H proteins. Based on bristle phenotypes, the predominant isoform Hp150 appears to be of particular importance. In contrast, growth regulation and venation of the wing require the concomitant activity of both isoforms. Finally, the IRES dependent production of Hp120 during mitosis was verified in vivo. Together our data confirm IRES mediated translation of H protein in vivo, supporting strict regulation of Notch in different cellular settings.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ribosomas/metabolismo , Factores de Transcripción/genética , Regiones no Traducidas 3' , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero , Ingeniería Genética/métodos , Genoma de los Insectos , Hemicigoto , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitosis , Mutación , Biosíntesis de Proteínas , Isoformas de Proteínas/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Ribosomas/genética , Factores de Transcripción/metabolismo , Alas de Animales/crecimiento & desarrolloRESUMEN
Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo.
Asunto(s)
Proteínas de Drosophila/química , Drosophila melanogaster , Proteínas Represoras/química , Factores de Transcripción/química , Animales , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , ADN/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Termodinámica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members.
Asunto(s)
Ciclina G/metabolismo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores Notch/metabolismo , Transducción de Señal/genética , Alas de Animales/embriología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Alas de Animales/metabolismoRESUMEN
Size and weight control is a tightly regulated process, involving the highly conserved Insulin receptor/target of rapamycin (InR/TOR) signaling cascade. We recently identified Cyclin G (CycG) as an important modulator of InR/TOR signaling activity in Drosophila. cycG mutant flies are underweight and show a disturbed fat metabolism resembling TOR mutants. In fact, InR/TOR signaling activity is disturbed in cycG mutants at the level of Akt1, the central kinase linking InR and TORC1. Akt1 is negatively regulated by protein phosphatase PP2A. Notably the binding of the PP2A B'-regulatory subunit Widerborst (Wdb) to Akt1 is differentially regulated in cycG mutants, presumably by a direct interaction of CycG and Wdb. Since the metabolic defects of cycG mutant animals are abrogated by a concomitant loss of Wdb, CycG presumably influences Akt1 activity at the PP2A nexus. Here we show that Well rounded (Wrd), another B' subunit of PP2A in Drosophila, binds CycG similar to Wdb, and that its loss ameliorates some, but not all, of the metabolic defects of cycG mutants. We propose a model, whereby the binding of CycG to a particular B'-regulatory subunit influences the tissue specific activity of PP2A, required for the fine tuning of the InR/TOR signaling cascade in Drosophila.