RESUMO
Tumor necrosis factor receptors (TNFRs) control pleiotropic pro-inflammatory functions that range from apoptosis to cell survival. The ability to trigger a particular function will depend on the upstream cues, association with regulatory complexes, and downstream pathways. In Drosophila melanogaster, two TNFRs have been identified, Wengen (Wgn) and Grindelwald (Grnd). Although several reports associate these receptors with JNK-dependent apoptosis, it has recently been found that Wgn activates a variety of other functions. We demonstrate that Wgn is required for survival by protecting cells from apoptosis. This is mediated by dTRAF1 and results in the activation of p38 MAP kinase. Remarkably, Wgn is required for apoptosis-induced regeneration and is activated by the reactive oxygen species (ROS) produced following apoptosis. This ROS activation is exclusive for Wgn, but not for Grnd, and can occur after knocking down Eiger/TNFα. The extracellular cysteine-rich domain of Grnd is much more divergent than that of Wgn, which is more similar to TNFRs from other animals, including humans. Our results show a novel TNFR function that responds to stressors by ensuring p38-dependent regeneration.
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Regeneration after damage requires early signals to trigger the tissue repair machinery. Reactive oxygen species (ROS) act as early signals that are sensed by the MAP3 kinase Ask1, which in turn activates by phosphorylation the MAP kinases p38 and JNK. The sustained or high activation of these kinases can result in apoptosis, whereas short or low activation can promote regeneration. Using the Ask1-dependent regeneration program, we demonstrate in Drosophila wing that PI3K/Akt signaling is necessary for Ask1 to activate p38, but not JNK. In addition, nutrient restriction or mutations that target Ser83 of the Drosophila Ask1 protein, a PI3K/Akt-sensitive residue, block regeneration. However, these effects can be reversed by the ectopic activation of p38, but not of JNK. Our results demonstrate that Ask1 controls the activation of p38 through Ser83, and that the phosphorylation of p38 during regeneration is nutrient sensitive. This mechanism is important for discriminating between p38 and JNK in the cells involved in tissue repair and regenerative growth.
Assuntos
Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração , Asas de Animais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
During the process of regeneration, a switch in the transcription program occurs in cells that contribute to the reconstruction of the missing tissue. Early signals released upon damage are integrated into the chromatin of responding cells to change its activity and function. Changes in chromatin dynamics result in transcriptional reprogramming, this is the coordinated regulation of expression of a specific subset of genes required for the regeneration process. Here we summarize changes in gene expression and chromatin dynamics that occurs during the process of regeneration of Drosophila imaginal discs.
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Cromatina/metabolismo , Drosophila/genética , Discos Imaginais/efeitos dos fármacos , Regeneração/genética , AnimaisRESUMO
The seventh EMBO meeting on the Molecular and Cellular Basis of Regeneration and Tissue Repair took place in Valletta, Malta, in September 2018. Researchers from all over the world gathered together with the aim of sharing the latest advances in wound healing, repair and regeneration. The meeting covered a wide range of regeneration models and tissues, identification of regulatory genes and signals, and striking advances toward regenerative therapies. Here, we report some of the exciting topics discussed during this conference, highlighting important discoveries in regeneration and the perspectives for regenerative medicine.
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Medicina Regenerativa/métodos , Animais , Humanos , Regeneração/fisiologia , Cicatrização/fisiologiaRESUMO
How cells communicate to initiate a regenerative response after damage has captivated scientists during the last few decades. It is known that one of the main signals emanating from injured cells is the Reactive Oxygen Species (ROS), which propagate to the surrounding tissue to trigger the replacement of the missing cells. However, the link between ROS production and the activation of regenerative signaling pathways is not yet fully understood. We describe here the non-autonomous ROS sensing mechanism by which living cells launch their regenerative program. To this aim, we used Drosophila imaginal discs as a model system due to its well-characterized regenerative ability after injury or cell death. We genetically-induced cell death and found that the Apoptosis signal-regulating kinase 1 (Ask1) is essential for regenerative growth. Ask1 senses ROS both in dying and living cells, but its activation is selectively attenuated in living cells by Akt1, the core kinase component of the insulin/insulin-like growth factor pathway. Akt1 phosphorylates Ask1 in a secondary site outside the kinase domain, which attenuates its activity. This modulation of Ask1 activity results in moderate levels of JNK signaling in the living tissue, as well as in activation of p38 signaling, both pathways required to turn on the regenerative response. Our findings demonstrate a non-autonomous activation of a ROS sensing mechanism by Ask1 and Akt1 to replace the missing tissue after damage. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.
Assuntos
Proteínas de Drosophila/genética , Discos Imaginais/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Regeneração/genética , Animais , Apoptose/genética , Comunicação Celular/genética , Proliferação de Células/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Humanos , Discos Imaginais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genéticaRESUMO
One of the most important questions in regenerative biology is to unveil how and when genes change expression and trigger regeneration programs. The resetting of gene expression patterns during response to injury is governed by coordinated actions of genomic regions that control the activity of multiple sequence-specific DNA binding proteins. Using genome-wide approaches to interrogate chromatin function, we here identify the elements that regulate tissue recovery in Drosophila imaginal discs, which show a high regenerative capacity after genetically induced cell death. Our findings indicate there is global coregulation of gene expression as well as a regeneration program driven by different types of regulatory elements. Novel enhancers acting exclusively within damaged tissue cooperate with enhancers co-opted from other tissues and other developmental stages, as well as with endogenous enhancers that show increased activity after injury. Together, these enhancers host binding sites for regulatory proteins that include a core set of conserved transcription factors that control regeneration across metazoans.
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Drosophila/fisiologia , Regulação da Expressão Gênica , Regeneração/genética , Elementos de Resposta , Animais , Cromatina/genética , Sequência Conservada , Perfilação da Expressão Gênica , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional , TranscriptomaRESUMO
Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome.
Assuntos
Evolução Biológica , Fosfotransferases/genética , Fosfotransferases/metabolismo , Animais , Biomarcadores , Eucariotos/genética , Eucariotos/metabolismo , Duplicação Gênica , Humanos , Insetos , Filogenia , Células Procarióticas/metabolismo , Seleção Genética , Selênio/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Urocordados , VertebradosRESUMO
Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.
Assuntos
Proteínas de Drosophila/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Regeneração/genética , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais/crescimento & desenvolvimento , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética , Fatores de Transcrição/biossíntese , Asas de Animais/crescimento & desenvolvimento , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
The Drosophila transcription factor Cabut/dTIEG (Cbt) is a growth regulator, whose expression is modulated by different stimuli. Here, we determine Cbt association with chromatin and identify Yorkie (Yki), the transcriptional co-activator of the Hippo (Hpo) pathway as its partner. Cbt and Yki co-localize on common gene promoters, and the expression of target genes varies according to changes in Cbt levels. Down-regulation of Cbt suppresses the overgrowth phenotypes caused by mutations in expanded (ex) and yki overexpression, whereas its up-regulation promotes cell proliferation. Our results imply that Cbt is a novel partner of Yki that is required as a transcriptional co-activator in growth control.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Hormônios Juvenis/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Modelos Biológicos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
To understand the cellular parameters that govern Drosophila wing disc regeneration, we genetically eliminated specific stripes of the wing disc along the proximodistal axis and used vein and intervein markers to trace tissue regeneration. We found that veins could regenerate interveins and vice versa, indicating respecification of cell fates. Moreover, respecification occurred in cells close to the wound. The newly generated domains were intercalated to fill in the missing parts. This intercalation was driven by increased proliferation, accompanied by changes in the orientation of the cell divisions. This reorientation depended on Fat (Ft) and Crumbs (Crb), which acted, at least partly, to control the activity of the effector of the Hippo pathway, Yorkie (Yki). Increased Yki, which promotes proliferation, affected the final shape and size. Heterozygous ft or crb, which normally elicit size and shape defects in regenerated wings, could be rescued by yki heterozygosity. Thus, Ft and Crb act as sensors to drive cell orientation during intercalary regeneration and control Yki levels to ensure a proper balance between proliferation and cell reorientation. We propose a model based on intercalation of missing cell identities, in which a coordinated balance between orientation and proliferation is required for normal organ shape and size.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Drosophila/fisiologia , Discos Imaginais/fisiologia , Regeneração/fisiologia , Asas de Animais/fisiologia , Animais , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Proteínas de Drosophila/metabolismo , Processamento de Imagem Assistida por Computador , Discos Imaginais/citologia , Proteínas de Membrana/metabolismo , Microscopia Confocal , Modelos Biológicos , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Asas de Animais/citologia , Proteínas de Sinalização YAPRESUMO
In a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development we found ß amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease. We analyzed Appl expression in the eye imaginal disc and found that is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, we have analyzed Appl null mutants and found reduced preference for UV light, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, we examined whether Ras1/MAPK is responsible for the high Appl expression. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. We show that Appl is directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. We also found that zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Proteínas ras/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas ras/genéticaRESUMO
BACKGROUND: PTOV1 is an adaptor protein with functions in diverse processes, including gene transcription and protein translation, whose overexpression is associated with a higher proliferation index and tumor grade in prostate cancer (PC) and other neoplasms. Here we report its interaction with the Notch pathway and its involvement in PC progression. METHODS: Stable PTOV1 knockdown or overexpression were performed by lentiviral transduction. Protein interactions were analyzed by co-immunoprecipitation, pull-down and/or immunofluorescence. Endogenous gene expression was analyzed by real time RT-PCR and/or Western blotting. Exogenous promoter activities were studied by luciferase assays. Gene promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). In vivo studies were performed in the Drosophila melanogaster wing, the SCID-Beige mouse model, and human prostate cancer tissues and metastasis. The Excel package was used for statistical analysis. RESULTS: Knockdown of PTOV1 in prostate epithelial cells and HaCaT skin keratinocytes caused the upregulation, and overexpression of PTOV1 the downregulation, of the Notch target genes HEY1 and HES1, suggesting that PTOV1 counteracts Notch signaling. Under conditions of inactive Notch signaling, endogenous PTOV1 associated with the HEY1 and HES1 promoters, together with components of the Notch repressor complex. Conversely, expression of active Notch1 provoked the dismissal of PTOV1 from these promoters. The antagonist role of PTOV1 on Notch activity was corroborated in the Drosophila melanogaster wing, where human PTOV1 exacerbated Notch deletion mutant phenotypes and suppressed the effects of constitutively active Notch. PTOV1 was required for optimal in vitro invasiveness and anchorage-independent growth of PC-3 cells, activities counteracted by Notch, and for their efficient growth and metastatic spread in vivo. In prostate tumors, the overexpression of PTOV1 was associated with decreased expression of HEY1 and HES1, and this correlation was significant in metastatic lesions. CONCLUSIONS: High levels of the adaptor protein PTOV1 counteract the transcriptional activity of Notch. Our evidences link the pro-oncogenic and pro-metastatic effects of PTOV1 in prostate cancer to its inhibitory activity on Notch signaling and are supportive of a tumor suppressor role of Notch in prostate cancer progression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Homeodomínio/biossíntese , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Drosophila melanogaster , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/patologia , Receptores Notch/biossíntese , Transdução de Sinais/genética , Fatores de Transcrição HES-1 , Ativação Transcricional/genéticaRESUMO
Regeneration is vital for many organisms, enabling them to repair injuries and adapt to environmental changes. The mechanisms underlying regeneration are complex and involve coordinated events at the cellular and molecular levels. Moreover, while some species exhibit remarkable regenerative capabilities, others, like mammals, have limited regenerative potential. Central to this process is the regulation of gene expression, and among the numerous genes involved, MYC emerges as a regulator of relevant processes during regeneration with roles conserved in several species, including Drosophila. This mini-review aims to provide valuable insights into the regeneration process in flies, focusing on significant organs where the role of MYC has been identified: from the imaginal discs, where MYC regulates cell growth, structure, and proliferation, to the gut, where it maintains the balance between renewal and differentiation of stem cells, and the central nervous system, where it influences the activities of neural stem cells and the interaction between glia and neuronal cells. By emphasizing the molecular mechanisms regulated by MYC, its significance in controlling regeneration mechanisms, and its conserved role in flies, we aim to offer valuable insights into the utility of Drosophila as a model for studying regeneration. Moreover, unraveling MYC's function in Drosophila during regeneration may help translate findings into the mechanisms underlying human tissue repair.
RESUMO
Understanding adipose metabolism is essential for addressing obesity and related health concerns. However, the ethical and scientific pressure to animal testing, aligning with the 3Rs, has triggered the implementation of diverse alternative models for analysing anomalies in adipose metabolism. In this review, we will address this issue from various perspectives. Traditional adipocyte cell cultures, whether animal or human-derived, offer a fundamental starting point. These systems have their merits but may not fully replicate in vivo complexity. Established cell lines are valuable for high-throughput screening but may lack the authenticity of primary-derived adipocytes, which closely mimic native tissue. To enhance model sophistication, spheroids have been introduced. These three-dimensional cultures better mimicking the in vivo microenvironment, enabling the study of intricate cell-cell interactions, gene expression, and metabolic pathways. Organ-on-a-chip (OoC) platforms take this further by integrating multiple cell types into microfluidic devices, simulating tissue-level functions. Adipose-OoC (AOoC) provides dynamic environments with applications spanning drug testing to personalized medicine and nutrition. Beyond in vitro models, genetically amenable organisms (Caenorhabditis elegans, Drosophila melanogaster, and zebrafish larvae) have become powerful tools for investigating fundamental molecular mechanisms that govern adipose tissue functions. Their genetic tractability allows for efficient manipulation and high-throughput studies. In conclusion, a diverse array of research models is crucial for deciphering adipose metabolism. By leveraging traditional adipocyte cell cultures, primary-derived cells, spheroids, AOoCs, and lower organism models, we bridge the gap between animal testing and a more ethical, scientifically robust, and human-relevant approach, advancing our understanding of adipose tissue metabolism and its impact on health.
Assuntos
Drosophila melanogaster , Peixe-Zebra , Animais , Humanos , Tecido Adiposo/metabolismo , Adipócitos/metabolismo , Técnicas de Cultura de Células/métodos , Obesidade/metabolismoRESUMO
The discovery of functional long non-coding RNAs (lncRNAs) changed their initial concept as transcriptional noise. LncRNAs have been identified as regulators of multiple biological processes, including chromatin structure, gene expression, splicing, mRNA degradation, and translation. However, functional studies of lncRNAs are hindered by the usual lack of phenotypes upon deletion or inhibition. Here, we used Drosophila imaginal discs as a model system to identify lncRNAs involved in development and regeneration. We examined a subset of lncRNAs expressed in the wing, leg, and eye disc development. Additionally, we analyzed transcriptomic data from regenerating wing discs to profile the expression pattern of lncRNAs during tissue repair. We focused on the lncRNA CR40469, which is upregulated during regeneration. We generated CR40469 mutant flies that developed normally but showed impaired wing regeneration upon cell death induction. The ability of these mutants to regenerate was restored by the ectopic expression of CR40469. Furthermore, we found that the lncRNA CR34335 has a high degree of sequence similarity with CR40469 and can partially compensate for its function during regeneration in the absence of CR40469. Our findings point to a potential role of the lncRNA CR40469 in trans during the response to damage in the wing imaginal disc.
RESUMO
Regeneration and tissue repair allow damaged or lost body parts to be replaced. After injury or fragmentation of Drosophila imaginal discs, regeneration leads to the development of normal adult structures. This process is likely to involve a combination of cell rearrangement and compensatory proliferation. However, the detailed mechanisms underlying these processes are poorly understood. We have established a system to allow temporally restricted induction of cell death in situ. Using Gal4/Gal80 and UAS-rpr constructs, targeted ablation of a region of the disc could be performed and regeneration monitored without the requirement for microsurgical manipulation. Using a ptc-Gal4 construct to drive rpr expression in the wing disc resulted in a stripe of dead cells in the anterior compartment flanking the anteroposterior boundary, whereas a sal-Gal4 driver generated a dead domain that includes both anterior and posterior cells. Under these conditions, regenerated tissues were derived from the damaged compartment, suggesting that compartment restrictions are preserved during regeneration. Our studies reveal that during regeneration the live cells bordering the domain in which cell death was induced first display cytoskeletal reorganisation and apical-to-basal closure of the epithelium. Then, proliferation begins locally in the vicinity of the wound and later more extensively in the affected compartment. Finally, we show that regeneration of genetically ablated tissue requires JNK activity. During cell death-induced regeneration, the JNK pathway is activated at the leading edges of healing tissue and not in the apoptotic cells, and is required for the regulation of healing and regenerative growth.
Assuntos
Morte Celular/fisiologia , Drosophila melanogaster , Embrião não Mamífero , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem da Célula , Proliferação de Células , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Mitose/fisiologia , Asas de Animais/anatomia & histologia , Asas de Animais/embriologia , Asas de Animais/fisiologiaRESUMO
An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila/genética , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Genoma de Inseto , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Metilação , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Modulation of signalling pathways can trigger different cellular responses, including differences in cell fate. This modulation can be achieved by controlling the pathway activity with great precision to ensure robustness and reproducibility of the specification of cell fate. The development of the photoreceptor R7 in the Drosophila melanogaster retina has become a model in which to investigate the control of cell signalling. During R7 specification, a burst of Ras small GTPase (Ras) and mitogen-activated protein kinase (MAPK) controlled by Sevenless receptor tyrosine kinase (Sev) is required. Several cells in each ommatidium express sev. However, the spatiotemporal expression of the boss ligand and the action of negative regulators of the Sev pathway will restrict the R7 fate to a single cell. The Drosophila suppressor of cytokine signalling 36E (SOCS36E) protein contains an SH2 domain and acts as a Sev signalling attenuator. By contrast, downstream of receptor kinase (Drk), the fly homolog of the mammalian Grb2 adaptor protein, which also contains an SH2 domain, acts as a positive activator of the pathway. Here, we apply the Förster resonance energy transfer (FRET) assay to transfected Drosophila S2 cells and demonstrate that Sev binds directly to either the suppressor protein SOCS36E or the adaptor protein Drk. We propose a mechanistic model in which the competition between these two proteins for binding to the same docking site results in either attenuation of the Sev transduction in cells that should not develop R7 photoreceptors or amplification of the Ras-MAPK signal only in the R7 precursor.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Drosophila melanogaster , Transferência Ressonante de Energia de Fluorescência , Fosforilação , Transdução de Sinais , Transfecção , Domínios de Homologia de srcRESUMO
Many animals display a capacity to regenerate tissues or even a complete body. One of the main goals of regenerative biology is to identify the genes and genetic networks necessary for this process. Drosophila offers an ideal model system for such studies. The wide range of genetic and genomic approaches available for use in flies has helped in initiating the deciphering of the mechanisms underlying regeneration, and the results may be applicable to other organisms, including mammals. Moreover, most models of regeneration require experimental manipulation, whereas in Drosophila discrete domains can be ablated by genetically induced methods. Here, we present a summary of current research into imaginal disc regeneration and discuss the power of this tissue as a tool for understanding the genetics of regeneration.
Assuntos
Drosophila melanogaster , Embrião não Mamífero , Regeneração/fisiologia , Animais , Padronização Corporal/fisiologia , Proliferação de Células , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Epigênese Genética , Epitélio/fisiologia , CicatrizaçãoRESUMO
One of the main topics in regeneration biology is the nature of the early signals that trigger the damage response. Recent advances in Drosophila point to the MAP3 kinase Ask1 as a molecular hub that integrates several signals at the onset of regeneration. It has been discovered that reactive oxygen species (ROS) produced in damaged imaginal discs and gut epithelia will activate the MAP3 kinase Ask1. Severely damaged and apoptotic cells produce an enormous amount of ROS, which ensures their elimination by activating Ask1 and in turn the pro-apoptotic function of JNK. However, this creates an oxidative stress environment with beneficial effects that is sensed by neighboring healthy cells. This environment, in addition to the Pi3K/Akt nutrient sensing pathway, can be integrated into Ask1 to launch regeneration. Ultimately the activity of Ask1 depends on these and other inputs and modulates its signaling to achieve moderate levels of p38 and low JNK signaling and thus promote survival and regeneration. This model based on the dual function of Ask1 for early response to damage is discussed here.