RESUMO
Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs we queried publicly available transcriptome data of over 180 highly inbred fly lines for dilp expression and used dilp expression as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. We found that IPC-specific depletion of most genes resulted in differences in expression of one or more of the dilps. We then elaborated further on one of the candidate genes with the strongest effect on dilp expression, Homothorax, a transcription factor known for its role in eye development. We found that Homothorax and its binding partner Extradenticle are involved in regulating dilp2, -3 and -5 expression and that genetic depletion of both TFs shows phenotypes associated with reduced insulin signalling. Furthermore, we provide evidence that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, we showed that this expression level-based GWAS approach identified genetic regulators implicated in IPC function and dilp expression.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Estudo de Associação Genômica Ampla , Insulina/genética , Insulina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Current rates of habitat degradation and climate change are causing unprecedented declines in global biodiversity. Studies on vertebrates highlight how conservation genomics can be effective in identifying and managing threatened populations, but it is unclear how vertebrate-derived metrics of genomic erosion translate to invertebrates, with their markedly different population sizes and life histories. The Black-veined White butterfly (Aporia crataegi) was extirpated from Britain in the 1920s. Here, we sequenced historical DNA from 17 specimens collected between 1854 and 1924 to reconstruct demography and compare levels of genomic erosion between extirpated British and extant European mainland populations. We contrast these results using modern samples of the Common Blue butterfly (Polyommatus icarus); a species with relatively stable demographic trends in Great Britain. We provide evidence for bottlenecks in both these species around the period of post-glacial colonization of the British Isles. Our results reveal different demographic histories and Ne for both species, consistent with their fates in Britain, likely driven by differences in life history, ecology and genome size. Despite a difference, by an order of magnitude, in historical effective population sizes (Ne), reduction in genome-wide heterozygosity in A. crataegi was comparable to that in P. icarus. Symptomatic of A. crataegi's disappearance were marked increases in runs-of-homozygosity (RoH), potentially indicative of recent inbreeding, and accumulation of putatively mildly and weakly deleterious variants. Our results provide a rare glimpse of genomic erosion in a regionally extinct insect and support the potential use of genomic erosion metrics in identifying invertebrate populations or species in decline.
RESUMO
Apoptosis, a conserved form of programmed cell death, shows interspecies differences that may reflect evolutionary diversification and adaptation, a notion that remains largely untested. Among insects, the most speciose animal group, the apoptotic pathway has only been fully characterized in Drosophila melanogaster, and apoptosis-related proteins have been studied in a few other dipteran and lepidopteran species. Here, we studied the apoptotic pathway in the aphid Acyrthosiphon pisum, an insect pest belonging to the Hemiptera, an earlier-diverging and distantly related order. We combined phylogenetic analyses and conserved domain identification to annotate the apoptotic pathway in A. pisum and found low caspase diversity and a large expansion of its inhibitory part, with 28 inhibitors of apoptosis (IAPs). We analyzed the spatiotemporal expression of a selected set of pea aphid IAPs and showed that they are differentially expressed in different life stages and tissues, suggesting functional diversification. Five IAPs are specifically induced in bacteriocytes, the specialized cells housing symbiotic bacteria, during their cell death. We demonstrated the antiapoptotic role of these five IAPs using heterologous expression in a tractable in vivo model, the Drosophila melanogaster developing eye. Interestingly, IAPs with the strongest antiapoptotic potential contain two BIR and two RING domains, a domain association that has not been observed in any other species. We finally analyzed all available aphid genomes and found that they all show large IAP expansion, with new combinations of protein domains, suggestive of evolutionarily novel aphid-specific functions.
Assuntos
Afídeos/citologia , Afídeos/fisiologia , Apoptose/fisiologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Animais , Animais Geneticamente Modificados , Caspases/química , Caspases/metabolismo , Drosophila melanogaster/genética , Olho/citologia , Olho/patologia , Regulação da Expressão Gênica , Genoma de Inseto , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas de Insetos/genética , Filogenia , Domínios ProteicosRESUMO
Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-ß/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription.
Assuntos
Infecções Bacterianas/metabolismo , Drosophila/genética , Drosophila/microbiologia , Transdução de Sinais , Animais , Infecções Bacterianas/genética , Proliferação de Células , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Enterócitos/metabolismo , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Intestinos/citologia , Intestinos/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Pectobacterium carotovorum/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas/metabolismo , Células-Tronco/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição/genética , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Quinase do Linfoma Anaplásico , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Sistemas CRISPR-Cas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise â¼ 5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such "bivalent" chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.
Assuntos
Padronização Corporal/genética , Drosophila melanogaster/embriologia , Discos Imaginais/embriologia , Fatores de Transcrição/genética , Asas de Animais/embriologia , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/genética , Interferência de RNA , RNA Polimerase II/genética , RNA Interferente PequenoRESUMO
During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network's topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a "dimer cloud" that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit's dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks.
Assuntos
Relógios Biológicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal , Dimerização , Retroalimentação Fisiológica , Modelos Biológicos , Fenótipo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Somitos/citologia , Somitos/embriologia , Somitos/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes.
Assuntos
Redes Reguladoras de Genes , Técnicas Analíticas Microfluídicas , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sequência Consenso , DNA/metabolismo , Drosophila melanogaster/genética , Biblioteca Gênica , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.
Assuntos
Drosophila melanogaster/genética , Expressão Gênica , Variação Genética , Genoma , Desequilíbrio Alélico/genética , Animais , Mapeamento Cromossômico , Mutação INDEL , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genéticaRESUMO
Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation, we need convenient tools to identify the proteins that bind to them. Here we describe the development and validation of a high-throughput yeast one-hybrid platform, which enables screening of DNA elements versus an array of full-length, sequence-verified clones containing over 85% of predicted Drosophila transcription factors. Using six well-characterized regulatory elements, we identified 33 transcription factor-DNA interactions of which 27 were previously unidentified. To simultaneously validate these interactions and locate the binding sites of involved transcription factors, we implemented a powerful microfluidics-based approach that enabled us to retrieve DNA-occupancy data for each transcription factor throughout the respective target DNA elements. Finally, we biologically validated several interactions and identified two new regulators of sine oculis gene expression and hence eye development.
Assuntos
DNA/genética , DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ensaios de Triagem em Larga Escala , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Animais , Automação , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Microfluídica , Fases de Leitura Aberta , Reprodutibilidade dos Testes , Fatores de Transcrição/genéticaRESUMO
The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.
Assuntos
Elementos Facilitadores Genéticos , Redes Reguladoras de Genes , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Genes Reporter , Luciferases , Camundongos , Microfluídica , Células NIH 3T3 , Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Transcrição/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.
Assuntos
Clonagem Molecular , Análise de Sequência de DNA/métodos , Software , Animais , DNA Complementar/química , Drosophila melanogaster/genética , Ensaios de Triagem em Larga Escala , Internet , Fases de Leitura Aberta , Interface Usuário-ComputadorRESUMO
Insulin/insulin-like growth factor (IGF) signaling constitutes an evolutionarily conserved pathway that controls growth, energy homeostasis, and longevity. In Drosophila melanogaster, key components of this pathway are the insulin-like peptides (Dilps). The major source of Dilps is a cluster of large neurons in the brain, the insulin-producing cells (IPCs). The genetic control of IPC development and function is poorly understood. Here, we demonstrate that the Pax6 homolog Eyeless is required in the IPCs to control their differentiation and function. Loss of eyeless results in phenotypes associated with loss of insulin signaling, including decreased animal size and increased carbohydrate levels in larval hemolymph. We show that mutations in eyeless lead to defective differentiation and morphologically abnormal IPCs. We also demonstrate that Eyeless controls IPC function by the direct transcriptional control of one of the major Dilps, dilp5. We propose that Eyeless has an evolutionarily conserved role in IPCs with remarkable similarities to the role of vertebrate Pax6 in beta cells of the pancreas.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas do Olho , Proteínas de Homeodomínio , Insulina/biossíntese , Insulinas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição Box Pareados , Proteínas Repressoras , Animais , Sequência de Bases , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Insulinas/genética , Masculino , Dados de Sequência Molecular , Mutação/genética , Fator de Transcrição PAX6 , Transdução de Sinais , Transcrição Gênica/genéticaRESUMO
Pax6 genes encode evolutionarily highly conserved transcription factors that are required for eye and brain development. Despite the characterization of mutations in Pax6 homologs in a range of organisms, and despite functional studies, it remains unclear what the relative importance is of the various parts of the Pax6 protein. To address this, we have studied the Drosophila Pax6 homolog eyeless. Specifically, we have generated new eyeless alleles, each with single missense mutations in one of the four domains of the protein. We show that these alleles result in abnormal eye and brain development while maintaining the OK107 eyeless GAL4 activity from which they were derived. We performed in vivo functional rescue experiments by expressing in an eyeless-specific pattern Eyeless proteins in which either the paired domain, the homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain phenotypes was only observed when full-length Eyeless was expressed, while all deletion constructs failed to rescue. These data, along with the phenotypes observed in the four newly characterized eyeless alleles, demonstrate the requirement for an intact Eyeless protein for normal Drosophila eye and brain development. They also suggest that some endogenous functions may be obscured in ectopic expression experiments.
Assuntos
Encéfalo/crescimento & desenvolvimento , Olho Composto de Artrópodes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Mutação de Sentido Incorreto , Mutação Puntual , Alelos , Animais , Células Cultivadas , Cruzamentos Genéticos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Teste de Complementação Genética , Genótipo , Cabeça/crescimento & desenvolvimento , Masculino , Fenótipo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.
Assuntos
Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica , Microscopia de Fluorescência , Mapas de Interação de Proteínas/genética , Elementos Reguladores de Transcrição , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoAssuntos
Sequência de Bases , Primers do DNA , Software , Animais , Drosophila melanogaster/genética , GenomaRESUMO
A balanced diet of macronutrients is critical for animal health. A lack of specific elements can have profound effects on behavior, reproduction, and lifespan. Here, we used Drosophila to understand how the brain responds to carbohydrate deprivation. We found that serine protease homologs (SPHs) are enriched among genes that are transcriptionally regulated in flies deprived of carbohydrates. Stimulation of neurons expressing one of these SPHs, Scarface (Scaf), or overexpression of scaf positively regulates feeding on nutritious sugars, whereas inhibition of these neurons or knockdown of scaf reduces feeding. This modulation of food intake occurs only in sated flies while hunger-induced feeding is unaffected. Furthermore, scaf expression correlates with the presence of sugar in the food. As Scaf and Scaf neurons promote feeding independent of the hunger state, and the levels of scaf are positively regulated by the presence of sugar, we conclude that scaf mediates the hedonic control of feeding.
Assuntos
Encéfalo/metabolismo , Metabolismo dos Carboidratos , Proteínas de Drosophila/metabolismo , Comportamento Alimentar , Serina Proteases/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Carboidratos da Dieta/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Serina Proteases/genéticaRESUMO
The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5' intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica , Estudos de Associação Genética , Pigmentação/genética , Interferência de RNA , Técnicas do Sistema de Duplo-Híbrido , Animais , Drosophila/metabolismo , Ecdisona/metabolismo , Elementos Facilitadores Genéticos , Estudos de Associação Genética/métodos , Testes Genéticos , Mutação , Fenótipo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Angiotensin-converting enzyme, a member of the M2 metalloprotease family, and endothelin-converting enzyme, a member of the M13 family, are key components in the regulation of blood pressure and electrolyte balance in mammals. From this point of view, they serve as important drug targets. Recently, the involvement of these enzymes in the development of Alzheimer's disease was discovered. The existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. Most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. Therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. This chapter reviews the structural and functional aspects of ACE and ECE and will particularly focus on these enzyme homologues in invertebrates.
Assuntos
Ácido Aspártico Endopeptidases , Evolução Molecular , Peptidil Dipeptidase A , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Pressão Sanguínea/fisiologia , Enzimas Conversoras de Endotelina , Humanos , Insetos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Sanguessugas , Metaloendopeptidases , Metaloproteases/química , Metaloproteases/metabolismo , Metamorfose Biológica/fisiologia , Dados de Sequência Molecular , Sistema Nervoso/fisiopatologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Reprodução/fisiologia , Alinhamento de Sequência , Homologia de Sequência , Homologia Estrutural de ProteínaRESUMO
Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities are highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extends even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities function in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells.