RESUMEN
The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.
Asunto(s)
Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Transcriptoma/genética , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Cromatina/genética , Análisis por Conglomerados , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/metabolismo , Humanos , Larva/genética , Larva/crecimiento & desarrollo , Modelos Genéticos , Anotación de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Pupa/genética , Pupa/crecimiento & desarrollo , ARN no Traducido/genética , Análisis de Secuencia de ARNRESUMEN
Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.
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Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Transcriptoma/genética , Empalme Alternativo/genética , Animales , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Femenino , Masculino , Anotación de Secuencia Molecular , Tejido Nervioso/metabolismo , Especificidad de Órganos , Poli A/genética , Poliadenilación , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Caracteres Sexuales , Estrés Fisiológico/genéticaRESUMEN
We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.
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Drosophila/genética , Variación Genética , MicroARNs/genética , ARN Interferente Pequeño/genética , Animales , Secuencia de Bases , Línea Celular , Biología Computacional/métodos , Expresión Génica , Sitios Genéticos , Células Germinativas , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/química , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/química , Alineación de SecuenciaRESUMEN
Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Transcripción Genética/genética , Empalme Alternativo/genética , Animales , Secuencia de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Exones/genética , Femenino , Genes de Insecto/genética , Genoma de los Insectos/genética , Masculino , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Edición de ARN/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Pequeño no Traducido/análisis , ARN Pequeño no Traducido/genética , Análisis de Secuencia , Caracteres SexualesRESUMEN
We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals.
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Línea Celular , Biología Evolutiva/métodos , Drosophila/genética , Animales , Drosophila/crecimiento & desarrollo , GenómicaRESUMEN
The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Receptores de Esteroides/genética , Factores de Transcripción/genética , Activación Transcripcional , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hormonas de Insectos/genética , Hormonas de Insectos/metabolismo , Muda , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.
Asunto(s)
Drosophila melanogaster/genética , Variación Genética , Transcripción Genética , Animales , Línea Celular , Análisis por Conglomerados , Exones , Femenino , Perfilación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. Here we report that TRR is a histone methyltransferases capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. We propose that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/efectos de los fármacos , Drosophila/embriología , Ecdisona/farmacología , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animales , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Ojo/embriología , Ojo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog , N-Metiltransferasa de Histona-Lisina/genética , Masculino , Metilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptores de Esteroides/metabolismoRESUMEN
Following publication of the original article [1], the authors reported the following errors.
RESUMEN
Given the power of Drosophila genetics, it may seem surprising to discover that many fly researchers are turning to Drosophila cell culture as an experimental system. However, as we will show in this chapter, there are many benefits to be gained by using cell lines as a complement to studies in a tissue and developmental context in the fly. Moreover, one can argue that Drosophila cell culture, in itself, provides an excellent model system for the study of many fundamental questions in molecular and cellular biology. In this review, we offer a summary of techniques that should be useful to researchers in the Drosophila community working with fly cell lines. These include techniques for growing and maintaining cell lines, transient and stable transfection, RNA interference, imaging, immunostaining, fluorescence-activated cell sorting, and for the isolation of RNA and protein from fly cells.
Asunto(s)
Biología Evolutiva/métodos , Biología Molecular/métodos , Interferencia de ARN , Animales , Línea Celular , Línea Celular Transformada , Núcleo Celular/metabolismo , Separación Celular , Drosophila melanogaster , Citometría de Flujo , Técnicas Genéticas , Microscopía Fluorescente/métodos , ARN/metabolismo , ARN Bicatenario/metabolismo , TransgenesRESUMEN
Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.
Asunto(s)
Baculoviridae/genética , Drosophila/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Línea Celular , Drosophila/citología , Drosophila/genética , Expresión Génica , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Transfección/métodosRESUMEN
Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.
Asunto(s)
Drosophila/genética , Drosophila/metabolismo , Regulación de la Expresión Génica , Hormonas/metabolismo , Transducción de Señal , Animales , Línea Celular , Análisis por Conglomerados , Ecdisona/metabolismo , Ecdisona/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas/farmacología , Isoformas de Proteínas , Receptores de Esteroides/metabolismo , Esteroides/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , TranscriptomaRESUMEN
The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the ecdysone receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Ecdisona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Dominantes , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Conformación Proteica , Isoformas de Proteínas , Receptores de Esteroides/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu(2+)-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays-a major emphasis of cell-based studies.
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Drosophila melanogaster/genética , Marcación de Gen/métodos , Genoma de los Insectos , Mutagénesis Insercional , Animales , Línea Celular , Células Clonales , Ingeniería Genética , Marcadores Genéticos , Integrasas/metabolismo , TransgenesRESUMEN
BACKGROUND: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. RESULTS: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). CONCLUSION: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.
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Línea Celular , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Evolución Molecular , Dosificación de Gen , Animales , Supervivencia Celular , ADN/análisis , Proteínas de Drosophila/genética , Femenino , Aptitud Genética , Variación Genética , Masculino , MicroARNs/genética , Proteínas Tirosina Quinasas Receptoras/genética , Selección Genética , Análisis de Secuencia de ADN , Cromosomas Sexuales/genética , Técnicas de Cultivo de TejidosRESUMEN
Most commonly used Drosophila cell lines adhere only loosely to the substrate. For some purposes (e.g., time-lapse photography and cytological examination of living cells), it is desirable to fix the growing cells more firmly to the substrate. As described in this protocol, this can be accomplished by treating the substrate with poly-L-lysine, with no obvious detrimental effect on the growth of the cells.
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Técnicas de Cultivo de Célula/métodos , Drosophila melanogaster/citología , Polilisina/farmacología , Animales , Células Cultivadas , Propiedades de Superficie/efectos de los fármacosRESUMEN
INTRODUCTIONPermanent Drosophila cell lines derived from mixed embryonic tissues including the most commonly used lines, S2 and Kc, have been available for ~30 yr. More recently, lines derived from specific tissues, imaginal discs, and the larval central nervous system have come into use. Although cultured cells were originally used by Drosophilists mainly as convenient sources of DNA or carrier RNA, that situation has changed, as an armamentarium of techniques for using the cells has slowly but steadily evolved. Most investigators use Drosophila cell lines as hosts for transformation experiments. The goal may be to characterize a promoter, to investigate the role of a transcription factor, to overexpress a polypeptide, or to do something more novel. This article provides an organized collection of pointers to published protocols.
RESUMEN
During metamorphosis, the reorganization of the nervous system of Drosophila melanogaster proceeds in part through remodeling of larval neurons. In this study, we used in-vitro imaging techniques and immunocytochemistry to track the remodeling of the thoracic ventral neurosecretory cells. Axons of these neurons prune their larval arbors early in metamorphosis and a larger, more extensive adult arbor is established via branch outgrowth. Expression of EcR dominant negative constructs and an EcR inverted repeat construct resulted in pruning defects of larval axon arbors and a lack of filopodia during pruning, but showed variable effects on outgrowth depending on the construct expressed. Cells expressing either UAS-EcR-B1(W650A) or UAS-EcR-A(W650A) lacked filopodia during the outgrowth period and formed a poorly branched, larval-like arbor in the adult. Cells expressing UAS-EcR-B1(F645A), UAS-EcR-B2(W650A) or UAS-IR-EcR (core) showed moderate filopodial activity and normal, albeit reduced, adult-like branching during outgrowth. These results are consistent with the role of activation versus derepression via EcR for successive phases of neuronal remodeling and suggest that functional ecdysone receptor is necessary for some, but not all, remodeling events.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas de Insectos/metabolismo , Neuronas/metabolismo , Receptores de Esteroides/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisona/metabolismo , Genes de Insecto , Proteínas de Insectos/genética , Metamorfosis Biológica , Microscopía por Video , Modelos Biológicos , Mutación , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Receptores de Esteroides/genética , Transducción de Señal , Factores de TiempoRESUMEN
Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, we performed whole genome expression analysis on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Ecdisteroides/metabolismo , Metamorfosis Biológica/fisiología , Nucleosomas/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Cromosómicas no Histona/genética , Drosophila , Proteínas de Drosophila/genética , Regulación de la Expresión Génica/fisiología , Larva/fisiología , Mamíferos/genética , Mamíferos/metabolismo , Mutación , Receptores de Esteroides/biosíntesis , Receptores de Esteroides/genéticaRESUMEN
The three Drosophila EcR isoforms differ only at their N termini; thus, they share the conserved ligand-binding domain transcriptional activation function (AF2) and only differ in the unconserved A/B region, which contains a second, isoform-specific, activation function (AF1). We have developed a dominant-negative mutant EcR (EcR-DN), expressed it in flies with the GAL4/UAS system, and used it to block ecdysone signaling in eight tissues or groups of tissues. Localized EcR-DN arrests ecdysone-dependent development in the target cells and often--because of a molting checkpoint--arrests development globally. Simultaneously expressing individual wild-type EcR isoforms in the same target tissues suppresses the EcR-DN phenotype and identifies the rescuing isoform as sufficient to support the development of the target. Every isoform, and even an N-terminal truncated EcR that lacks any AF1, supports development in the fat body, eye discs, salivary glands, EH-secreting neurosecretory cells and in the dpp expression domain, implying that AF1 is dispensable in these tissues. By contrast, only EcR-A is able to support development in the margins of the wing discs, and only EcR-B2 can do so in the larval epidermis and the border cells of the developing egg chamber. In light of our results, the simplest explanations for the widespread spatial and temporal variations in EcR isoform titers appear untenable.