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1.
Elife ; 132024 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-39221782

RESUMEN

The initially homogeneous epithelium of the early Drosophila embryo differentiates into regional subpopulations with different behaviours and physical properties that are needed for morphogenesis. The factors at top of the genetic hierarchy that control these behaviours are known, but many of their targets are not. To understand how proteins work together to mediate differential cellular activities, we studied in an unbiased manner the proteomes and phosphoproteomes of the three main cell populations along the dorso-ventral axis during gastrulation using mutant embryos that represent the different populations. We detected 6111 protein groups and 6259 phosphosites of which 3398 and 3433 respectively, were differentially regulated. The changes in phosphosite abundance did not correlate with changes in host protein abundance, showing phosphorylation to be a regulatory step during gastrulation. Hierarchical clustering of protein groups and phosphosites identified clusters that contain known fate determinants such as Doc1, Sog, Snail and Twist. The recovery of the appropriate known marker proteins in each of the different mutants we used validated the approach, but also revealed that two mutations that both interfere with the dorsal fate pathway, Toll10B and serpin27aex do this in very different manners. Diffused network analyses within each cluster point to microtubule components as one of the main groups of regulated proteins. Functional studies on the role of microtubules provide the proof of principle that microtubules have different functions in different domains along the DV axis of the embryo.

2.
Cell ; 182(6): 1490-1507.e19, 2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32916131

RESUMEN

Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. Here we demonstrate that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, we find that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). We combine targeted metabolomics and in vivo genetic screening to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD+ sensor, we show that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD+ regeneration. Our work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Carcinogénesis/metabolismo , Transformación Celular Neoplásica/metabolismo , Dinámicas Mitocondriales , NAD/metabolismo , Células Madre Neoplásicas/metabolismo , Células-Madre Neurales/metabolismo , Fosforilación Oxidativa , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Carcinogénesis/genética , Carcinogénesis/patología , Transformación Celular Neoplásica/patología , Ciclo del Ácido Cítrico/genética , Biología Computacional , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glucólisis/genética , Espectrometría de Masas , Metabolómica , Microscopía Electrónica de Transmisión , Familia de Multigenes , Células-Madre Neurales/patología , Consumo de Oxígeno/genética , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Análisis de la Célula Individual , Transcriptoma/genética
3.
Development ; 146(6)2019 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-30923056

RESUMEN

Cell type specification during early nervous system development in Drosophila melanogaster requires precise regulation of gene expression in time and space. Resolving the programs driving neurogenesis has been a major challenge owing to the complexity and rapidity with which distinct cell populations arise. To resolve the cell type-specific gene expression dynamics in early nervous system development, we have sequenced the transcriptomes of purified neurogenic cell types across consecutive time points covering crucial events in neurogenesis. The resulting gene expression atlas comprises a detailed resource of global transcriptome dynamics that permits systematic analysis of how cells in the nervous system acquire distinct fates. We resolve known gene expression dynamics and uncover novel expression signatures for hundreds of genes among diverse neurogenic cell types, most of which remain unstudied. We also identified a set of conserved long noncoding RNAs (lncRNAs) that are regulated in a tissue-specific manner and exhibit spatiotemporal expression during neurogenesis with exquisite specificity. lncRNA expression is highly dynamic and demarcates specific subpopulations within neurogenic cell types. Our spatiotemporal transcriptome atlas provides a comprehensive resource for investigating the function of coding genes and noncoding RNAs during crucial stages of early neurogenesis.


Asunto(s)
Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Sistema Nervioso/embriología , Neurogénesis/genética , ARN Largo no Codificante/genética , Animales , Linaje de la Célula , Drosophila melanogaster/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Hibridación Fluorescente in Situ , Neuroglía/fisiología , Filogenia , Transcriptoma
4.
RNA Biol ; 16(1): 69-81, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30582411

RESUMEN

During Drosophila melanogaster embryogenesis, tight regulation of gene expression in time and space is required for the orderly emergence of specific cell types. While the general importance of microRNAs in regulating eukaryotic gene expression has been well-established, their role in early neurogenesis remains to be addressed. In this survey, we investigate the transcriptional dynamics of microRNAs and their target transcripts during neurogenesis of Drosophila melanogaster. To this end, we use the recently developed DIV-MARIS protocol, a method for enriching specific cell types from the Drosophila embryo in vivo, to sequence cell type-specific transcriptomes. We generate dedicated small and total RNA-seq libraries for neuroblasts, neurons and glia cells at early (6-8 h after egg laying (AEL)) and late (18-22 h AEL) stage. This allows us to directly compare these transcriptomes and investigate the potential functional roles of individual microRNAs with spatiotemporal resolution genome-wide, which is beyond the capabilities of existing in situ hybridization methods. Overall, we identify 74 microRNAs that are significantly differentially expressed between the three cell types and the two developmental stages. In all cell types, predicted target genes of down-regulated microRNAs show a significant enrichment of Gene Ontology terms related to neurogenesis. We also investigate how microRNAs regulate the transcriptome by targeting transcription factors and find many candidate microRNAs with putative roles in neurogenesis. Our survey highlights the roles of microRNAs as regulators of differentiation and glioneurognesis in the fruit fly and provides distinct starting points for dedicated functional follow-up studies.


Asunto(s)
Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Neurogénesis/genética , Interferencia de ARN , ARN Mensajero/genética , Transcriptoma , Animales , Linaje de la Célula/genética , Biología Computacional/métodos , Drosophila/embriología , Desarrollo Embrionario/genética , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Especificidad de Órganos/genética
5.
Nat Commun ; 9(1): 4472, 2018 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-30367057

RESUMEN

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general feature of all eukaryotic cis regulatory elements. To address this, here we define cis regulatory elements in C. elegans, D. melanogaster and H. sapiens and investigate the determinants of their transcription directionality. In all three species, we find that divergent transcription is initiated from two separate core promoter sequences and promoter regions display competition between histone modifications on the + 1 and -1 nucleosomes. In contrast, promoter directionality, sequence composition surrounding promoters, and positional enrichment of chromatin states, are different across species. Integrative models of H3K4me3 levels and core promoter sequence are highly predictive of promoter and enhancer directionality and support two directional classes, skewed and balanced. The relative importance of features to these models are clearly distinct for promoters and enhancers. Differences in regulatory architecture within and between metazoans are therefore abundant, arguing against a unified eukaryotic model.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética , Animales , Caenorhabditis elegans/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Código de Histonas , Humanos , Modelos Genéticos , Nucleosomas/metabolismo
6.
Genome Biol ; 18(1): 199, 2017 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-29070071

RESUMEN

Transcriptional enhancers regulate spatio-temporal gene expression. While genomic assays can identify putative enhancers en masse, assigning target genes is a complex challenge. We devised a machine learning approach, McEnhancer, which links target genes to putative enhancers via a semi-supervised learning algorithm that predicts gene expression patterns based on enriched sequence features. Predicted expression patterns were 73-98% accurate, predicted assignments showed strong Hi-C interaction enrichment, enhancer-associated histone modifications were evident, and known functional motifs were recovered. Our model provides a general framework to link globally identified enhancers to targets and contributes to deciphering the regulatory genome.


Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Aprendizaje Automático , Animales , Desoxirribonucleasa I , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Desarrollo Embrionario/genética , Genes Reporteros , Código de Histonas , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo
7.
Science ; 358(6360): 194-199, 2017 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-28860209

RESUMEN

By the onset of morphogenesis, Drosophila embryos consist of about 6000 cells that express distinct gene combinations. Here, we used single-cell sequencing of precisely staged embryos and devised DistMap, a computational mapping strategy to reconstruct the embryo and to predict spatial gene expression approaching single-cell resolution. We produced a virtual embryo with about 8000 expressed genes per cell. Our interactive Drosophila Virtual Expression eXplorer (DVEX) database generates three-dimensional virtual in situ hybridizations and computes gene expression gradients. We used DVEX to uncover patterned expression of transcription factors and long noncoding RNAs, as well as signaling pathway components. Spatial regulation of Hippo signaling during early embryogenesis suggests a mechanism for establishing asynchronous cell proliferation. Our approach is suitable to generate transcriptomic blueprints for other complex tissues.


Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Comunicación Celular , Proteínas de Drosophila/genética , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética
8.
EMBO J ; 35(1): 1-3, 2016 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-26567170

RESUMEN

Cell fate decisions require the deployment of distinct transcriptional programmes - how this is controlled and orchestrated is a key question from basic developmental biology to regenerative medicine. In this issue of The EMBO Journal, Pataskar and Jung et al (Pataskar et al, 2015) demonstrate how the transcription factor NeuroD1 acts genome-wide to elicit a specific neurogenic programme, including differentiation and migration. Much of that activity is due to NeuroD1 acting as a pioneer factor. NeuroD1 is able to bind its targets within repressive chromatin and can induce a more open chromatin state amenable to cell type­specific regulation.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neuronas/fisiología , Factores de Transcripción/metabolismo , Animales
9.
J Biol Chem ; 290(33): 20284-94, 2015 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-26152724

RESUMEN

In response to fasting or hyperglycemia, the pancreatic ß-cell alters its output of secreted insulin; however, the pathways governing this adaptive response are not entirely established. Although the precise role of microRNAs (miRNAs) is also unclear, a recurring theme emphasizes their function in cellular stress responses. We recently showed that miR-184, an abundant miRNA in the ß-cell, regulates compensatory proliferation and secretion during insulin resistance. Consistent with previous studies showing miR-184 suppresses insulin release, expression of this miRNA was increased in islets after fasting, demonstrating an active role in the ß-cell as glucose levels lower and the insulin demand ceases. Additionally, miR-184 was negatively regulated upon the administration of a sucrose-rich diet in Drosophila, demonstrating strong conservation of this pathway through evolution. Furthermore, miR-184 and its target Argonaute2 remained inversely correlated as concentrations of extracellular glucose increased, underlining a functional relationship between this miRNA and its targets. Lastly, restoration of Argonaute2 in the presence of miR-184 rescued suppression of miR-375-targeted genes, suggesting these genes act in a coordinated manner during changes in the metabolic context. Together, these results highlight the adaptive role of miR-184 according to glucose metabolism and suggest the regulatory role of this miRNA in energy homeostasis is highly conserved.


Asunto(s)
Glucosa/metabolismo , Islotes Pancreáticos/fisiología , MicroARNs/fisiología , Animales , Proteínas Argonautas/metabolismo , Línea Celular , Homeostasis/fisiología , Islotes Pancreáticos/metabolismo , Ratones , MicroARNs/genética , Mitocondrias/metabolismo
10.
Genes Dev ; 28(2): 167-81, 2014 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-24402316

RESUMEN

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ∼25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, we identified 106 genes that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of "Snail-activated" genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, we show that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which we demonstrate is essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development.


Asunto(s)
Drosophila/genética , Drosophila/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Animales , Drosophila/embriología , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Unión Proteica , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist/metabolismo
11.
Nat Protoc ; 7(5): 978-94, 2012 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-22538849

RESUMEN

This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.


Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Drosophila/genética , Histonas/metabolismo , Animales , Sitios de Unión , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Elementos de Facilitación Genéticos , Citometría de Flujo , Regulación de la Expresión Génica , Biblioteca de Genes , Genes Reporteros , Histonas/química , Sonicación , Factores de Transcripción/química , Factores de Transcripción/metabolismo
12.
Nat Genet ; 44(2): 148-56, 2012 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-22231485

RESUMEN

Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type-specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks.


Asunto(s)
Drosophila melanogaster/embriología , Elementos de Facilitación Genéticos , Animales , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo
13.
Nature ; 462(7269): 65-70, 2009 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-19890324

RESUMEN

Development requires the establishment of precise patterns of gene expression, which are primarily controlled by transcription factors binding to cis-regulatory modules. Although transcription factor occupancy can now be identified at genome-wide scales, decoding this regulatory landscape remains a daunting challenge. Here we used a novel approach to predict spatio-temporal cis-regulatory activity based only on in vivo transcription factor binding and enhancer activity data. We generated a high-resolution atlas of cis-regulatory modules describing their temporal and combinatorial occupancy during Drosophila mesoderm development. The binding profiles of cis-regulatory modules with characterized expression were used to train support vector machines to predict five spatio-temporal expression patterns. In vivo transgenic reporter assays demonstrate the high accuracy of these predictions and reveal an unanticipated plasticity in transcription factor binding leading to similar expression. This data-driven approach does not require previous knowledge of transcription factor sequence affinity, function or expression, making it widely applicable.


Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Modelos Genéticos , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Inteligencia Artificial , Inmunoprecipitación de Cromatina , Secuencia Conservada/genética , Bases de Datos Genéticas , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Reporteros/genética , Mesodermo/embriología , Mesodermo/metabolismo , Unión Proteica , Factores de Tiempo
14.
Genome Biol ; 9(11): 240, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19012800

RESUMEN

Many essential transcription factors have conserved roles in regulating biological programs, yet their genomic occupancy can diverge significantly. A new study demonstrates that such variations are primarily due to cis-regulatory sequences, rather than differences between the regulators or nuclear environments.


Asunto(s)
Evolución Molecular , Redes Reguladoras de Genes , Factores de Transcripción/genética , Animales , ARN Polimerasas Dirigidas por ADN , Humanos , Especificidad de la Especie , Transcripción Genética
15.
Development ; 134(13): 2415-24, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17522157

RESUMEN

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.


Asunto(s)
Anopheles/embriología , Anopheles/metabolismo , Evolución Biológica , Tipificación del Cuerpo , Animales , Anopheles/citología , Anopheles/genética , Diferenciación Celular , Linaje de la Célula , Forma de la Célula , Drosophila melanogaster , Ectodermo/citología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hormonas de Insectos/genética , Hormonas de Insectos/metabolismo , Mesodermo/citología , Neuronas/citología , Pirazoles , Membrana Serosa/citología , Transducción de Señal , Factores de Tiempo
16.
PLoS Comput Biol ; 3(5): e84, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17500585

RESUMEN

Formation of spatial gene expression patterns in development depends on transcriptional responses mediated by gene control regions, enhancers. Here, we explore possible responses of enhancers to overlapping gradients of antagonistic transcriptional regulators in the Drosophila embryo. Using quantitative models based on enhancer structure, we demonstrate how a pair of antagonistic transcription factor gradients with similar or even identical spatial distributions can lead to the formation of distinct gene expression domains along the embryo axes. The described mechanisms are sufficient to explain the formation of the anterior and the posterior knirps expression, the posterior hunchback expression domain, and the lateral stripes of rhomboid expression and of other ventral neurogenic ectodermal genes. The considered principles of interaction between antagonistic gradients at the enhancer level can also be applied to diverse developmental processes, such as domain specification in imaginal discs, or even eyespot pattern formation in the butterfly wing.


Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Drosophila/fisiología , Drosophila/embriología , Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Biológicos , Factores de Transcripción/fisiología , Adaptación Fisiológica/fisiología , Animales , Simulación por Computador , Secuencias Reguladoras de Ácidos Nucleicos/fisiología
17.
Genes Dev ; 21(4): 385-90, 2007 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17322397

RESUMEN

Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. We determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. Our analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Tipificación del Cuerpo/genética , Inmunoprecipitación de Cromatina , Proteínas de Drosophila/análisis , Drosophila melanogaster/genética , Embrión no Mamífero/química , Embrión no Mamífero/metabolismo , Elementos de Facilitación Genéticos , Proteínas Nucleares/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/análisis , Factores de Transcripción de la Familia Snail , Factores de Transcripción/análisis , Proteína 1 Relacionada con Twist/análisis
18.
Dev Cell ; 11(6): 895-902, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17141163

RESUMEN

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D. mel.) and the mosquito Anopheles gambiae (A. gam.), the midline is narrow and spans just 1-2 cells. However, in the honeybee, Apis mellifera (A. mel.), the ventral midline is broad and encompasses 5-6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D. mel., but it is activated solely by Twi in A. mel. We suggest that the Twi-only mode of regulation--and the broad ventral midline--represents the ancestral form of CNS patterning in Holometabolous insects.


Asunto(s)
Anopheles/embriología , Abejas/embriología , Evolución Biológica , Sistema Nervioso Central/citología , Drosophila melanogaster/embriología , Embrión no Mamífero , Animales , Anopheles/genética , Anopheles/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Abejas/genética , Abejas/metabolismo , Tipificación del Cuerpo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos , Larva/citología , Larva/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transcripción Genética , Transgenes/fisiología , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo
19.
Curr Biol ; 16(13): 1358-65, 2006 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-16750631

RESUMEN

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.


Asunto(s)
Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Genes del Desarrollo , Sistema Nervioso/embriología , Animales , Secuencia de Bases , Simulación por Computador , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Elementos de Facilitación Genéticos , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Sistema Nervioso/metabolismo , Neurregulinas/genética , Neurregulinas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Alineación de Secuencia , Factores de Transcripción de la Familia Snail , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo
20.
Mol Cell Biol ; 24(2): 912-23, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14701761

RESUMEN

Telomeres are synthesized by telomerase, a specialized reverse transcriptase, which contains a template in its intrinsic RNA component. In Kluyveromyces lactis, the repeats synthesized by the wild-type telomerase are 25 nucleotides (nt) in length and uniform in sequence. To determine the role of the 5-nt repeats defining the ends of the K. lactis telomerase RNA template in telomerase translocation, we have made mutations in and around them and observed their effects on telomere length and the sequence of newly made telomeric repeats. These template mutations typically result in telomeres that are shorter than those of wild-type cells. The mismatches between the telomerase template and the telomeric tip that occur after telomerase-mediated incorporation of the mutations are normally not removed. Instead, the mutations lead to the synthesis of aberrant repeats that range in size from 31 to 13 bp. Therefore, the specificity with which the telomeric tip aligns with the telomere is critical for the production of the uniform repeats seen in K. lactis. In addition, the region immediately 3' of the template may play an important role in translocation of the enzyme.


Asunto(s)
Kluyveromyces/enzimología , Telomerasa/metabolismo , Disparidad de Par Base , Secuencia de Bases , Transporte Biológico Activo , ADN Bacteriano/genética , Genes Bacterianos , Kluyveromyces/genética , Datos de Secuencia Molecular , Mutación , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Telomerasa/genética , Secuencias Repetidas Terminales
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