RESUMEN
Identifying functional enhancer elements in metazoan systems is a major challenge. Large-scale validation of enhancers predicted by ENCODE reveal false-positive rates of at least 70%. We used the pregrastrula-patterning network of Drosophila melanogaster to demonstrate that loss in accuracy in held-out data results from heterogeneity of functional signatures in enhancer elements. We show that at least two classes of enhancers are active during early Drosophila embryogenesis and that by focusing on a single, relatively homogeneous class of elements, greater than 98% prediction accuracy can be achieved in a balanced, completely held-out test set. The class of well-predicted elements is composed predominantly of enhancers driving multistage segmentation patterns, which we designate segmentation driving enhancers (SDE). Prediction is driven by the DNA occupancy of early developmental transcription factors, with almost no additional power derived from histone modifications. We further show that improved accuracy is not a property of a particular prediction method: after conditioning on the SDE set, naïve Bayes and logistic regression perform as well as more sophisticated tools. Applying this method to a genome-wide scan, we predict 1,640 SDEs that cover 1.6% of the genome. An analysis of 32 SDEs using whole-mount embryonic imaging of stably integrated reporter constructs chosen throughout our prediction rank-list showed >90% drove expression patterns. We achieved 86.7% precision on a genome-wide scan, with an estimated recall of at least 98%, indicating high accuracy and completeness in annotating this class of functional elements.
Asunto(s)
Proteínas de Drosophila , Embrión no Mamífero/embriología , Desarrollo Embrionario/fisiología , Elementos de Facilitación Genéticos/fisiología , Análisis de Secuencia de ADN , Factores de Transcripción , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estudio de Asociación del Genoma Completo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
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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
Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.
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Empalme Alternativo , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas de Unión al ARN/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Animales , Proteínas de Drosophila/metabolismo , Exones , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Interferencia de ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARN , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
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Drosophila melanogaster/genética , Genoma , Animales , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Biología Computacional , Mapeo Contig , Secuenciación de Nucleótidos de Alto Rendimiento , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Cromosomas Politénicos , Mapeo RestrictivoRESUMEN
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.
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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
Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.
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Biología Computacional , Drosophila melanogaster/genética , Genoma de los Insectos/genética , Regiones Promotoras Genéticas , Regiones no Traducidas 3'/genética , Animales , Mapeo Cromosómico , Drosophila melanogaster/embriología , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Sitio de Iniciación de la TranscripciónRESUMEN
Synthetic biology is creating genetically engineered organisms at an increasing rate for many potentially valuable applications, but this potential comes with the risk of misuse or accidental release. To begin to address this issue, we have developed a system called GUARDIAN that can automatically detect signatures of engineering in DNA sequencing data, and we have conducted a blinded test of this system using a curated Test and Evaluation (T&E) data set. GUARDIAN uses an ensemble approach based on the guiding principle that no single approach is likely to be able to detect engineering with perfect accuracy. Critically, ensembling enables GUARDIAN to detect sequence inserts in 13 target organisms with a high degree of specificity that requires no subject matter expert (SME) review.
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ADN , Análisis de Secuencia de ADN , ADN/genéticaRESUMEN
Transcription factors (TFs) play a key role in development and in cellular responses to the environment by activating or repressing the transcription of target genes in precise spatial and temporal patterns. In order to develop a catalog of target genes of Drosophila melanogaster TFs, the modERN consortium systematically knocked down the expression of TFs using RNAi in whole embryos followed by RNA-seq. We generated data for 45 TFs which have 18 different DNA-binding domains and are expressed in 15 of the 16 organ systems. The range of inactivation of the targeted TFs by RNAi ranged from log2fold change -3.52 to +0.49. The TFs also showed remarkable heterogeneity in the numbers of candidate target genes identified, with some generating thousands of candidates and others only tens. We present detailed analysis from five experiments, including those for three TFs that have been the focus of previous functional studies (ERR, sens, and zfh2) and two previously uncharacterized TFs (sens-2 and CG32006), as well as short vignettes for selected additional experiments to illustrate the utility of this resource. The RNA-seq datasets are available through the ENCODE DCC (http://encodeproject.org) and the Sequence Read Archive (SRA). TF and target gene expression patterns can be found here: https://insitu.fruitfly.org. These studies provide data that facilitate scientific inquiries into the functions of individual TFs in key developmental, metabolic, defensive, and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks during embryogenesis.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interferencia de ARN , Factores de Transcripción/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas de Unión al ADN/genéticaRESUMEN
Microbacterium sp. BDGP8 is a species of facultative anaerobic gram-positive bacterium of the family Microbacteriaceae. The complete genome consists of a single circular chromosome of 3,293,567 bp with a G + C content of 69.84% and two plasmids of 49,365 bp and 32,884 bp.
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Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.
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Proteínas de Drosophila , Mapas de Interacción de Proteínas , Animales , Mapas de Interacción de Proteínas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mapeo de Interacción de Proteínas/métodos , Técnicas del Sistema de Dos HíbridosRESUMEN
The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.
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Atrazina/toxicidad , Drosophila melanogaster/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Interacciones Microbiota-Huesped/efectos de los fármacos , Insecticidas/toxicidad , Acetobacter/genética , Acetobacter/metabolismo , Animales , Drosophila melanogaster/microbiología , Femenino , Inactivación Metabólica , MasculinoRESUMEN
Lactobacillus brevis Oregon-R-modENCODE strain BDGP6 was isolated from the gut of Drosophila melanogaster for functional host-microbial interaction studies. The bacterial chromosome is a single circular DNA molecule of 2,785,111 bp with a G+C content of 46%.
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Citrobacter freundii is a species of facultative anaerobic Gram-negative bacteria of the family Enterobacteriaceae The complete genome is composed of a single chromosomal circle of 4,957,773 bp with a G+C content of 52%.
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High-content image acquisition is generally limited to cells grown in culture, requiring complex hardware and preset imaging modalities. Here we report an open source software package, OpenHiCAMM (Open Hi Content Acquisition for µManager), that provides a flexible framework for integration of generic microscope-associated robotics and image processing with sequential work-flows. As an example, we imaged Drosophila embryos, detecting the embryos at low resolution, followed by re-imaging the detected embryos at high resolution, suitable for computational analysis and screening. The OpenHiCAMM package is easy to use and adapt for automating complex microscope image tasks. It expands our abilities for high-throughput image-based screens to a new range of biological samples, such as organoids, and will provide a foundation for bioimaging systems biology.
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Acetobacter pomorum Oregon-R-modENCODE strain BDGP5 was isolated from Drosophila melanogaster for functional host-microbe interaction studies. The complete genome is composed of a single chromosomal circle of 2,848,089 bp, with a G+C content of 53% and three plasmids of 131,455 bp, 19,216 bp, and 9,160 bp.
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Lactobacillus plantarum Oregon-R-modENCODE strain BDGP2 was isolated from the gut of Drosophila melanogaster for functional host microbial interaction studies. The complete genome comprised a single circular genome of 3,407,160 bp, with a G+C content of 44%, and four plasmids.
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Acetobacter tropicalis Oregon-R-modENCODE strain BDGP1 was isolated from Drosophila melanogaster for functional host-microbe interaction studies. The complete genome comprises a single chromosomal circle of 3,988,649 bp with a G+C content of 56% and a conjugative plasmid of 151,013 bp.
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Enterococcus durans Oregon-R-modENCODE strain BDGP3 was isolated from the Drosophila melanogaster gut for functional host-microbe interaction studies. The complete genome is composed of a single circular genome of 2,983,334 bp, with a G+C content of 38%, and a single plasmid of 5,594 bp.
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Bacillus kochii Oregon-R-modENCODE strain BDGP4 was isolated from the gut of Drosophila melanogaster for functional host microbial interaction studies. The complete genome comprised a single chromosomal circle of 4,557,232 bp with a G+C content of 37% and a single plasmid of 137,143 bp.
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Here, we document a collection of â¼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.