RESUMO
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.
Assuntos
Proteínas de Drosophila , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Análise de Sequência de DNA , Fatores de Transcrição , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estudo de Associação Genômica Ampla , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Transcriptoma/genética , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Cromatina/genética , Análise por Conglomerados , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Modelos Genéticos , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , RNA não Traduzido/genética , Análise de Sequência de RNARESUMO
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.
Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Feminino , Masculino , Anotação de Sequência Molecular , Tecido Nervoso/metabolismo , Especificidade de Órgãos , Poli A/genética , Poliadenilação , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Estresse Fisiológico/genéticaRESUMO
Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set ofDrosophilaearly embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identified 21 principal patterns (PP). Providing a compact yet biologically interpretable representation ofDrosophilaexpression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. The performance of PP with theDrosophiladata suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.
Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica/fisiologia , Redes Reguladoras de Genes/fisiologia , Modelos Genéticos , Animais , Drosophila melanogasterRESUMO
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.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcrição Gênica/genética , Processamento Alternativo/genética , Animais , Sequência de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Éxons/genética , Feminino , Genes de Insetos/genética , Genoma de Inseto/genética , Masculino , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Edição de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/genética , Análise de Sequência , Caracteres SexuaisRESUMO
In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.
Assuntos
DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Feminino , Genoma de Inseto/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Ligação Proteica/genéticaRESUMO
A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the modERN (model organism Encyclopedia of Regulatory Networks) consortium that systematically assayed TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). We describe key features of these datasets, comprising 604 TFs identifying 3.6M sites in the fly and 350 TFs identifying 0.9 M sites in the worm. Applying a machine learning model to these data identifies sets of TFs with a prominent role in promoting target gene expression in specific cell types. TF binding data are available through the ENCODE Data Coordinating Center and at https://epic.gs.washington.edu/modERNresource, which provides access to processed and summary data, as well as widgets to probe cell type-specific TF-target relationships. These data are a rich resource that should fuel investigations into TF function during development.
RESUMO
Cis-regulatory modules that drive precise spatial-temporal patterns of gene expression are central to the process of metazoan development. We describe a new computational strategy to annotate genomic sequences based on their "pattern generating potential" and to produce quantitative descriptions of transcriptional regulatory networks at the level of individual protein-module interactions. We use this approach to convert the qualitative understanding of interactions that regulate Drosophila segmentation into a network model in which a confidence value is associated with each transcription factor-module interaction. Sequence information from multiple Drosophila species is integrated with transcription factor binding specificities to determine conserved binding site frequencies across the genome. These binding site profiles are combined with transcription factor expression information to create a model to predict module activity patterns. This model is used to scan genomic sequences for the potential to generate all or part of the expression pattern of a nearby gene, obtained from available gene expression databases. Interactions between individual transcription factors and modules are inferred by a statistical method to quantify a factor's contribution to the module's pattern generating potential. We use these pattern generating potentials to systematically describe the location and function of known and novel cis-regulatory modules in the segmentation network, identifying many examples of modules predicted to have overlapping expression activities. Surprisingly, conserved transcription factor binding site frequencies were as effective as experimental measurements of occupancy in predicting module expression patterns or factor-module interactions. Thus, unlike previous module prediction methods, this method predicts not only the location of modules but also their spatial activity pattern and the factors that directly determine this pattern. As databases of transcription factor specificities and in vivo gene expression patterns grow, analysis of pattern generating potentials provides a general method to decode transcriptional regulatory sequences and networks.
Assuntos
Padronização Corporal , Biologia Computacional/métodos , Drosophila/embriologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Padronização Corporal/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Modelos Genéticos , Ligação Proteica , Software , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
Discovery of temporal and spatial patterns of gene expression is essential for understanding the regulatory networks and development in multicellular organisms. We analyzed the images from our large-scale spatial expression data set of early Drosophila embryonic development and present a comprehensive computational image analysis of the expression landscape. For this study, we created an innovative virtual representation of embryonic expression patterns using an elliptically shaped mesh grid that allows us to make quantitative comparisons of gene expression using a common frame of reference. Demonstrating the power of our approach, we used gene co-expression to identify distinct expression domains in the early embryo; the result is surprisingly similar to the fate map determined using laser ablation. We also used a clustering strategy to find genes with similar patterns and developed new analysis tools to detect variation within consensus patterns, adjacent non-overlapping patterns, and anti-correlated patterns. Of the 1800 genes investigated, only half had previously assigned functions. The known genes suggest developmental roles for the clusters, and identification of related patterns predicts requirements for co-occurring biological functions.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Processamento de Sinais Assistido por Computador , Biologia de Sistemas , Animais , Análise por Conglomerados , Gráficos por Computador , Bases de Dados Genéticas , Drosophila/embriologia , Redes Reguladoras de Genes , Fatores de TempoRESUMO
We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.
Assuntos
Drosophila melanogaster , Neurônios/metabolismo , Neurociências/métodos , Recombinação Genética/genética , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica/métodos , Encéfalo/citologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de InsetosRESUMO
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.
Assuntos
Atrazina/toxicidade , Drosophila melanogaster/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Inseticidas/toxicidade , Acetobacter/genética , Acetobacter/metabolismo , Animais , Drosophila melanogaster/microbiologia , Feminino , Inativação Metabólica , MasculinoRESUMO
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%.
RESUMO
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.
RESUMO
To develop a catalog of regulatory sites in two major model organisms, Drosophila melanogaster and Caenorhabditis elegans, the modERN (model organism Encyclopedia of Regulatory Networks) consortium has systematically assayed the binding sites of transcription factors (TFs). Combined with data produced by our predecessor, modENCODE (Model Organism ENCyclopedia Of DNA Elements), we now have data for 262 TFs identifying 1.23 M sites in the fly genome and 217 TFs identifying 0.67 M sites in the worm genome. Because sites from different TFs are often overlapping and tightly clustered, they fall into 91,011 and 59,150 regions in the fly and worm, respectively, and these binding sites span as little as 8.7 and 5.8 Mb in the two organisms. Clusters with large numbers of sites (so-called high occupancy target, or HOT regions) predominantly associate with broadly expressed genes, whereas clusters containing sites from just a few factors are associated with genes expressed in tissue-specific patterns. All of the strains expressing GFP-tagged TFs are available at the stock centers, and the chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center and also through a simple interface (http://epic.gs.washington.edu/modERN/) that facilitates rapid accessibility of processed data sets. These data will facilitate a vast number of scientific inquiries into the function of individual TFs in key developmental, metabolic, and defense and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks and globally across the life spans of these two key model organisms.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Bases de Dados Genéticas , Drosophila/genética , Drosophila/metabolismo , Estudo de Associação Genômica Ampla , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina , Estudo de Associação Genômica Ampla/métodos , Modelos Biológicos , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
The Stubble-stubbloid (Sb-sbd) gene is required for ecdysone-regulated epithelial morphogenesis of imaginal tissues during Drosophila metamorphosis. Mutations in Sb-sbd are associated with defects in apical cell shape changes critical for the evagination of the leg imaginal disc and with defects in assembly and extension of parallel actin bundles in growing mechanosensory bristles. The Sb-sbd gene encodes a type II transmembrane serine protease (TTSP). Here we use a Sb-sbd transgenic construct to rescue both bristle and leg morphogenesis defects in Sb-sbd mutations. Molecular characterization of Sb-sbd mutations and rescue experiments with wild-type and modified Sb-sbd transgenic constructs show that the protease domain is required for both leg and bristle functions. Truncated proteins that express the noncatalytic domains without the protease have dominant effects in bristles but not in legs. Leg morphogenesis, but not bristle growth, is sensitive to Sb-sbd overexpression. Antibody localization of the Sb-sbd protein shows apical expression in elongating legs. Sb-sbd protein is found in the base and shaft in budding bristles and then concentrates at the growing tip when bristles are elongating rapidly. We propose a model whereby Sb-sbd helps coordinate proteolytic modification of extracellular matrix attachments with cytoskeletal changes in both legs and bristles.
Assuntos
Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/genética , Extremidades/crescimento & desenvolvimento , Proteínas de Membrana/genética , Serina Endopeptidases/genética , Animais , Animais Geneticamente Modificados , Citoesqueleto/enzimologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Análise Mutacional de DNA , Drosophila/enzimologia , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Matriz Extracelular/enzimologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Extremidades/fisiologia , Genes Dominantes , Hidrólise , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Mutação , Fenótipo , Polimorfismo Genético , Serina Endopeptidases/química , Serina Endopeptidases/fisiologia , Relação Estrutura-Atividade , TransgenesRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.