RESUMEN
BACKGROUND: Genome-wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman primates (NHPs), and humans. However, these data were obtained from cross-sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within-subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model. METHODS: Two cohorts of cynomolgus macaques (n = 23) underwent a schedule-induced polydipsia procedure to establish EtOH self-administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH-naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA-Seq paired analysis, which allowed for correction of individual baseline differences in gene expression. RESULTS: A total of 675 genes were significantly down-regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up-regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll-like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites. CONCLUSIONS: The data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro- and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.
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Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/metabolismo , Etanol/administración & dosificación , Perfilación de la Expresión Génica/métodos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/fisiología , Animales , Expresión Génica , Macaca fascicularis , Masculino , AutoadministraciónRESUMEN
This is the first description of the relationship between chronic ethanol self-administration and the brain transcriptome in a non-human primate (rhesus macaque). Thirty-one male animals self-administered ethanol on a daily basis for over 12 months. Gene transcription was quantified with RNA-Seq in the central nucleus of the amygdala (CeA) and cortical Area 32. We constructed coexpression and cosplicing networks, and we identified areas of preservation and areas of differentiation between regions and network types. Correlations between intake and transcription included largely distinct gene sets and annotation categories across brain regions and between expression and splicing; positive and negative correlations were also associated with distinct annotation groups. Membrane, synaptic and splicing annotation categories were over-represented in the modules (gene clusters) enriched in positive correlations (CeA); our cosplicing analysis further identified the genes affected only at the exon inclusion level. In the CeA coexpression network, we identified Rab6b, Cdk18 and Igsf21 among the intake-correlated hubs, while in the Area 32, we identified a distinct hub set that included Ppp3r1 and Myeov2. Overall, the data illustrate that excessive ethanol self-administration is associated with broad expression and splicing mechanisms that involve membrane and synapse genes.
Asunto(s)
Consumo de Bebidas Alcohólicas/genética , Encéfalo/metabolismo , Depresores del Sistema Nervioso Central/administración & dosificación , Etanol/administración & dosificación , Consumo de Bebidas Alcohólicas/metabolismo , Animales , Calcineurina/genética , Núcleo Amigdalino Central/metabolismo , Corteza Cerebral/metabolismo , Quinasas Ciclina-Dependientes/genética , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Macaca mulatta , Masculino , Proteínas del Tejido Nervioso/genética , Empalme del ARN , Autoadministración , Proteínas de Unión al GTP rab/genéticaRESUMEN
BACKGROUND: The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. RESULTS: We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. CONCLUSIONS: Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding the utilization of the CC populations in the study of complex traits. This report is also the first to establish guidelines in defining and identifying strain-specific splicing across different mouse strains.
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Ratones Endogámicos/genética , Empalme del ARN/genética , Transcriptoma , Animales , Genoma , Ratones , Anotación de Secuencia Molecular , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine- related molecules distinct from other lung-resident CD8+ T cells, such as interferon-γ- and IL-12- receptors. These data highlight MAIT cells' predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.
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Antiinfecciosos , Células T Invariantes Asociadas a Mucosa , Antibacterianos , Linfocitos T CD8-positivos , Citocinas , Humanos , PulmónRESUMEN
This review summarizes the proceedings of a symposium presented at the "Alcoholism and Stress: A Framework for Future Treatment Strategies" conference held in Volterra, Italy on May 9-12, 2017. Psychiatric diseases, including alcohol-use disorders (AUDs), are influenced through complex interactions of genes, neurobiological pathways, and environmental influences. A better understanding of the common neurobiological mechanisms underlying an AUD necessitates an integrative approach, involving a systematic assessment of diverse species and phenotype measures. As part of the World Congress on Stress and Alcoholism, this symposium provided a detailed account of current strategies to identify mechanisms underlying the development and progression of AUDs. Dr. Sean Farris discussed the integration and organization of transcriptome and postmortem human brain data to identify brain regional- and cell type-specific differences related to excessive alcohol consumption that are conserved across species. Dr. Brien Riley presented the results of a genome-wide association study of DSM-IV alcohol dependence; although replication of genetic associations with alcohol phenotypes in humans remains challenging, model organism studies show that COL6A3, KLF12, and RYR3 affect behavioral responses to ethanol, and provide substantial evidence for their role in human alcohol-related traits. Dr. Rob Williams expanded upon the systematic characterization of extensive genetic-genomic resources for quantifying and clarifying phenotypes across species that are relevant to precision medicine in human disease. The symposium concluded with Dr. Robert Hitzemann's description of transcriptome studies in a mouse model selectively bred for high alcohol ("binge-like") consumption and a non-human primate model of long-term alcohol consumption. Together, the different components of this session provided an overview of systems-based approaches that are pioneering the experimental prioritization and validation of novel genes and gene networks linked with a range of behavioral phenotypes associated with stress and AUDs.
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Consumo de Bebidas Alcohólicas/genética , Trastornos Relacionados con Alcohol/genética , Animales , Colágeno Tipo VI/genética , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Macaca , Ratones , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
BACKGROUND: Recent highly publicized cases of premature patient assignment into clinical trials, resulting from non-reproducible omics analyses, have prompted many to call for a more thorough examination of translational omics and highlighted the critical need for transparency and reproducibility to ensure patient safety. The use of workflow platforms such as Galaxy and Taverna have greatly enhanced the use, transparency and reproducibility of omics analysis pipelines in the research domain and would be an invaluable tool in a clinical setting. However, the use of these workflow platforms requires deep domain expertise that, particularly within the multi-disciplinary fields of translational and clinical omics, may not always be present in a clinical setting. This lack of domain expertise may put patient safety at risk and make these workflow platforms difficult to operationalize in a clinical setting. In contrast, semantic workflows are a different class of workflow platform where resultant workflow runs are transparent, reproducible, and semantically validated. Through semantic enforcement of all datasets, analyses and user-defined rules/constraints, users are guided through each workflow run, enhancing analytical validity and patient safety. METHODS: To evaluate the effectiveness of semantic workflows within translational and clinical omics, we have implemented a clinical omics pipeline for annotating DNA sequence variants identified through next generation sequencing using the Workflow Instance Generation and Specialization (WINGS) semantic workflow platform. RESULTS: We found that the implementation and execution of our clinical omics pipeline in a semantic workflow helped us to meet the requirements for enhanced transparency, reproducibility and analytical validity recommended for clinical omics. We further found that many features of the WINGS platform were particularly primed to help support the critical needs of clinical omics analyses. CONCLUSIONS: This is the first implementation and execution of a clinical omics pipeline using semantic workflows. Evaluation of this implementation provides guidance for their use in both translational and clinical settings.
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Biología Computacional/métodos , Flujo de Trabajo , Humanos , Neoplasias/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
MODULEWRITER is a PERL object relational mapping (ORM) tool that automatically generates database specific application programming interfaces (APIs) for SQL databases. The APIs consist of a package of modules providing access to each table row and column. Methods for retrieving, updating and saving entries are provided, as well as other generally useful methods (such as retrieval of the highest numbered entry in a table). MODULEWRITER provides for the inclusion of user-written code, which can be preserved across multiple runs of the MODULEWRITER program.
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Biología Computacional/métodos , Sistemas de Administración de Bases de Datos , Redes de Comunicación de Computadores , Bases de Datos como Asunto , Programas InformáticosRESUMEN
DNA arrays have become the immediate choice in the analysis of large-scale expression measurements. Understanding the expression pattern of genes provide functional information on newly identified genes by computational approaches. Gene expression pattern is an indicator of the state of the cell, and abnormal cellular states can be inferred by comparing expression profiles. Since co-regulated genes, and genes involved in a particular pathway, tend to show similar expression patterns, clustering expression patterns has become the natural method of choice to differentiate groups. However, most methods based on cluster analysis suffer from the usual problems (i) dead units, and (ii) the problem of determining the correct number of clusters (k) needed to classify the data. Selecting the k has been an open problem of pattern recognition and statistics for decades. Since clustering reveals similar patterns present in the data, fixing this number strongly influences the quality of the result. While there is no theoretical solution to this problem, the number of clusters can be decided by a heuristic clustering algorithm called rival penalized competitive learning (RPCL). We present a novel implementation of RPCL that transforms the correct number of clusters problem to the tractable problem of clustering based on the degree of similarity. This is biologically significant since our implementation clusters functionally co-regulated genes and genes that present similar patterns of expression. This new approach reveals potential genes that are co-involved in a biological process. This implementation of the RPCL algorithm is useful in differentiating groups involved in concerted functional regulation and helps to progressively home into patterns, which are closely similar.
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Algoritmos , Perfilación de la Expresión Génica/estadística & datos numéricos , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Ciclo Celular/genética , Análisis por Conglomerados , Simulación por Computador , Regulación de la Expresión Génica/genética , Riñón/metabolismo , Saccharomyces cerevisiae/genética , Factores de TiempoRESUMEN
The computational recognition of precise splice junctions is a challenge faced in the analysis of newly sequenced genomes. This is challenging due to the fact that the distribution of sequence patterns in these regions is not always distinct. Our objective is to understand the sequence signatures at the splice junctions, not simply to create an artificial recognition system. We use a combination of a neural network based calliper randomization approach and an information theoretic based feature selection approach for this purpose. This has been done in an effort to understand regions that harbor information content and to extract features relevant for the prediction of splice junctions. The analysis using the neural network based calliper randomization approach revealed regions important in the internal representation of the network model. The calliper approach captured both correlated as well as independently important features. The feature selection approach captures features that are independently informative. The two different methods can capture features with different properties. Comparative analysis of the results using both the methods help to infer about the kind of information present in the region.
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Empalme Alternativo/genética , Inteligencia Artificial , Exones , Intrones , Modelos Genéticos , Modelos Teóricos , Red Nerviosa , Reproducibilidad de los ResultadosRESUMEN
RNA-Seq allows one to examine only gene expression as well as expression of noncoding RNAs, alternative splicing, and allele-specific expression. With this increased sensitivity and dynamic range, there are computational and statistical considerations that need to be contemplated, which are highly dependent on the biological question being asked. We highlight these to provide an overview of their importance and the impact they can have on downstream interpretation of the brain transcriptome.
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Encéfalo/metabolismo , Expresión Génica/fisiología , ARN/genética , Transcriptoma/genética , Animales , Procesamiento Automatizado de Datos , Perfilación de la Expresión Génica , Humanos , Modelos Estadísticos , ARN/metabolismoRESUMEN
Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.
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Carcinoma de Células Escamosas/genética , Reparación del ADN/genética , Genoma Humano/genética , Heterocromatina/genética , Tasa de Mutación , Neoplasias Cutáneas/genética , Transcripción Genética , Empaquetamiento del ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Células Germinativas/metabolismo , Humanos , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Alternative splicing is a major contributor to genomic complexity, disease, and development. Previous studies have captured some of the characteristics that distinguish alternative splicing from constitutive splicing. However, most published work only focuses on skipped exons and/or a single species. Here we take advantage of the highly curated data in the MAASE database (see related paper in this issue) to analyze features that characterize different modes of splicing. Our analysis confirms previous observations about alternative splicing, including weaker splicing signals at alternative splice sites, higher sequence conservation surrounding orthologous alternative exons, shorter exon length, and more frequent reading frame maintenance in skipped exons. In addition, our study reveals potentially novel regulatory principles underlying distinct modes of alternative splicing and a role of a specific class of repeat elements (transposons) in the origin/evolution of alternative exons. These features suggest diverse regulatory mechanisms and evolutionary paths for different modes of alternative splicing.
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Empalme Alternativo , Biología Computacional , Empalme del ARN , Animales , Bases de Datos Genéticas , Evolución Molecular , Exones , Genoma , Genoma Humano , Humanos , Ratones , Sistemas de Lectura , Secuencias Reguladoras de Ácidos Nucleicos , RetroelementosRESUMEN
Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication.