BayGO: Bayesian analysis of ontology term enrichment in microarray data

BACKGROUND: The search for enriched (aka over-represented or enhanced) ontology terms in a list of genes obtained from microarray experiments is becoming a standard procedure for a system-level analysis. This procedure tries to summarize the information focussing on classification designs such as Gene Ontology, KEGG pathways, and so on, instead of focussing on individual genes. Although it is well known in statistics that association and significance are distinct concepts, only the former approach has been used to deal with the ontology term enrichment problem. RESULTS: BayGO implements a Bayesian approach to search for enriched terms from microarray data. The R source-code is freely available at in three versions: Linux, which can be easily incorporated into pre-existent pipelines; Windows, to be controlled interactively; and as a web-tool. The software was validated using a bacterial heat shock response dataset, since this stress triggers known system-level responses. CONCLUSION: The Bayesian model accounts for the fact that, eventually, not all the genes from a given category are observable in microarray data due to low intensity signal, quality filters, genes that were not spotted and so on. Moreover, BayGO allows one to measure the statistical association between generic ontology terms and differential expression, instead of working only with the common significance analysis

Integration and visualization of systems biology data in context of the genome

<p>Abstract</p> <p>Background</p> <p>High-density tiling arrays and new sequencing technologies are generating rapidly increasing volumes of transcriptome and protein-DNA interaction data. Visualization and exploration of this data is critical to understanding the regulatory logic encoded in the genome by which the cell dynamically affects its physiology and interacts with its environment.</p> <p>Results</p> <p>The Gaggle Genome Browser is a cross-platform desktop program for interactively visualizing high-throughput data in the context of the genome. Important features include dynamic panning and zooming, keyword search and open interoperability through the Gaggle framework. Users may bookmark locations on the genome with descriptive annotations and share these bookmarks with other users. The program handles large sets of user-generated data using an in-process database and leverages the facilities of SQL and the R environment for importing and manipulating data.</p> <p>A key aspect of the Gaggle Genome Browser is interoperability. By connecting to the Gaggle framework, the genome browser joins a suite of interconnected bioinformatics tools for analysis and visualization with connectivity to major public repositories of sequences, interactions and pathways. To this flexible environment for exploring and combining data, the Gaggle Genome Browser adds the ability to visualize diverse types of data in relation to its coordinates on the genome.</p> <p>Conclusions</p> <p>Genomic coordinates function as a common key by which disparate biological data types can be related to one another. In the Gaggle Genome Browser, heterogeneous data are joined by their location on the genome to create information-rich visualizations yielding insight into genome organization, transcription and its regulation and, ultimately, a better understanding of the mechanisms that enable the cell to dynamically respond to its environment.</p

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

<p>Abstract</p> <p>Background</p> <p><it>Xylella fastidiosa</it>, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, <it>X. fastidiosa </it>needs to cope with nitrogen limitation in its natural habitat.</p> <p>Results</p> <p>In this work, we performed a whole-genome microarray analysis of the <it>X. fastidiosa </it>nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the <it>rpoN </it>null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ⁵⁴-dependent manner. A more complete picture of the σ⁵⁴regulon was achieved by combining the transcriptome data with an <it>in silico </it>search for potential σ⁵⁴-dependent promoters, using a position weight matrix approach. One of these σ⁵⁴-predicted binding sites, located upstream of the <it>glnA </it>gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ⁵⁴-dependent promoter.</p> <p>Conclusions</p> <p>Together, these results show that nitrogen starvation causes intense changes in the <it>X. fastidiosa </it>transcriptome and some of these differentially expressed genes belong to the σ⁵⁴regulon.</p

Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease.

Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD.FRT was funded by a BEPE-FAPESP Fellowship (2012/09241-8). SR and HL are funded by the Biotechnology and Biological Science Research Council (BB/J007846/1). DK is funded by ERC (309756), MRC (U105192732) and the Lister Institute for Preventive Medicine. TETM is funded by the Marie Curie ITN “UPStream.”This is the final version of the article. It first appeared from Portland Press via https://doi.org/10.1042/BCJ2016038

Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis

Abstract Background Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable. Results We have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control. Conclusions Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems

Prevalence of transcription promoters within archaeal operons and coding sequences

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements

Iron Deficiency Generates Oxidative Stress and Activation of the SOS Response in Caulobacter crescentus

In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage

Global gene expression analysis of Xylella fastidiosa under environmental stress conditions

Xylella fastidiosa é uma bactéria fitopatogênica, responsável por doenças em diversas plantas de importância econômica. Diversas cepas têm sido estudadas, porém, pouco se sabe a respeito da resposta a estresses ambientais em X. fastidiosa. Utilizando a tecnologia de microarranjos de DNA, verificou-se a resposta global aos estresses térmico, salino e osmótico em nível de transcrição. Os experimentos foram realizados em séries temporais, os perfis de expressão gênica dos genes diferencialmente expressos foram agrupados e validados por RT-PCR quantitativo. No choque térmico, 261 genes foram induzidos (9,7%) e 222 genes foram reprimidos (8,3%). Dentre os genes altamente induzidos, destacam-se os que codificam proteínas de choque térmico (Hsps), que previnem a desnaturação e a formação de agregados protéicos ou degradam polipeptídeos irreversivelmente desnaturados. A partir da determinação do início de transcrição de seis genes altamente induzidos no choque térmico, propôs-se um consenso para promotores dependentes do fator sigma alternativo que controla a resposta ao choque térmico, sigma32. Observou-se também a indução de genes relacionados ao estresse extracitoplasmático, que são regulados pelo fator sigma alternativo sigmaE. No choque osmótico e salino, os genes codificando a maioria das Hsps foram reprimidos na exposição prolongada a esses estresses, indicando que a resposta não é mediada por sigma32 ou sigmaE. Dos 142 genes induzidos tanto no estresse salino como osmótico, 57% codificam proteínas hipotéticas ou hipotéticas conservadas, indicando uma possível função na resposta a estes estresses. Observou-se a repressão de genes relacionados à síntese protéica e ao metabolismo intermediário nos três estresses analisados, além da indução de genes relacionados à virulência como toxinas e adesinas, revelando a complexa rede de genes envolvida na resposta a estresses ambientais. Para auxiliar a análise de dados de microarranjos de DNA, foram desenvolvidas três ferramentas de bioinformática: HTself, utilizada na determinação de genes diferencialmente expressos; BayGO, utilizada na análise categorias funcionais altamente representadas dentre os genes de interesse e SpotWhatR, uma plataforma que integra programas utilizados nas diversas etapas da análise e pré-processamento de dados de microarranjos, com uma interface de fácil utilização. Estas ferramentas foram utilizadas com sucesso e estão disponíveis livremente para outros pesquisadores.Xylella fastidiosa is a phytopathogenic bacterium responsible for diseases in many economically important crops. Although different strains have been studied, little is known about X. fastidiosa stress responses. To investigate X. fastidiosa genes involved in heat, salt and osmotic shock responses, we performed a whole genome microarray analysis in time-course experiments. The expression profiles of the differentially expressed genes were grouped and their expression patterns were validated by quantitative RT-PCR experiments. During heat shock, 261 genes were induced (9.7%) and 222 genes were repressed (8.3%). Among the differentially expressed genes, the ones presenting the highest induction ratios encode heat shock proteins (Hsps), which prevents protein misfolding and aggregation or promote the degradation of the irreversibly denatured polypeptides. We determined the transcription start sites of six heat shock inducible genes and analyzed their promoter regions, which allowed us to propose a putative consensus for sigma32 promoters in X. fastidiosa. We also observed the induction of genes related to the extracytoplasmic stress response, that are regulated by the alternative sigma factor sigmaE. During prolongued exposure to salt and osmotic stress, genes encoding most of the Hsps were repressed, indicating that the response is not mediated by sigma32 or sigmaE. Among the 142 genes induced by both salt and osmotic stress, 57% encode hypothetical or conserved hypothetical proteins, indicating a possible role of these genes in the stress response. In addition, we observed the repression of genes related to protein biosynthesis and intermediary metabolism during the three stresses tested, besides the induction of genes related to virulence such as toxins and adhesins, revealing the complex network of genes that work together in response to environmental stresses. To facilitate the microarray data analysis process, we developed three bioinformatics tools: HTself, which is used to determine the differentially expressed genes; BayGO, which aims at finding over-represented gene categories and SpotWhatR, a system that integrates programs used in different steps of microarray data analysis in a user-friendly interface. These tools were successfully used and are freely available to the research community

doi:10.1093/dnares/dsi007 HTself: Self–Self Based Statistical Test for Low Replication Microarray Studies

Different statistical methods have been used to classify a gene as differentially expressed in microarray experiments. They usually require a number of experimental observations to be adequately applied. However, many microarray experiments are constrained to low replication designs for different reasons, from financial restrictions to scarcely available RNA samples. Although performed in a high-throughput framework, there are few experimental replicas for each gene to allow the use of traditional or state-of-art statistical methods. In this work, we present a web-based bioinformatics tool that deals with real-life problems concerning low replication experiments. It uses an empirically derived criterion to classify a gene as differentially expressed by combining two widely accepted ideas in microarray analysis: self–self experiments to derive intensity-dependent cutoffs and non-parametric estimation techniques. To help laboratories without a bioinformatics infrastructure, we implemented the tool in a user-friendly websit