Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Thermal evolution of gene expression profiles in Drosophila subobscura

Background: Despite its pervasiveness, the genetic basis of adaptation resulting in variation directly or indirectly related to temperature (climatic) gradients is poorly understood. By using 3-fold replicated laboratory thermal stocks covering much of the physiologically tolerable temperature range for the temperate (i.e., cold tolerant) species Drosophila subobscura we have assessed whole-genome transcriptional responses after three years of thermal adaptation, when the populations had already diverged for inversion frequencies, pre-adult life history components, and morphological traits. Total mRNA from each population was compared to a reference pool mRNA in a standard, highly replicated two-colour competitive hybridization experiment using cDNA microarrays.Results: A total of 306 (6.6%) cDNA clones were identified as 'differentially expressed' (following a false discovery rate correction) after contrasting the two furthest apart thermal selection regimes (i.e., 13°C vs . 22°C), also including four previously reported candidate genes for thermotolerance in Drosophila (Hsp26, Hsp68, Fst, and Treh). On the other hand, correlated patterns of gene expression were similar in cold- and warm-adapted populations. Analysis of functional categories defined by the Gene Ontology project point to an overrepresentation of genes involved in carbohydrate metabolism, nucleic acids metabolism and regulation of transcription among other categories. Although the location of differently expressed genes was approximately at random with respect to chromosomes, a physical mapping of 88 probes to the polytene chromosomes of D. subobscura has shown that a larger than expected number mapped inside inverted chromosomal segments.Conclusion: Our data suggest that a sizeable number of genes appear to be involved in thermal adaptation in Drosophila, with a substantial fraction implicated in metabolism. This apparently illustrates the formidable challenge to understanding the adaptive evolution of complex trait variation. Furthermore, some clustering of genes within inverted chromosomal sections was detected. Disentangling the effects of inversions will be obviously required in any future approach if we want to identify the relevant candidate genes.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

Concordance among digital gene expression, microarrays, and qPCR when measuring differential expression of microRNAs.

Profiling microRNA (miRNA) expression is of widespread interest given the critical role of miRNAs in many cellular functions. Profiling can be achieved via hybridization-based (microarrays), sequencing-based, or amplification-based (quantitative reverse transcription-PCR, qPCR) technologies. Among these, microarrays face the significant challenge of accurately distinguishing between mature and immature miRNA forms, and different vendors have developed different methods to meet this challenge. Here we measure differential miRNA expression using the Affymetrix, Agilent, and Illumina microarray platforms, as well as qPCR (Applied Biosystems) and ultra high-throughput sequencing (Illumina). We show that the differential expression measurements are more divergent when the three types of microarrays are compared than when the Agilent microarray, qPCR, and sequencing technology measurements are compared, which exhibit a good overall concordance.

Evolution of gene expression in fire ants: the effects of developmental stage, caste, and species.

Ants provide remarkable examples of equivalent genotypes developing into divergent and discrete phenotypes. Diploid eggs can develop either into queens, which specialize in reproduction, or workers, which participate in cooperative tasks such as building the nest, collecting food, and rearing the young. In contrast, the differentiation between males and females generally depends upon whether eggs are fertilized, with fertilized (diploid) eggs giving rise to females and unfertilized (haploid) eggs giving rise to males. To obtain a comprehensive picture of the relative contributions of gender (sex), caste, developmental stage, and species divergence to gene expression evolution, we investigated gene expression patterns in pupal and adult queens, workers, and males of two species of fire ants, Solenopsis invicta and S. richteri. Microarray hybridizations revealed that variation in gene expression profiles is influenced more by developmental stage than by caste membership, sex, or species identity. The second major contributor to variation in gene expression was the combination of sex and caste. Although workers and queens share equivalent diploid nuclear genomes, they have highly distinctive patterns of gene expression in both the pupal and the adult stages, as might be expected given their extraordinary level of phenotypic differentiation. Overall, the difference in the proportion of differentially expressed genes was greater between workers and males than between workers and queens or queens and males, consistent with the fact that workers and males share neither gender nor reproductive capability. Moreover, between-species comparisons revealed that the greatest difference in gene expression patterns occurred in adult workers, a finding consistent with the fact that adult workers most directly experience the distinct external environments characterizing the different habitats occupied by the two species. Thus, much of the evolution of gene expression in ants may occur in the worker caste, despite the fact that these individuals are largely or completely sterile. Analyses of gene expression evolution revealed a combination of positive selection and relaxation of stabilizing selection as important factors driving the evolution of such genes.

Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages.

The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.

GSVA: gene set variation analysis for microarray and RNA-seq data

Gene set enrichment (GSE) analysis is a popular framework for condensing information from gene expression profiles into a pathway or signature summary. The strengths of this approach over single gene analysis include noise and dimension reduction, as well as greater biological interpretability. As molecular profiling experiments move beyond simple case-control studies, robust and flexible GSE methodologies are needed that can model pathway activity within highly heterogeneous data sets. To address this challenge, we introduce Gene Set Variation Analysis (GSVA), a GSE method that estimates variation of pathway activity over a sample population in an unsupervised manner. We demonstrate the robustness of GSVA in a comparison with current state of the art sample-wise enrichment methods. Further, we provide examples of its utility in differential pathway activity and survival analysis. Lastly, we show how GSVA works analogously with data from both microarray and RNA-seq experiments. GSVA provides increased power to detect subtle pathway activity changes over a sample population in comparison to corresponding methods. While GSE methods are generally regarded as end points of a bioinformatic analysis, GSVA constitutes a starting point to build pathway-centric models of biology. Moreover, GSVA contributes to the current need of GSE methods for RNA-seq data. GSVA is an open source software package for R which forms part of the Bioconductor project and can be downloaded at http://www.bioconductor.org.

Copy number variants, diseases and gene expression.

Copy number variation (CNV) has recently gained considerable interest as a source of genetic variation likely to play a role in phenotypic diversity and evolution. Much effort has been put into the identification and mapping of regions that vary in copy number among seemingly normal individuals in humans and a number of model organisms, using bioinformatics or hybridization-based methods. These have allowed uncovering associations between copy number changes and complex diseases in whole-genome association studies, as well as identify new genomic disorders. At the genome-wide scale, however, the functional impact of CNV remains poorly studied. Here we review the current catalogs of CNVs, their association with diseases and how they link genotype and phenotype. We describe initial evidence which revealed that genes in CNV regions are expressed at lower and more variable levels than genes mapping elsewhere, and also that CNV not only affects the expression of genes varying in copy number, but also have a global influence on the transcriptome. Further studies are warranted for complete cataloguing and fine mapping of CNVs, as well as to elucidate the different mechanisms by which they influence gene expression.

Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under Accession Nos E-MTAB-990 and E-MTAB-1026. © 2013 Swiss Institute of Bioinformatics. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Gene expression profiling reveals consistent differences between clinical samples of human leukaemias and their model cell lines.

Microarray gene expression profiles of fresh clinical samples of chronic myeloid leukaemia in chronic phase, acute promyelocytic leukaemia and acute monocytic leukaemia were compared with profiles from cell lines representing the corresponding types of leukaemia (K562, NB4, HL60). In a hierarchical clustering analysis, all clinical samples clustered separately from the cell lines, regardless of leukaemic subtype. Gene ontology analysis showed that cell lines chiefly overexpressed genes related to macromolecular metabolism, whereas in clinical samples genes related to the immune response were abundantly expressed. These findings must be taken into consideration when conclusions from cell line-based studies are extrapolated to patients.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.

The direct effects of tacrolimus and cyclosporin A on isolated human islets: A functional, survival and gene expression study.

The use of immunosuppressive drugs in transplanted patients is associated with the development of diabetes, possibly due to β-cell toxicity. To better understand the mechanisms leading to post-transplant diabetes, we investigated the actions of prolonged exposure of isolated human islets to therapeutical levels of tacrolimus (Tac) or cyclosporin A (CsA). Islets were isolated from the pancreas of multiorgan donors by enzymatic digestion and density gradient centrifugation. Functional, survival and molecular studies were then performed after 4 days of incubation with therapeutical concentrations of Tac or  CsA. Glucose-induced insulin secretion was significantly decreased in Tac, but not in CsA exposed islets, which was associated with a reduction of the amount of insulin granules as shown by electron microscopy. The percentage of apoptotic β-cells was higher in Tac than CsA exposed islets. Microarray experiments followed by Gene Set Enrichment Analysis revealed that gene expression was more markedly affected upon Tac treatment. In conclusion, Tac and CsA affect features of beta-cell differently, with several changes occurring at the molecular level.

CleanEx: a database of heterogeneous gene expression data based on a consistent gene nomenclature and linked to an improved annotation system

Résumé: L'automatisation du séquençage et de l'annotation des génomes, ainsi que l'application à large échelle de méthodes de mesure de l'expression génique, génèrent une quantité phénoménale de données pour des organismes modèles tels que l'homme ou la souris. Dans ce déluge de données, il devient très difficile d'obtenir des informations spécifiques à un organisme ou à un gène, et une telle recherche aboutit fréquemment à des réponses fragmentées, voir incomplètes. La création d'une base de données capable de gérer et d'intégrer aussi bien les données génomiques que les données transcriptomiques peut grandement améliorer la vitesse de recherche ainsi que la qualité des résultats obtenus, en permettant une comparaison directe de mesures d'expression des gènes provenant d'expériences réalisées grâce à des techniques différentes. L'objectif principal de ce projet, appelé CleanEx, est de fournir un accès direct aux données d'expression publiques par le biais de noms de gènes officiels, et de représenter des données d'expression produites selon des protocoles différents de manière à faciliter une analyse générale et une comparaison entre plusieurs jeux de données. Une mise à jour cohérente et régulière de la nomenclature des gènes est assurée en associant chaque expérience d'expression de gène à un identificateur permanent de la séquence-cible, donnant une description physique de la population d'ARN visée par l'expérience. Ces identificateurs sont ensuite associés à intervalles réguliers aux catalogues, en constante évolution, des gènes d'organismes modèles. Cette procédure automatique de traçage se fonde en partie sur des ressources externes d'information génomique, telles que UniGene et RefSeq. La partie centrale de CleanEx consiste en un index de gènes établi de manière hebdomadaire et qui contient les liens à toutes les données publiques d'expression déjà incorporées au système. En outre, la base de données des séquences-cible fournit un lien sur le gène correspondant ainsi qu'un contrôle de qualité de ce lien pour différents types de ressources expérimentales, telles que des clones ou des sondes Affymetrix. Le système de recherche en ligne de CleanEx offre un accès aux entrées individuelles ainsi qu'à des outils d'analyse croisée de jeux de donnnées. Ces outils se sont avérés très efficaces dans le cadre de la comparaison de l'expression de gènes, ainsi que, dans une certaine mesure, dans la détection d'une variation de cette expression liée au phénomène d'épissage alternatif. Les fichiers et les outils de CleanEx sont accessibles en ligne (http://www.cleanex.isb-sib.ch/). Abstract: The automatic genome sequencing and annotation, as well as the large-scale gene expression measurements methods, generate a massive amount of data for model organisms. Searching for genespecific or organism-specific information througout all the different databases has become a very difficult task, and often results in fragmented and unrelated answers. The generation of a database which will federate and integrate genomic and transcriptomic data together will greatly improve the search speed as well as the quality of the results by allowing a direct comparison of expression results obtained by different techniques. The main goal of this project, called the CleanEx database, is thus to provide access to public gene expression data via unique gene names and to represent heterogeneous expression data produced by different technologies in a way that facilitates joint analysis and crossdataset comparisons. A consistent and uptodate gene nomenclature is achieved by associating each single gene expression experiment with a permanent target identifier consisting of a physical description of the targeted RNA population or the hybridization reagent used. These targets are then mapped at regular intervals to the growing and evolving catalogues of genes from model organisms, such as human and mouse. The completely automatic mapping procedure relies partly on external genome information resources such as UniGene and RefSeq. The central part of CleanEx is a weekly built gene index containing crossreferences to all public expression data already incorporated into the system. In addition, the expression target database of CleanEx provides gene mapping and quality control information for various types of experimental resources, such as cDNA clones or Affymetrix probe sets. The Affymetrix mapping files are accessible as text files, for further use in external applications, and as individual entries, via the webbased interfaces . The CleanEx webbased query interfaces offer access to individual entries via text string searches or quantitative expression criteria, as well as crossdataset analysis tools, and crosschip gene comparison. These tools have proven to be very efficient in expression data comparison and even, to a certain extent, in detection of differentially expressed splice variants. The CleanEx flat files and tools are available online at: http://www.cleanex.isbsib. ch/.

Genome-wide transcriptional responses to shade: Linking shade avoidance and auxin

Plants have the ability to use the composition of incident light as a cue to adapt development and growth to their environment. Arabidopsis thaliana as well as many crops are best adapted to sunny habitats. When subjected to shade, these plants exhibit a variety of physiological responses collectively called shade avoidance syndrome (SAS). It includes increased growth of hypocotyl and petioles, decreased growth rate of cotyledons and reduced branching and crop yield. These responses are mainly mediated by phytochrome photoreceptors, which exist either in an active, far-red light (FR) absorbing or an inactive, red light (R) absorbing isoform. In direct sunlight, the R to FR light (R/FR) ratio is high and converts the phytochromes into their physiologically active state. The phytochromes interact with downstream transcription factors such as PHYTOCHROME INTERACTING FACTOR (PIF), which are subsequently degraded. Light filtered through a canopy is strongly depleted in R, which result in a low R/FR ratio and renders the phytochromes inactive. Protein levels of downstream transcription factors are stabilized, which initiates the expression of shade-induced genes such as HFR1, PIL1 or ATHB-2. In my thesis, I investigated transcriptional responses mediated by the SAS in whole Arabidopsis seedlings. Using microarray and chromatin immunoprecipitation data, we identified genome-wide PIF4 and PIF5 dependent shade regulated gene as well as putative direct target genes of PIF5. This revealed evidence for a direct regulatory link between phytochrome signaling and the growth promoting phytohormone auxin (IAA) at the level of biosynthesis, transport and signaling. Subsequently, it was shown, that free-IAA levels are upregulated in response to shade. It is assumed that shade-induced auxin production takes predominantly place in cotyledons of seedlings. This implies, that IAA is subsequently transported basipetally to the hypocotyl and enhances elongation growth. The importance of auxin transport for growth responses has been established by chemical and genetic approaches. To gain a better understanding of spatio-temporal transcriptional regulation of shade-induce auxin, I generated in a second project, an organ specific high throughput data focusing on cotyledon and hypocotyl of young Arabidopsis seedlings. Interestingly, both organs show an opposite growth regulation by shade. I first investigated the spatio-transcriptional regulation of auxin re- sponsive gene, in order to determine how broad gene expression pattern can be explained by the hypothesized movement of auxin from cotyledons to hypocotyls in shade. The analysis suggests, that several genes are indeed regulated according to our prediction and others are regulated in a more complex manner. In addition, analysis of gene families of auxin biosynthetic and transport components, lead to the identification of essential family members for shade-induced growth re- sponses, which were subsequently experimentally confirmed. Finally, the analysis of expression pattern identified several candidate genes, which possibly explain aspects of the opposite growth response of the different organs.