Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

Clinical Application of Prognostic Gene Expression Signature in Fusion Gene-Negative Rhabdomyosarcoma: A Report from the Children's Oncology Group.

PURPOSE: Pediatric rhabdomyosarcoma (RMS) has two common histologic subtypes: embryonal (ERMS) and alveolar (ARMS). PAX-FOXO1 fusion gene status is a more reliable prognostic marker than alveolar histology, whereas fusion gene-negative (FN) ARMS patients are clinically similar to ERMS patients. A five-gene expression signature (MG5) previously identified two diverse risk groups within the fusion gene-negative RMS (FN-RMS) patients, but this has not been independently validated. The goal of this study was to test whether expression of the MG5 metagene, measured using a technical platform that can be applied to routine pathology material, would correlate with outcome in a new cohort of patients with FN-RMS. EXPERIMENTAL DESIGN: Cases were taken from the Children's Oncology Group (COG) D9803 study of children with intermediate-risk RMS, and gene expression profiling for the MG5 genes was performed using the nCounter assay. The MG5 score was correlated with clinical and pathologic characteristics as well as overall and event-free survival. RESULTS: MG5 standardized score showed no significant association with any of the available clinicopathologic variables. The MG5 signature score showed a significant correlation with overall (N = 57; HR, 7.3; 95% CI, 1.9-27.0; P = 0.003) and failure-free survival (N = 57; HR, 6.1; 95% CI, 1.9-19.7; P = 0.002). CONCLUSIONS: This represents the first, validated molecular prognostic signature for children with FN-RMS who otherwise have intermediate-risk disease. The capacity to measure the expression of a small number of genes in routine pathology material and apply a simple mathematical formula to calculate the MG5 metagene score provides a clear path toward better risk stratification in future prospective clinical trials. Clin Cancer Res; 21(20); 4733-9. ©2015 AACR.

Impact of a cis-associated gene expression SNP on chromosome 20q11.22 on bipolar disorder susceptibility, hippocampal structure and cognitive performance.

BackgroundBipolar disorder is a highly heritable polygenic disorder. Recent enrichment analyses suggest that there may be true risk variants for bipolar disorder in the expression quantitative trait loci (eQTL) in the brain.AimsWe sought to assess the impact of eQTL variants on bipolar disorder risk by combining data from both bipolar disorder genome-wide association studies (GWAS) and brain eQTL.MethodTo detect single nucleotide polymorphisms (SNPs) that influence expression levels of genes associated with bipolar disorder, we jointly analysed data from a bipolar disorder GWAS (7481 cases and 9250 controls) and a genome-wide brain (cortical) eQTL (193 healthy controls) using a Bayesian statistical method, with independent follow-up replications. The identified risk SNP was then further tested for association with hippocampal volume (n = 5775) and cognitive performance (n = 342) among healthy individuals.ResultsIntegrative analysis revealed a significant association between a brain eQTL rs6088662 on chromosome 20q11.22 and bipolar disorder (log Bayes factor = 5.48; bipolar disorder P = 5.85×10(-5)). Follow-up studies across multiple independent samples confirmed the association of the risk SNP (rs6088662) with gene expression and bipolar disorder susceptibility (P = 3.54×10(-8)). Further exploratory analysis revealed that rs6088662 is also associated with hippocampal volume and cognitive performance in healthy individuals.ConclusionsOur findings suggest that 20q11.22 is likely a risk region for bipolar disorder; they also highlight the informative value of integrating functional annotation of genetic variants for gene expression in advancing our understanding of the biological basis underlying complex disorders, such as bipolar disorder.

Functional impact of genetic variation on gene expression

Numerous links between genetic variants and phenotypes are known and genome-wide association studies dramatically increased the number of genetic variants associated with traits during the last decade. However, how changes in the DNA perturb the molecular mechanisms and impact on the phenotype of an organism remains elusive. Studies suggest that many traitassociated variants are in the non-coding region of the genome and probably act through regulation of gene expression. During my thesis I investigated how genetic variants affect gene expression through gene regulatory mechanisms. The first chapter was a collaborative project with a pharmaceutical company, where we investigated genome-wide copy number variation (CNVs) among Cynomolgus monkeys (Macaca fascicularis) used in pharmaceutical studies, and associated them to changes in gene expression. We found substantial copy number variation and identified CNVs linked to tissue-specific expression changes of proximal genes. The second and third chapters focus on genetic variation in humans and its effects on gene regulatory mechanisms and gene expression. The second chapter studies two human trios, where the allelic effects of genetic variation on genome-wide gene expression, protein-DNA binding and chromatin modifications were investigated. We found abundant allele specific activity across all measured molecular phenotypes and show extended coordinated behavior among them. In the third chapter, we investigated the impact of genetic variation on these phenotypes in 47 unrelated individuals. We found that chromatin phenotypes are organized into local variable modules, often linked to genetic variation and gene expression. Our results suggest that chromatin variation emerges as a result of perturbations of cis-regulatory elements by genetic variants, leading to gene expression changes. The work of this thesis provides novel insights into how genetic variation impacts gene expression by perturbing regulatory mechanisms. -- De nombreux liens entre variations génétiques et phénotypes sont connus. Les études d'association pangénomique ont considérablement permis d'augmenter le nombre de variations génétiques associées à des phénotypes au cours de la dernière décennie. Cependant, comprendre comment ces changements perturbent les mécanismes moléculaires et affectent le phénotype d'un organisme nous échappe encore. Des études suggèrent que de nombreuses variations, associées à des phénotypes, sont situées dans les régions non codantes du génome et sont susceptibles d'agir en modifiant la régulation d'expression des gènes. Au cours de ma thèse, j'ai étudié comment les variations génétiques affectent les niveaux d'expression des gènes en perturbant les mécanismes de régulation de leur expression. Le travail présenté dans le premier chapitre est un projet en collaboration avec une société pharmaceutique. Nous avons étudié les variations en nombre de copies (CNV) présentes chez le macaque crabier (Macaca fascicularis) qui est utilisé dans les études pharmaceutiques, et nous les avons associées avec des changements d'expression des gènes. Nous avons découvert qu'il existe une variabilité substantielle du nombre de copies et nous avons identifié des CNVs liées aux changements d'expression des gènes situés dans leur voisinage. Ces associations sont présentes ou absentes de manière spécifique dans certains tissus. Les deuxième et troisième chapitres se concentrent sur les variations génétiques dans les populations humaines et leurs effets sur les mécanismes de régulation des gènes et leur expression. Le premier se penche sur deux trios humains, père, mère, enfant, au sein duquel nous avons étudié les effets alléliques des variations génétiques sur l'expression des gènes, les liaisons protéine-ADN et les modifications de la chromatine. Nous avons découvert que l'activité spécifique des allèles est abondante abonde dans tous ces phénotypes moléculaires et nous avons démontré que ces derniers ont un comportement coordonné entre eux. Dans le second, nous avons examiné l'impact des variations génétiques de ces phénotypes moléculaires chez 47 individus, sans lien de parenté. Nous avons observé que les phénotypes de la chromatine sont organisés en modules locaux, qui sont liés aux variations génétiques et à l'expression des gènes. Nos résultats suggèrent que la variabilité de la chromatine est due à des variations génétiques qui perturbent des éléments cis-régulateurs, et peut conduire à des changements dans l'expression des gènes. Le travail présenté dans cette thèse fournit de nouvelles pistes pour comprendre l'impact des différentes variations génétiques sur l'expression des gènes à travers les mécanismes de régulation.

Individual differences in gene expression : insights from transcriptional control of the CSL gene and from human population studies

CSL is a key transcription factor, mostly acting as a repressor, which has been shown to have a highly context-dependent function. While known as the main effector of Notch signaling, it can also exert Notch-independent functions. The downstream effects of the Notch/CSL signaling pathway and its involvement in several biological processes have been intensively studied. We recently showed that CSL is important to maintain skin homeostasis, as its specific deletion in mouse dermal fibroblasts -or downmodulation in human stromal fibroblasts- creates an inducing environment for squamous cell carcinoma (SCC) development, possibly due to the conversion of stromal fibroblasts into cancer associated fibroblasts (CAFs). Despite the wide interest in CSL as a transcriptional regulator, the mechanism of its own regulation has so far been neglected. We show here that CSL expression levels differ between individuals, and correlate among others with genes involved in DNA damage response. Starting from this finding we show that in dermal fibroblasts CSL is under transcriptional control of stress inducers such as UVA irradiation and Reactive Oxygen Species (ROS) induction, and that a main player in CSL transcriptional regulation is the transcription factor p53. In a separate line of work, we focused on individual variability, studying the differences in gene expression between human populations in various cancer types, particularly focusing on the Caucasian and African populations. It is indeed widely known that these populations have different incidences and mortalities for various cancers, and response to cancer treatment may also vary between them. We show here several genes that are differentially expressed and could be of interest in the study of population differences in cancer. -- CSL est un facteur de transcription agissant essentiellement comme répresseur, et qui a une fonction hautement dépendant du contexte. C'est l'effecteur principal de la voie de signalisation de Notch, mais il peut également exercer ses fonctions dans une façon Notch- indépendante. Nous avons récemment montré que CSL est important pour maintenir l'homéostasie de la peau. Sa suppression spécifique dans les fibroblastes dermiques de la souris ou dans les fibroblastes stromales humaines crée un environnement favorable pour le développement du carcinome épidermoïde (SCC), probablement en raison de la conversion des fibroblastes en fibroblastes associé au cancer (CAF). Malgré le grand intérêt de CSL comme régulateur transcriptionnel, le mécanisme de sa propre régulation a été jusqu'ici négligée. Nous montrons ici que dans les fibroblastes dermiques CSL est sous le contrôle transcriptionnel de facteurs de stress tels que l'irradiation UVA et l'induction des ROS dont p53 est l'acteur principal de cette régulation. Nous montrons aussi que les niveaux d'expression de CSL varient selon les individus, en corrélation avec d'autres gènes impliqués dans la réponse aux dommages de l'ADN. Dans une autre axe de recherche, concernant la variabilité individuelle, nous avons étudié les différences dans l'expression des gènes dans différents types de cancer entre les populations humaines, en se concentrant particulièrement sur les populations africaines et caucasiennes. Il est en effet bien connu que ces populations montrent des variations dans l'incidence des cancers, la mortalité, ainsi que pour les réponses au traitement. Nous montrons ici plusieurs gènes qui sont exprimés différemment et pourraient être digne d'intérêt dans l'étude du cancer au sein de différentes populations.

Comparison of differential gene expression to water stress among bacteria with relevant pollutant-degradation properties.

Resistance to semi-dry environments has been considered a crucial trait for superior growth and survival of strains used for bioaugmentation in contaminated soils. In order to compare water stress programmes, we analyse differential gene expression among three phylogenetically different strains capable of aromatic compound degradation: Arthrobacter chlorophenolicus A6, Sphingomonas wittichii RW1 and Pseudomonas veronii 1YdBTEX2. Standardized laboratory-induced water stress was imposed by shock exposure of liquid cultures to water potential decrease, induced either by addition of solutes (NaCl, solute stress) or by addition of polyethylene glycol (matric stress), both at absolute similar stress magnitudes and at those causing approximately similar decrease of growth rates. Genome-wide differential gene expression was recorded by micro-array hybridizations. Growth of P. veronii 1YdBTEX2 was the most sensitive to water potential decrease, followed by S. wittichii RW1 and A. chlorophenolicus A6. The number of genes differentially expressed under decreasing water potential was lowest for A. chlorophenolicus A6, increasing with increasing magnitude of the stress, followed by S. wittichii RW1 and P. veronii 1YdBTEX2. Gene inspection and gene ontology analysis under stress conditions causing similar growth rate reduction indicated that common reactions among the three strains included diminished expression of flagellar motility and increased expression of compatible solutes (which were strain-specific). Furthermore, a set of common genes with ill-defined function was found between all strains, including ABC transporters and aldehyde dehydrogenases, which may constitute a core conserved response to water stress. The data further suggest that stronger reduction of growth rate of P. veronii 1YdBTEX2 under water stress may be an indirect result of the response demanding heavy NADPH investment, rather than the presence or absence of a suitable stress defence mechanism per se.