Inferring the Transcriptional Landscape of Bovine Skeletal Muscle by Integrating Co-Expression Networks

Background: Despite modern technologies and novel computational approaches, decoding causal transcriptional regulation remains challenging. This is particularly true for less well studied organisms and when only gene expression data is available. In muscle a small number of well characterised transcription factors are proposed to regulate development. Therefore, muscle appears to be a tractable system for proposing new computational approaches. Methodology/Principal Findings: Here we report a simple algorithm that asks "which transcriptional regulator has the highest average absolute co-expression correlation to the genes in a co-expression module?" It correctly infers a number of known causal regulators of fundamental biological processes, including cell cycle activity (E2F1), glycolysis (HLF), mitochondrial transcription (TFB2M), adipogenesis (PIAS1), neuronal development (TLX3), immune function (IRF1) and vasculogenesis (SOX17), within a skeletal muscle context. However, none of the canonical pro-myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6 and MEF2C) were linked to muscle structural gene expression modules. Co-expression values were computed using developing bovine muscle from 60 days post conception (early foetal) to 30 months post natal (adulthood) for two breeds of cattle, in addition to a nutritional comparison with a third breed. A number of transcriptional landscapes were constructed and integrated into an always correlated landscape. One notable feature was a 'metabolic axis' formed from glycolysis genes at one end, nuclear-encoded mitochondrial protein genes at the other, and centrally tethered by mitochondrially-encoded mitochondrial protein genes. Conclusions/Significance: The new module-to-regulator algorithm complements our recently described Regulatory Impact Factor analysis. Together with a simple examination of a co-expression module's contents, these three gene expression approaches are starting to illuminate the in vivo transcriptional regulation of skeletal muscle development

RBR E3 ubiquitin ligases: new structures, new insights, new questions

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. INTRODUCTIO

Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated ∼617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein – two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-κB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment

Investigating the Developmental Toxicity of Legacy, Emergent, and Current Use Environmental Toxicants Using the Zebrafish Model System

Environmental toxicants are chemical or physical agents that enter the environment and can have adverse health effects on populations of humans. Chemical contamination of rivers, lakes, and ground water is a significant public health concern; 23% of private wells are contaminated with at least one significant environmental toxicant and chemical spill incidences can have severe consequences to human health and the environment. Even low doses of chemicals in the environment can have an effect on human health, and it is suggested by the Developmental Origins of Health and Disease (DOHaD) hypothesis that embryonic exposure to toxicants alters health status and disease prevalence later in life. Therefore, it is important to study the legacy, emergent, and current-use chemicals that are found in low levels in drinking water sources to assess the risk of early exposure. The first chapter of this dissertation introduces the problem of drinking water contamination and provides a background on the zebrafish biomedical model used in this dissertation as well as the zebrafish neurotransmitter systems and methods used to evaluate neurotoxicity. The second chapter is focused on the legacy toxicant, trichloroethylene (TCE). TCE, an industrial solvent and degreaser, is an environmental toxicant that contaminates over half of Superfund sites. In the study described in Chapter 2, the zebrafish model was used to evaluate the acute developmental toxicity of near regulatory concentrations of TCE by monitoring survivability, percent hatching, morphological measurements, and neurobehavior. The percent survival was not significantly different between treatments, but the 500 ppb treatment had increased hatching at 48 hpf. Morphological measurements indicated that the 500 ppb treatment had significantly larger head length to body length ratios and significantly shorter brains compared to the controls. No significant differences were observed during the evaluation of neurobehavior. The expression of four cytochrome p450 enzymes was also evaluated and the expression of cyp24a1 was significantly downregulated, suggesting altered vitamin D3 metabolism. The reported alterations suggest that developmental TCE toxicity is still a concern near regulatory concentrations and that TCE should remain a priority environmental toxicant. The third chapter describes the acute toxicity of the emergent chemical mixtures in the methylcyclohexanemethanol (MCHM) family. In January 2014, a chemical mixture containing crude MCHM contaminated the water supply of Charleston, West Virginia. Although the mixture was later identified as a mix of crude MCHM and stripped propylene glycol phenyl ethers, initial risk assessment focused on 4-MCHM, the predominant component of crude MCHM. The mixture’s exact composition and the toxicity differences between 4-MCHM, crude MCHM, and the tank mixture were unknown. In the studies described in Chapter 3, the chemical compositions of crude MCHM and the tank mixture were analyzed via GC/MS and found to be different from each other and the reported composition of crude MCHM. The percent mortality and percent hatch, larval morphology alterations, and larval visual motor response test were used to establish toxicity profiles for each of the chemicals or mixtures. The acute toxicity differed between 4-MCHM, crude MCHM and the tank mixture with significant differences in survival, hatching, morphology, and locomotion. The acute toxicity increased as the complexity of the mixture increased, but the developmental toxicity was greatest with the 4-MCHM parent compound. Additionally, behavior was changed at levels as low as the short-term screening level of 1 ppm, suggesting a need for further research into human health risks. The final toxicant of interest is the current use herbicide, atrazine (ATZ). ATZ is the second most commonly used pesticide and frequently contaminates rural and urban water sources. Exposure to ATZ is linked to endocrine disruption, cancer, changes in genome methylation, and alterations in neurochemistry and behavior. The studies described in Chapter 4 focus on ATZ related developmental neurotoxicity while the studies described in Chapter 5 examine the later life effects on the nervous system of embryonic ATZ exposure. In Chapter 4, the relative expression of six genes was monitored throughout a developmental time course to determine normal expression and to determine if developmental exposure to 0, 0.3, 3, or 30 ppb ATZ altered expression. One gene, cyp17a1, had dynamic expression during development that was not related to ATZ exposure, but ttc3 and tpd52l1 both had ATZ related changes in relative gene expression. The brain lengths were measured and larval behavior was monitored to determine physical and functional outcomes of developmental ATZ exposure. Increased brain length was observed in larvae exposed to 0.3 ppb ATZ, while hypoactivity was observed larvae with developmental exposure to 30 ppb ATZ. Proteomic analysis identified 28 proteins with a significant label-free quantification intensity value and the proteins were associated with pathways related to organism and organ system development, intracellular signaling, protein handling and degradation, epigenetic regulation of gene expression, and cancer. The results suggest that the effects of developmental ATZ exposure are broad, time sensitive, and involve the interaction of many pathways. In Chapter 5, the studies use adult zebrafish aged 9 to 14 months to tests the hypothesis that embryonic exposure to low levels of ATZ results in age and sex-specific changes in behavior, the adult brain transcriptome, and adult body and brain pathology, according to the DOHaD hypothesis. At age 9 months, a novel tank test, a light-dark box, and an open field test evaluated adult behavior; microarray analysis investigated ATZ related differences in gene expression; and brain histopathology investigated neuropathology. At 14 months, the body length, weight, and brain weight was measured to evaluate effects of ATZ on mature body and brain size. The 9 month adult behavioral tests found nonmonotonic, sex-specific behavior changes, with male zebrafish having decreased activity and female zebrafish having increased signs of anxiety. Microarray analysis identified sex-specific transcriptomic alterations, with females having altered expression of genes in pathways related to cancer and organismal injury and males having altered gene expression in organismal development and reproductive system development and function pathways. Genes with persistant altered expression were also identified. Adult zebrafish also had nonmonotonic, sex-specific alterations in body length, body weight, and brain weight. This study suggests that developmental exposure to ATZ does cause sex-specific alterations in adult neural function later in life. In summary, developmental exposure to legacy, emergent, and current use environmental toxicants can alter neurodevelopment and later life neural function in zebrafish, suggesting a greater need for human health risk assessments

Study of how excessive follicular non-esterified fatty acids impact programming in porcine early embryo and granulosa cells

Le développement des follicules, des ovocytes et des embryons sont des processus biologiques qui contrôlent la possibilité de devenir un organisme vivant. La maturation des ovocytes est un événement tout à fait unique avec deux divisions asymétriques, la méiose, mais également caractérisée par l'arrêt de la transcription et le stockage des transcrits et des protéines nécessaires au développement embryonnaire précoce. Comme nous le savons, le liquide folliculaire forme partie du micro environnement autour de l'ovocyte et influence la compétence de l'ovocyte. Cependant, la composition du liquide folliculaire peut changer sous divers statuts nutritionnels, tels que l'obésité chez l'homme et un bilan énergétique négatif chez les vaches et les truies. Ces modifications moléculaires du liquide folliculaire induites par les conditions métaboliques maternelles peuvent affecter la qualité des ovocytes, le développement embryonnaire et peuvent par la suite provoquer des troubles métaboliques et autres maladies chez les prochaines générations. Ces dernières années, de nouvelles études se sont concentrées sur les effets de contextes nutritionnels anormaux, p.ex., niveaux élevés d'acides gras non estérifiés (AGNE), sur les cellules de la granulosa, les cellules du cumulus, les ovocytes et le développement précoce de l'embryon chez diverses espèces, notamment chez l'homme, la souris et le bétail. Dans cette thèse, le porc a été utilisé comme modèle expérimental. Le porc est un excellent modèle pour étudier l'impact de la nutrition sur la fonction ovarienne chez l'homme, puisqu'il existe de nombreuses similitudes entre les porcs et l'homme telles que la physiologie digestive et la durée approximative de la maturation in vitro (IVM). En outre, les niveaux élevés d'AGNE induits par la nutrition existent également pendant la lactation chez les porcs. Enfin, les embryons de porc peuvent être générés in vitro et leur étude n'est pas soumise aux mêmes limitations éthiques que les humains. Pour mieux comprendre les effets de niveaux élevés d'AGNE au cours de la maturation des ovocytes chez le porc, des ovocytes porcins ont été exposés au milieu de maturation in vitro contenant une combinaison de niveaux élevés d'AGNE. Des puces à ADN ont été utilisées pour obtenir des informations globales sur les schémas transcriptomiques et de méthylation de l'ADN des blastocystes provenant des ovocytes exposés aux AGNE. Afin de mieux mimer un environnement folliculaire fonctionnel lors de la maturation, des ovocytes exposés aux AGNE ont été co-cultivés ou non avec des cellules de la granulosa porcine issues de follicules de taille moyenne (diamètre entre 3 et 5 mm). Pour capturer la réponse des cellules de la granulosa aux AGNE, les informations transcriptomiques des cellules de la granulosa co-cultivées ont également été étudiées. Le travail réalisé dans cette thèse nous a permis de mettre en évidence et d'expliquer les impacts de niveaux élevés d'AGNE folliculaires sur les blastocystes issus d'ovocytes exposés aux AGNE et les cellules de la granulosa co-cultivées. Les ovocytes exposés aux AGNE ont moins de potentiel de se développer en blastocystes, et les blastocystes dérivés ont montré une inflammation, plus d'apoptose, et des changements métaboliques des schémas transcriptomiques et de méthylation de l'ADN. La méthylation différente de l'ADN ouvre la possibilité de transmettre les impacts induits par les AGNE aux générations suivantes. Lorsque les voies affectées des blastocystes provenant d'ovocytes avec coculture de cellules de la granulosa ont été comparées à celles sans coculture, davantage de voies anti-inflammatoires ont été observées dans des conditions de coculture, suggérant le rôle protecteur des cellules de la granulosa dans la diminution des effets néfastes des AGNE sur les ovocytes. En examinant le transcriptome des cellules de la granulosa avec ou sans AGNE, nous avons observé que les cellules de la granulosa étaient capables d'atténuer les effets néfastes des AGNE sur les ovocytes par la régulation du métabolisme, des facteurs anti-inflammatoires et l'inhibition de la transition épithéliale-mésenchymateuse. Le dernier chapitre résume le métabolisme des acides gras dans les ovocytes, les cellules folliculaires et les embryons, et traite également des effets néfastes de l'exposition aux AGNE pendant la maturation in vitro sur les ovocytes, les cellules folliculaires et les embryons. Les informations dérivées des résultats mettent en évidence les effets néfastes des niveaux élevés d'AGNE dans le liquide folliculaire induits par l'état nutritionnel maternelle avec un aperçu global des schémas transcriptomiques et de méthylation de l'ADN chez les porcs. Ensuite, le rôle des cellules de la granulosa dans la protection des ovocytes envers l'environnement défavorable des acides gras en excès est proposé. Les connaissances acquises donnent une idée du métabolisme du niveau élevé d'acides gras dans les follicules et suggèrent l'importance d'une concentration appropriée d'acide gras pendant la folliculogenèse et la maturation des ovocytes.The development of follicles, oocytes, and embryos are the biological processes that control the possibility to become a living organism. The oocyte maturation is a quite unique event with two asymmetric cleavages, the meiosis, but also characterized by the arrest of transcription and the storage of transcripts and proteins required for early embryonic development. As we know, the follicular fluid is part of the microenvironment surrounding the oocyte and influences the oocyte competence. However, the composition of follicular fluid may change under various nutrition statuses, such as obesity in humans and negative energy balance in cows and sows. These maternal metabolic conditions induce follicular fluid changes may affect oocyte quality, embryonic development, and subsequently cause metabolic problems and diseases in the next generations. In recent years, new studies focused on the effects of abnormal nutrition contexts, e.g., elevated levels of non-esterified fatty acids (NEFAs), on granulosa cells, cumulus cells, oocytes, and early embryo development in various species including humans, mice, and cattle. In this thesis, the pig was used as the experimental model. The pig is an excellent model to study the impacts of nutrition on human ovarian function since there are many similarities between pigs and humans, such as digestive physiology and approximate length of in vitro maturation (IVM). Besides, the nutrition-induced elevated levels of NEFA also exist during the lactation in pigs. Finally, porcine embryos can be generated in vitro and are not subjected to the same ethical limitations as human embryos. To better understand the effects of elevated levels of NEFAs during oocyte maturation in pigs, porcine oocytes were exposed to an in vitro maturation medium containing a combination of high level of NEFAs. Microarrays were used to draw global information of the transcriptomic and DNA methylation patterns of blastocysts originated from NEFAs' exposed oocytes. To better mimic a functional follicular environment during maturation, oocytes exposed to NEFAs were co-cultured or not with porcine granulosa cells from medium size follicles (diameter between 3 and 5 mm). To capture the response of granulosa cells to NEFAs, the transcriptomic information of the cocultured granulosa cells was also investigated. The work completed in this thesis allowed us to highlight and explain the impacts of elevated levels of follicular NEFAs on the blastocysts originated from NEFAs-exposed oocytes and the cocultured granulosa cells. NEFAs-exposed oocytes have less potential to develop into blastocysts, and the derived blastocysts showed inflammation, more apoptosis, and metabolic changes both in transcriptomic and DNA methylation patterns. The altered DNA methylation opens the possibility of transmitting the NEFAs-induced impacts to subsequent generations. When the affected pathways of blastocysts originated from oocytes cocultured with granulosa cells were compared to those without coculture, more anti-inflammatory pathways were observed in coculture condition, suggesting the protective role of granulosa cells in decreasing the detrimental effects of NEFAs on oocytes. Looking at the granulosa cell transcriptome with or without NEFAs, we observed that the granulosa cells were able to attenuate the adverse effects of NEFAs on oocytes through regulation of metabolism, anti-inflammatory factors, and inhibition of the epithelial-mesenchymal transition. The last chapter summarizes the metabolism of fatty acids in oocytes, follicular cells, and embryos, and also discusses the adverse impacts of exposure to NEFAs during IVM on oocytes, follicular cells, and embryos. The information derived from the results highlights the detrimental impacts of maternal nutrition-induced elevated NEFAs levels in follicular fluid with a global overview of the transcriptomic and DNA methylation patterns in pigs. Then the role of granulosa cells in protecting the oocytes from the adverse environment of excess fatty acids is proposed. The acquired knowledge provides the idea about the metabolism of the high level of fatty acids in follicles and suggests the importance of a suitable concentration of fatty acids during folliculogenesis and oocyte maturation

Analisi della varianza di dati di espressione genica

Per il diabete mellito la distinzione tra gruppi fenotipici, difficilmente deducibile dalle differenze tra le medie di espressione, può essere definita dalle differenze tra le varianze delle distribuzioni dei valori di espressione. Il lavoro di tesi vuole valutare quali geni siano differenzialmente espressi tra 3 fenotipi(normali, intolleranti al glucosio e diabetici) tramite due procedure: analisi standard sui dati di espressione(SAM, GSEA); analisi sui dati della varianz

Phosphatidylinositol-3 kinase/AKT and Fos like protein 1 regulation of trophoblast differentiation

Trophoblast cells are one of the earliest lineages to develop in the mammalian embryo. Trophoblast giant cells are the first trophoblast lineage to differentiate and have endocrine and invasive properties. This study focus on the role of the PI3K/AKT signaling pathway in trophoblast giant cell gene expression, steroidogenic potential and invasiveness. Gene profiling of trophoblast populations in the stem, differentiated or differentiated with modified PI3K/AKT signaling states identify genes regulated by PI3K/AKT. These genes are involved in the endocrine and invasive functions of trophoblast and most are upregulated during trophoblast differentiation. Further analysis shows that modification of the PI3K/AKT singling pathway decreases the invasive phenotype of trophoblast and expression of Fos like antigen 1 (FOSL1 or FRA1) a key transcriptional regulator of trophoblast invasion related genes. In summary, we show that FRA1 is a mediator of the PI3K/AKT signaling pathway in regulating gene expression and trophoblast invasion

A comprehensive functional analysis of tissue specificity of human gene expression

<p>Abstract</p> <p>Background</p> <p>In recent years, the maturation of microarray technology has allowed the genome-wide analysis of gene expression patterns to identify tissue-specific and ubiquitously expressed ('housekeeping') genes. We have performed a functional and topological analysis of housekeeping and tissue-specific networks to identify universally necessary biological processes, and those unique to or characteristic of particular tissues.</p> <p>Results</p> <p>We measured whole genome expression in 31 human tissues, identifying 2374 housekeeping genes expressed in all tissues, and genes uniquely expressed in each tissue. Comprehensive functional analysis showed that the housekeeping set is substantially larger than previously thought, and is enriched with vital processes such as oxidative phosphorylation, ubiquitin-dependent proteolysis, translation and energy metabolism. Network topology of the housekeeping network was characterized by higher connectivity and shorter paths between the proteins than the global network. Ontology enrichment scoring and network topology of tissue-specific genes were consistent with each tissue's function and expression patterns clustered together in accordance with tissue origin. Tissue-specific genes were twice as likely as housekeeping genes to be drug targets, allowing the identification of tissue 'signature networks' that will facilitate the discovery of new therapeutic targets and biomarkers of tissue-targeted diseases.</p> <p>Conclusion</p> <p>A comprehensive functional analysis of housekeeping and tissue-specific genes showed that the biological function of housekeeping and tissue-specific genes was consistent with tissue origin. Network analysis revealed that tissue-specific networks have distinct network properties related to each tissue's function. Tissue 'signature networks' promise to be a rich source of targets and biomarkers for disease treatment and diagnosis.</p

Transcriptional control of prostate cancer metabolism

242 p.La transformación y la progresión tumoral van acompañados de cambios metabólicos. Los principales co-reguladores metabólicos son capaces de orquestar la modulación de múltiples rutas metabólicas a través de la regulación de programas transcripcionales. En esta tesis hemos mostrado que el co-regulador transcripcional y metabólico PGC1 suprime la progresión tumoral y metastática en el cáncer de próstata. El análisis bioinformatico de los principales co-reguladores metabólicos reveló a PGC1 como factor principalmente alterado en cáncer de próstata, cuya expresión disminuía y estaba asociada a la progresión tumoral. Usando modelos de ratón y xenotransplantes, demostramos la inhibición de la progresión tumoral y metástasis por parte de PGC1. La integración de análisis metabólicos y transcriptómicos reveló que PGC1 activa un programa transcripcional dependiente del factor de transcripción ERR, el cual inducía un estado catabólico responsable de la supresión metastática. Además, hemos observado que la expresión de este co-regulador altera la remodelación del citoesqueleto, induciendo cambios en la morfología celular. Finalmente, una marca genética basada en la activación del programa transcripcional (PGC1-ERR) exhibe potencial pronóstico en cáncer de próstata. El uso tanto de la marca genética como del estudio metabólico realizado podría permitir la estratificación de pacientes y el desarrollo de nuevas terapias en el cáncer de próstata.CICbioGUN