Dissertationen zum Thema „Non coding variations“

The genetic and molecular bases of most of the human diseases have become one of the main goals of the human biology in the last decades. To be able to unveil the genetic variations and the affected cellular processes associated with a specific disease is crucial in order to generate accurate diagnosis and further therapies. The Next Generation Sequencing (NGS) revolution, with the associated reduction in time and costs of sequencing, has allowed the scientist to access large number of human genomes to their biomedical studies. The study of genetic disorders, cancer in particular, has benefit from NGS identifying genetic variations associated with a given disorder. All these new results, some of them in regions with unknown function, have generated a double challenge in the scientific community. Firstly, detect as much as possible all the different variants associated with a disease, in some complex diseases several. Secondly, to understand the functional impact those modifications are causing in the cell. Regarding the first challenge, this thesis contributes in the identification of genetic modifications throw the development of a bioinformatics tool named SMUFIN (Moncunill et al. 2014). SMUFIN can detect somatic variants related with tumour development and progression in a quickly and effective way. Not limited to the software development, several tumours has been analysed and their somatic variants characterized. These tumours include mantel cell lymphoma, paediatric medulloblastoma and chronic lymphocytic leukaemia (Moncunill et al. 2014; Puente et al. 2015). In the evaluation of the functional impact, the thesis also includes a method, RELA, to determine when these annotated variants play a regulatory role as enhancers or promoters (Gonzalez et al. 2012). Combined with other available data and a spread methodology to unveil regulatory regions evaluation of variants affecting regulatory regions have been performed in chronic lymphocytic leukaemia (details included in the thesis discussion). To sum up, this thesis cover with methodology and provide bioinformatics tools to perform a complete genomic analysis of genetic variants in biomedicine studies. It includes from the identification of variants for each of the patients to the evaluation of their functional impact in the disease development and progression. This kind of approach is currently common in the research laboratories and it will be part of the healthcare system in a close future to diagnose and classify patients.
El estudio de las bases genéticas y moleculares de las patologías humanas ha constituido el centro de atención de gran parte de la investigación en biología durante las últimas décadas con el fin último de comprender los procesos celulares alterados en cada caso y la posibilidad de generar protocolos de diagnosis y terapias específicas. Con la llegada de la denominada Next Generation Sequencing (NGS) y su consiguiente reducción en tiempo y costes ha permitido el acceso a la secuenciación de numeroso genomas humanos en el entorno biomédico. El estudio de enfermedades genéticas, y del cáncer en particular, se ha visto enormemente favorecido al poder incorporar un importante número de genomas de pacientes a sus estudios y así poder identificar directamente las mutaciones asociadas a cada patología. A su vez, esta revolución junto con la capacidad de detectar modificaciones genéticas en regiones cuya función todavía se desconoce, ha generado un doble desafío en la comunidad científica: por un lado el análisis de variantes genéticas asociadas a cada tipo de enfermedad y, por el otro, el entender el impacto funcional que dichas modificaciones provocan en la célula. Esta tesis contribuye a solucionar estas limitaciones a través del desarrollo de una aplicación, SMUFIN (Moncunill et al. 2014), que permite de forma rápida y eficaz la identificación de variaciones somáticas asociadas al desarrollo o progresión de tumores. También se describen los resultados obtenidos relativos a la identificación y caracterización de las reorganizaciones cromosómicas en cáncer, así como los resultados obtenido en cuanto a sus mecanismos e impacto funcional (Puente et al. 2015). Además, como parte de la anotación genómica para la interpretación funcional de las variaciones detectadas, esta tesis incluye los resultados del desarrollo de estrategias y metodologías para la detección de regiones reguladoras en genomas de eucariotas (Gonzalez et al. 2012). En resumen esta tesis intenta cubrir y dotar de herramientas bionformáticas para completar los pasos necesarios para el análisis de genomas en biomedicina, desde que un grupo de pacientes son secuenciados hasta que sus diferentes variantes son identificadas y su impacto funcional determinado. Este tipo de análisis, que ahora esta ocurriendo en el campo de la investigación, pronto será una realidad y una rutina en el sistema sanitario.

L’expression des gènes passe par le processus de transcription dans le noyau des cellules eucaryotes qui produit les ARNs, intermédiaires indispensables pour former des protéines. La synthèse et le devenir des ARNs sont soumis à un contrôle complexe assuré par de nombreux acteurs incluant entre autres les séquences d'ADN non codantes régulatrices qui assurent une régulation spatio-temporelle fine de l’expression génique et les ribonucléoprotéines hétérogènes nucléaires (hnRNP) capables de lier les molécules d’ARN et de réguler leur maturation, leur stabilité et leur localisation.L'approche standard actuelle pour l'exploration moléculaire des patients atteints d'anomalies du développement (AD) et/ou de déficience intellectuelle (DI), déploie la combinaison de l’analyse chromosomique par puces à ADN, le test de l’X fragile, le séquençage d'exome, et plus récemment le séquençage du génome pour établir un diagnostic moléculaire. L’ensemble de ces approches comporte un rendement diagnostique inférieur à 50% pour les AD/DI. Cependant, les analyses peuvent parfois mettre en évidence la présence de variations de signification incertaine dans des gènes candidats, non encore impliqués en pathologie humaine. Des tests fonctionnels sont alors nécessaires afin d’établir une correcte corrélation génotype-phénotype. De cette manière, des variations pathogènes ont été identifiées au sein de deux gènes candidats codant des hnRNPs intervenant dans le métabolisme des ARNs : PTBP1 et PTBP2. Le but de cette première étude est de décrire le mécanisme cellulaire physiopathologique lié aux défauts transcriptionnels à l'origine de l’atteinte neurodéveloppementale syndromique (pour PTBP1) ou non syndromique (pour PTBP2) par des approches moléculaires fonctionnelles in vitro et in vivo dont le séquençage d'ARNs immunoprécipités (RIP-seq) dans une cohorte d’individus atteints.Dans certains cas, l’analyse génomique met en évidence la présence de variations de structure complexes, pouvant interrompre la séquence d’un gène sensible au dosage, modifier l’activité d’un enhancer ou encore exercer des effets de position sur l'expression génique en altérant les interactions enhancer/gène(s) cible(s). Ces communications moléculaires sont facilitées au sein de domaines d'association topologique (TADs) qui jouent un rôle important dans la régulation transcriptionnelle de manière tissu-spécifique. Par conséquent, toute variation de structure susceptible de réorganiser les TADs (fusion, mélange entre deux TADs ou même création) peut entraîner une altération de l’expression génique. Dans ce contexte, l'objectif du second travail de recherche est de caractériser, au moyen de la capture de conformation de chromosomes à haut débit (Hi-C), les remaniements complexes des patients capables de modifier la structure des TADs. Combinée avec d’autres techniques omiques comme le séquençage longs fragments, les études transcriptomiques ou encore épigénomiques, cette approche permet d'étudier les mécanismes moléculaires sous-jacents sur différents modèles cellulaires dérivés des individus affectés.Ces travaux de recherche mettent en évidence l'impact physiopathologique des variations génétiques ponctuelles et de structure sur les mécanismes de régulation transcriptionnelle et post-transcriptionnelle des gènes cibles et ouvrent la voie à de nouvelles hypothèses biologiques dans le cadre de la recherche translationnelle en pathologie humaine
Gene expression occurs through the transcription process in the nucleus of eukaryotic cells, which produces RNAs, essential intermediates for protein formation. RNA synthesis and fate are controlled by a complex network of factors, among which are regulatory non-coding DNA sequences that ensure precise spatio-temporal regulation of gene expression and heterogeneous nuclear ribonucleoproteins (hnRNP), able to bind RNA molecules and contributing to their maturation, stability, and localization.The current standard approach for molecular exploration of patients with developmental disorders (DD) and/or intellectual disabilities (ID) uses a combination of chromosomal analysis using DNA microarrays, fragile X testing, exome sequencing, and more recently, genome sequencing to establish a molecular diagnosis. These approaches yield a diagnostic yield of less than 50% for DD/ID. However, the analyses sometimes reveal the presence of variations of uncertain significance in candidate genes not yet implicated in human pathology. Functional tests are then necessary to establish a correct genotype-phenotype correlation. In this way, pathogenic variations have been identified in two candidate genes encoding hnRNPs involved in RNA metabolism: PTBP1 and PTBP2. The aim of this first study is to describe the cellular pathophysiological mechanism related to transcriptional defects causing syndromic (for PTBP1) or non-syndromic (for PTBP2) neurodevelopmental impairment using in vitro and in vivo functional molecular approaches including RNA immunoprecipitation sequencing (RIP-seq) in a cohort of affected individuals.In some cases, genomic analysis identifiy complex structural variations that can disrupt the sequence of a dosage-sensitive gene, alter the activity of an enhancer, or exert position effects on gene expression by altering enhancer/target gene interactions. These molecular communications are facilitated within topological associating domains (TADs), which play an important role in tissue-specific transcriptional regulation. Consequently, any structural variation that reorganizes TADs (fusion, shuffling or even new TAD) can lead to an alteration in gene expression. In this context, the goal of this second research project is to characterize, through high-throughput chromosome conformation capture (Hi-C), the complex rearrangements in patients reorganizing the structure of TADs. Combined with other omic techniques such as long fragment sequencing, transcriptomic or epigenomic analysis, this approach allows the study of the underlying molecular mechanisms on different cellular models derived from affected individuals.These research efforts highlight the physiopathological impact of punctual and structural genetic variations on the transcriptional and post-transcriptional regulatory mechanisms of target genes and pave the way for new biological hypotheses in the context of translational research in human pathology

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 101-107).
One of the fundamental goals of human genetics is to identify the genetic causes of human disease to ultimately design novel therapeutics. However, two challenges have become readily apparent. First, the majority of genomic regions associated with disease do not implicate protein-altering variants but might instead alter gene regulation, making interpretation and validation more difficult. Second, the genomic regions associated with disease explain a fraction of the variance of associated phenotypes, suggesting human diseases are highly polygenic and that many additional regions remain to be discovered and characterized. Here, we address these challenges by using functional annotation of the human genome spanning diverse data types: epigenomic profiles, gene regulatory circuitry, and biological pathways. We first develop a method to simultaneously select relevant genomic regions not yet associated with disease as well as select relevant functional annotations enriched in those regions. We show that both tissue-specific and shared regulatory regions are enriched for disease associations across eight common diseases. We then characterize specific genetic variants in the selected regions, the gene regulatory elements they reside in, the cellular contexts in which those elements are active, their upstream regulators, their downstream target genes, and the biological pathways they disrupt across eight common diseases. We show that disease associations are additionally enriched in regulatory motifs of relevant transcription factors and in relevant biological pathways. We finally investigate why predicted regulatory elements are enriched in disease-associated variants by framing the problem as Bayesian inference of hyperparameters in a structured sparse regression model. We propose an active sampling method to efficiently explore the hyperparameter space and avoid exponential scaling in the dimension of the hyperparameters. We show in simulation that our method can distinguish between possible explanations of the observed enrichments, and we characterize potential biases in the estimates. Together, our results can help guide the development of new models of disease and gene regulation and discovery of biologically meaningful, but currently undetectable regulatory loci underlying a number of common diseases.
by Abhishek Sarkar.
Ph. D.

La variation antigénique est un mécanisme d'évasion immunitaire utilisé par le parasite du paludisme Plasmodium falciparum pour établir une infection prolongée. Il empêche la clairance parasitaire en modifiant l’expression des antigènes variables de surface, codés par la famille de gènes appelés « var », comprenant 60 membres. Au stade sanguin, les parasites expriment les gènes var d'une manière mutuellement exclusive, avec un seul gène var actif à un moment donné, permettant ainsi au parasite d’échapper au système immunitaire qui reste naïf envers une sous-population de parasites. Malgré les multiples facteurs connus pour être impliqués dans l’inactivation des gènes var par défaut, tels que les modifications des histones et l'architecture nucléaire, un facteur d'activation spécifique n'a pas encore été identifié. Dans ce travail de thèse, une famille d'ARNnc riche en GC, transcrite par l’ARN polymérase III, a été explorée comme facteur régulateur candidat à l'activation des gènes var. La famille des ARNnc riches en GC se compose de 15 membres homologues, positionnés de façon adjacente aux gènes var situés au centre du chromosome. L'analyse d’expériences de FISH de l'ARN a révélé que l'ARNnc riche en GC cible le site d'expression du gène var en trans. De plus, la surexpression d'un membre des ARNnc riches en GC surpasse l'expression mutuellement exclusive des gènes var et induit la transcription d'un sous-ensemble spécifique de gènes var. Nous avons développé un système de dead Cas9 ciblant la transcription de tous les membres riches en GC. Il est frappant de constater que le blocage transcriptionnel de tous les membres riches en GC par interférence CRISPR entraîne une régulation à la baisse de la transcription des gènes var à un niveau basal. Nos données confirment le rôle de cet ARNnc dans le processus d'activation des gènes var. De plus, nous montrons que la famille de gènes de l'ARNnc riche en GC est une variante clonale, ce qui indique que cela peut déterminer les taux de commutation et l'ordre de commutation de la famille de gènes var. Nous avons développé un protocole ChIRP (chromatin isolation by RNA purification) robuste qui nous a permis d'identifier des régions potentielles de liaison à la chromatine et des partenaires interagissant avec cette famille d'ARNnc. La validation de plusieurs protéines candidates prometteuses obtenues par spectrométrie de masse est en cours. De plus, nous avons cherché à savoir si les loci géniques de ces ARNnc présentent des interactions à longue distance, à l'aide d'une technique de capture de conformation chromosomique. Nous avons obtenu la preuve que les loci géniques de ces ARNnc présentent des interactions à longue distance entre eux et peuvent donc jouer un rôle dans l'organisation spatiale des foci nucléaires. Cette étude fournit le premier lien fonctionnel entre la transcription de l’ARN Pol III et Pol II dans le processus d'expression mutuellement exclusive des gènes de virulence. De plus, l'exploration de cet ARNnc est une étape clé dans le dévoilement du processus d'évasion immunitaire et de la pathogenèse de P. falciparum
Antigenic variation is an immune evasion mechanism used by the malaria parasite Plasmodium falciparum to establish prolonged infection. It prevents parasite clearance by switching the display of variant surface antigens encoded by the 60-member var gene family. Blood stage parasites express var genes in a mutually exclusive manner, with a single var active at a given time, ensuring that the immune system remains naive to a parasite sub-population. Despite multiple factors known to be involved in default var gene silencing, such as histone modifications and nuclear architecture, a specific activation factor has not been identified yet. In this thesis work, a polymerase III-transcribed family of GC-rich ncRNA was explored as a candidate regulatory factor of var gene activation. The GC-rich ncRNA family consists of 15 homologous members positioned adjacent to chromosome-central var genes. RNA FISH analysis revealed that the GC-rich ncRNA is targeted to the var expression site in trans. Overexpression of one GC-rich ncRNA member overrules singular var expression and induces transcription of a specific subset of var genes. We developed a dead Cas9 system aiming to target transcription of all GC-rich members. Strikingly, transcriptional blocking of all GC-rich members by CRISPR interference results in down-regulation of var transcription to background levels. Our data support a role of this ncRNA in the activation process of var genes. Moreover, we show that the GC-rich ncRNA gene family is clonally variant, indicating that this may determine switch rate and switch order of the var gene family. We developed a robust ChIRP (chromatin isolation by RNA purification) protocol that allowed us to identify potential chromatin binding regions and interacting partners of this ncRNA family. The validation of several promising candidate proteins obtained my mass spectrometry is ongoing. Furthermore, we investigated whether the gene loci of these ncRNAs display long-range interactions using a chromosome conformation capture technique. We obtained evidence that the gene loci of these ncRNAs display long-range interactions between them and can thus play a role in nuclear spatial foci organization. This study provides the first functional link between Pol III and Pol II transcription in the process of mutually exclusive expression of virulence genes. Moreover, exploring this ncRNA is a key step in unveiling the process of immune evasion and pathogenesis of P. falciparum

A major virulence factor of the human malaria parasite Plasmodium falciparum is Plasmodium falciparum erythrocyte membrane protein 1(PfEMP-1). This protein is inserted into the erythrocyte membrane, giving cytoadherence properties. A family of genes called var, located sub-telomerically and in chromosome central clusters encode this protein. Var genes are expressed in a mutually exclusive manner, how this is controlled is unclear. A non-coding RNA (ncRNA) termed the GC-rich element (GRE) had been identified that is only located at the central clusters and is transcribed throughout the parasite lifecycle. A screen of the P. falciparum genome for novel ncRNAs identified ncRNAs from known classes. Novel transcripts were identified, but none in the proximity of var genes. We have investigated the role of the GRE in var gene regulation. A set of qRT-PCR primers have been designed and tested to follow var gene expression in the HB3 isolate, these are not cross-reactive with a published set for the 3D7 isolate. Alterations were made to the 3D7 set to remove cross-reactivity with HB3. Var gene expression was studied in 31 HB3 clones and progeny of the 3D7xHB3 genetic cross. Following var switching over five months in eleven HB3 clones showed that all of the clones ended up expressing var genes from the same central cluster on chromosome 4. GRE Transcription in these clones is linked to a specific class of var gene. Transcription from a single GRE locus occurs only when a var gene of the central UpsC class is expressed from the same cluster. Expression of other classes of var gene gives multiple transcripts from different GRE loci. Investigations into the in vitro binding properties of the GRE revealed an RNA:protein complex that can be resolved by electrophoresis. Proteomic analysis of the complex revealed predominantly ribosome proteins and translation factors.

L'annotation fonctionnelle de mutations somatiques est un point focal des études de génomique du cancer. Jusque récemment, la recherche s'est concentré sur des mutations dans la fraction codante du génome, pour lesquelles de puissants outils bioinformatiques ont été développés afin de distinguer des mutations délétères des mutations neutres. On identifie un nombre croissant de variants associés à des maladies dans le génome non-codant. L'interprétation des mutations non-codantes dans le cancer est donc devenue une tâche urgente. Des projets de grande envergure tels que ENCODE ont rendu possible l'interprétation fonctionnelle de variants dans les cancers. Plusieurs programmes ont été produits sur la base de ces informations fonctionnelles. Ces outilssont encore limités, notamment, une bas précision de la prédiction, le manque d'information de la mutation de cancer et biais de constatation importante. Dans le chapitre 2 de cette thèse, pour interpréter fonctionnellement les mutations non-codantes dans les cancers, nous avons développé deux modèles de forêts aléatoires indépendants, appelées SNP et SOM. Compte tenu de la combinaison de caractéristiques fonctionnelles à une position donnée du génome, le modèle SNP prédit la fraction de SNP rares (une mesure de la sélection négative), et le modèle SOM prédit la densité de mutations somatiques attendue à cette position. Nous avons appliqué nos deux modèles pour évaluer des clinvariant and HGMD variants asociés à des maladies, et un ensemble de SNP-contrôle aléatoires. Les résultats ont montré que les variants associés à des maladies ont des scores plus élevés que les SNP-contrôle avec le modèle SNP et inférieures avec le modèle SOM, confortant notre hypothèse selon laquelle la sélection négative, telle que mesurée par fraction de SNP rares et de densité de mutation somatiques, nous informe sur l'impact fonctionnel des mutations tumorales dans le génome non-codant. Jusqu'à présent, les chercheurs ont surtout considéré les gènes protéiques comme critiques dans l'initiation et la progression des cancers. Toutefois, des preuves récentes ont montré que les ARN non-codants, en particulier les lncRNAs, sont activement impliqués dans divers processus de cancer. Un chapitre de cette thèse est consacré à cette classe de transcripts non codants. Comme pour les gènes codants, il pourrait exister un grand nombre de lncRNAs driver de cancer. Le développement d'outils bioinformatiques pour identifier et hiérarchiser les lncRNA et autres ARN non-codants est devenu un important objet de recherche en oncologie.La dernière partie de cette thèse est consacrée à la mise en œuvre de méthodes pour découvrir des éléments non-codants potentiellement driver de cancer. Nous avons d'abord appliqué trois outils tierces, CADD, funSeq2, GWAVA, ainsi que nos modèles SNP et SOM, pour évaluer l'impact des mutations non-codantes dans tout le génome. Pour chaque locus, nous calculons la moyenne des scores de tous les variants observés à l'aide de l'un des modèles, et nous prenons au hasard le même nombre de variants et calculons leur score moyen 1 million de fois pour former une distribution nulle et obtenir une P-valeur pour ce locus. Pour valider notre hypothèse et notre modèle de permutation, nous avons testé ce système sur 452 gènes codants et 61 lncRNA liés au cancer, en utilisant des données de mutation somatique de cancer du foie, cancer du poumon, CLL et mélanome. Nous avons constaté que les lncRNAs et gènes codants associés au cancer avaient des valeurs-P significativement plus faibles que l'ensemble de lncRNAs et gènes codant. Appliquer ce test de permutation à des lncRNAs avec cinq systèmes de notation différents nous a permis de prioriser les centaines de candidats potentiellement liés au cancer.Ces candidats peuvent maintenant être soumis à validation expérimentale
Functional annotation of somatic mutations have been a consistent hotspot of cancer genomics studies. In the past, researchers preferentially focused on mutations in the coding fraction of the genome, for which ample bioinformatics tools were developed to distinguish cancer-driver mutations from neutral ones. In recent years, as an increasing number of variants were being identified as disease-associated in the non-coding genome, interpreting non-coding cancer mutations has become an urgent task. The completion of large scale projects such as ENCODE, has made functional interpretation of cancer variants achievable, and several programs were produced based on this functional information. However, there still exists some limitations as to these prediction tools, such as low prediction accuracy, lack of cancer mutation information and significant ascertainment bias. In chapter 2 of this thesis, in order to functionally interpret non-coding mutations in cancer, we developed two independent random forest models, referred to as SNP and SOM. Given a combination of features at a given genome positions, the SNP model predicts the expected fraction of rare SNPs (a measure of negative selection), and the SOM model predicts the expected mutation density at this position. We applied our two models to score these non-coding disease-associated clinvariant and HGMD variants and a set of random control SNPs. Results showed that disease-associated variants were scored higher than control SNPs with the SNP model and lower than control SNPs with the SOM model, supporting our hypothesis that purifying selection as measured by fraction of rare SNPs and mutation density is informative for the evaluation of the functional impact of cancer mutations in the non-coding genome. In the past, researchers have preferentially considered protein-coding genes as critical to the initiation and progression of cancers. However, recent evidences have shown that ncRNAs, in particular lncRNAs, are actively implicated in various cancer processes. A chapter of this thesis is devoted to this class of non-coding transcripts. Similar to protein coding genes, there might be a large number of lncRNAs with cancer-driving functions. The development of bioinformatics tools to prioritize them has become a new focus of research for computational oncologists.The last part of this thesis is devoted to the implementation of methods for discovering potential cancer-driving non-coding elements in lncRNA and protein-coding genes. We applied three scoring tools, CADD, funSeq2, GWAVA, together with our SNP and SOM scoring systems to prioritize cancer-associated elements using a permutation-based algorithm. For each locus, we compute the average score of all observed variants using one of the models, and we randomly take the same number of variants and compute their average score 1 million times to form a null distribution and obtain a P value for this locus. To validate our hypothesis and permutation model, we tested this system on 61 cancer-related lncRNA and 452 cancer genes using somatic mutation data from liver cancer, lung cancer, CLL and melanoma. We observed that both cancer lncRNAs and protein-coding genes had significantly lower average P values than total lncRNAs and protein-coding genes in all cases. Applying the permutation test to lncRNAs with five different scoring systems enabled us to prioritize hundreds to thousands of cancer-related lncRNA candidates. These candidates can be used for future experimental validation

Pseudomonas brassicacearum a la particularité de générer une diversité intraclonale aussi bien in vitro qu'en conditions naturelles dans la rhizosphère de plantes. Ce phénomène de variation phénotypique commun chez les bactéries est un processus d'adaptation aux environnements changeants. Des données de transcriptomique issues de puces à ADN, contenant aussi bien des séquences codantes que non codantes, nous ont permis d'identifier les gènes dont l'expression est altérée et surtout de relier ce phénomène à l'expression d'ARN non codants régulateurs (ARNnc) de type Rsm qui sont sous le contrôle du système à deux composants GacS/GacA. Nous avons montré que des mutations ponctuelles dans les gènes gacS ou gacA sont à l'origine de cette variation phénotypique et que l'expression de l'un des trois gènes rsmX, rsmY ou rsmZ permet de restaurer le phénotype de la souche sauvage. L'importance de ces ARNnc dans la survie de la bactérie aux fluctuations de son environnement est dénotée par la duplication de rsmX en un gène que nous avons nommé rsmX-2, dont la fonction a été validée. Nos données suggèrent une activation exclusive des gènes rsmX-1 et rsmX-2 par GacA et l'intervention de régulateurs additionnels dans le cas de rsmY et rsmZ. Au vu de la redondance fonctionnelle de ces quatre ARNnc, nous avons investigué leur niveau d'expression et leur stabilité dans différentes conditions de culture et montré des différences pour les quatre ARNnc. En réponse à une carence en nutriments, l'expression des ARNnc Rsm est fortement activée et atteint son maximum quand le ppGpp est détecté dans le milieu, suggérant un lien entre le système Gac/Rsm et la réponse « stringente »
The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonisation under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as non-coding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small non-coding RNA (ncRNA) genes rsmX, rsmY and rsmZ, which was characterized by down-regulation. Naturally occurring mutations in the GacS/GacA two-component system accounted for phenotypic switching. The importance of these ncRNAs in the survival of the bacteria to changing environments is denoted by the duplication of rsmX gene, which we called rsmX-2 and whose function has been validated. Our data suggest an exclusive activation of rsmX-1 and rsmX-2 genes by GacA and the involvement of additional regulators in the case of rsmY and rsmZ. Given the functional redundancy of these ncRNAs, we investigated their expression level and stability in different culture conditions and showed differences for the four ncRNAs. In response to nutrient depletion, the four ncRNAs expression is strongly activated and reaches its maximum when the ppGpp is detected in bacterial cells, suggesting a link between the Gac/Rsm system and the "stringent" response. Determining the level of each Rsm ncRNA, which is defined by a balance between synthesis and degradation of each transcript, shows the maintenance of a very important pool of RsmZ compared to other ncRNAs

Le parasite protozoaire Plasmodium falciparum est l'agent causal de la forme la plus mortelle de paludisme humain. Ce pathogène utilise l'expression monoallélique de molécules d'adhésion de surface variantes, codées par la famille de gènes var, pour échapper au système immunitaire de l'hôte et provoquer une pathogenèse. On ne sait toujours pas comment l'activation du gène var fonctionne au niveau moléculaire et si des facteurs environnementaux peuvent moduler l'expression du gène var. Notre laboratoire a montré qu'une famille de gènes d'ARN non codants transcrits par Pol III, appelée RUF6, agit comme un trans-activateur des gènes var. Une association physique entre l'ARNnc RUF6 transcrit et le locus du gène var actif a été observée par FISH. La répression transcriptionnelle de tous les RUF6 par une stratégie d'interférence CRISPR spécifique a entraîné une régulation négative de la transcription de toute la famille des gènes var, suggérant une fonction potentielle de type amplificateur pour l'expression des gènes var. Une compréhension de la façon dont l'ARNnc RUF6 médie l'activation du gène var fait défaut. Ici, nous avons développé un protocole robuste de découverte protéomique dirigée par l'ARN (ChIRP-MS) pour identifier les interactions in vivo des protéines ARNnc RUF6. Des oligonucléotides antisens biotinylés ont été utilisés pour purifier l'interactome d'ARNnc RUF6. La spectrométrie de masse a identifié plusieurs protéines enrichies de manière unique qui sont liées à la transcription génique, telles que les sous-unités d'ARN Pol II, les protéines d'assemblage des nucléosomes et un homologue de la Dead-Box Helicase 5 (DDX5). La purification par affinité de PfDDX5 a identifié plusieurs protéines trouvées à l'origine par notre protocole RUF6-ChIRP, validant la robustesse de la technique pour l'identification des interactomes d'ARNnc chez P. falciparum. Le déplacement inductible de PfDDX5 nucléaire a entraîné une importante régulation à la baisse du gène var actif. Notre travail identifie un complexe protéique RUF6 ARNnc qui interagit avec l'ARN Pol II pour soutenir l'expression du gène var. Nous postulons que l'hélicase DDX5 peut résoudre les structures secondaires G-quadruplex hautement enrichies en gènes var pour faciliter l'activation et la progression de la transcription. De plus, nous découvrons des facteurs environnementaux qui déclenchent une régulation négative de la transcription du gène var. Nous observons que la privation d'isoleucine et les concentrations élevées de MgCl2 dans le milieu inhibent les gènes transcrits par l'ARN polymérase III. Il est important de noter que cela inclut une famille de gènes ARNnc régulateurs spécifiques de P. falciparum (codée par la famille de gènes RUF6) qui est un régulateur clé de l'activation du gène var. Nous avons identifié un gène homologue à l'eucaryote Maf1 hautement conservé, en tant qu'effecteur négatif de la transcription de l'ARNnc RUF6. Des concentrations élevées de MgCl2 ont entraîné un déplacement de PfMaf1 cytoplasmique vers le compartiment nucléaire. Nous avons utilisé un système de dégradation inductible des protéines pour montrer que les stimuli externes dépendent de PfMaf1 pour déclencher une expression plus faible des gènes RUF6. Nos résultats indiquent une voie indépendante de TOR qui répond aux changements de l'environnement et réprime la transcription Pol III. Ce travail fournit des informations conceptuelles nouvelles et importantes sur la répression de la virulence du parasite dépendante de PfMaf1 qui peuvent être très pertinentes pour établir la persistance subclinique du parasite pendant la saison sèche. Pris ensemble, ces résultats aident à mieux comprendre la fonction et la régulation d'un ARNnc impliqué dans la régulation de la variation antigénique et de la pathogenèse chez P. falciparum. Notre validation de la technique ChIRP-MS permet de futures études dans l'identification des protéines de liaison à l'ARN pour les ARNnc dont la fonction [...]
The protozoan parasite Plasmodium falciparum is the causative agent of the deadliest form of human malaria. This pathogen uses monoallelic expression of variant surface adhesion molecules, encoded by the var gene family, to evade the host immune system and cause pathogenesis. It remains unclear how monoallelic expression of var gene activation works at the molecular level and if environmental factors can modulate var gene expression. Our laboratory showed a Pol III transcribed GC-rich non-coding RNA gene family, termed RUF6, acts as a trans-activator of var genes. A physical association between the transcribed RUF6 ncRNA and the active var gene locus was observed through FISH. Transcriptional repression of all RUF6 by a specific CRISPR interference strategy resulted in transcriptional down regulation of the entire var gene family, suggesting a potential enhancer-like function to var gene expression. An understanding of how RUF6 ncRNA mediates var gene activation is lacking. Here we developed a robust RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. Biotinylated antisense oligonucleotides were used to purify the RUF6 ncRNA interactome. Mass spectrometry identified several uniquely enriched proteins that are linked to gene transcription such as RNA Pol II subunits, nucleosome assembly proteins, and a homologue of the Dead-Box Helicase 5 (DDX5). Affinity purification of PfDDX5 identified several proteins originally found by our RUF6-ChIRP protocol, validating the robustness of the technique for the identification of ncRNA interactomes in P. falciparum. Inducible displacement of nuclear Pf-DDX5 resulted in the significant down-regulation of the active var gene. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression. We postulate that DDX5 helicase may resolve G-quadruplex secondary structures highly enriched in var genes to facilitate transcriptional activation and progression. Furthermore, we discovered environmental factors that trigger downregulation of var gene transcription. We observe that isoleucine starvation and high MgCl2 concentrations in the medium inhibit RNA Polymerase III transcribed genes. Importantly, this includes a P. falciparum-specific regulatory ncRNA gene family (encoded by the RUF6 gene family) that is a key regulator in var gene activation. We identified a homologous gene to the highly conserved eukaryotic Maf1, as a negative effector of RUF6 ncRNA transcription. Elevated MgCl2 concentrations led to a shift of cytoplasmic PfMaf1 to the nuclear compartment. We used an inducible protein degradation system to show that external stimuli depend on PfMaf1 to trigger lower expression of RUF6 genes. Our results point to a TOR independent pathway that responds to changes in the environment and represses Pol III transcription. This work provides new and important conceptual insights into PfMaf1-dependent repression of parasite virulence that may be highly relevant for establishing subclinical parasite persistence in the dry season. Taken together, these results help to better understand the function and regulation of a ncRNA involved in regulating the antigenic variation and pathogenesis in P. falciparum. Our validation of the ChIRP-MS technique allows for future studies in identifying RNA-binding proteins for ncRNAs whose function remains to be fully characterized

The studies in this thesis describe the application of genotyping and allele specific expression analysis to genetic studies. The role of the gene NPC1 in Triglyceride metabolism was explored in mouse models and in humans on the population level in study I. NPC1 was found to affect hepatic triglyceride metabolism, and to be relevant for controlling serum triglyceride levels in mice and potentially in humans. In study II the utility of the HapMap CEU samples was investigated for tagSNP selection in six European populations. The HapMap CEU was found to be representative for tagSNP selection in all populations while allele frequencies differed significantly in the sample from Kuusamo, Finland. In study III the power of Allele specific expression as a tool for the mapping of cis-regulatory variation was compared to standard eQTL analysis, ASE was found to be the more powerful type of analysis for a similar sample size. Finally ASE mapping was applied to regions reported to harbour long non-coding RNAs and associated SNPs were compared to published trait-associations. This revealed strong cis-regulatory SNPs of long non-coding RNAs with reported trait or disease associations.

Thesis (Ph. D.)--University of California, San Diego, 2006.
Title from first page of PDF file (viewed December 13, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 143-150).

Les rétrotransposons sont des éléments génétiques mobiles qui constituent presque la moitié de notre génome. Seule la sous-famille L1HS appartenant à la classe des Long Interspersed Element-1(LINE-1 ou L1) a gardé une capacité de mobilité autonome chez l’Homme. Leur mobilisation dans la lignée germinale, mais Aussi dans certains tissus somatiques, contribue à la diversité du génome humain ainsi qu’à certaines maladies comme le cancer. Ainsi, de nouvelles copies de L1 peuvent directement s'intégrer dans des séquences codantes ou régulatrices, et altérer leur fonction. De plus, les séquences L1 contiennent elles-mêmes plusieurs éléments cis-régulateurs et leur insertion à proximité ou dans un gène peut produire des altérations génétiques plus subtiles. Afin d'explorer l'ensemble de ces altérations à l'échelle du génome, nous avons développé un logiciel dédié à l’analyse des données de séquençage d'ARN qui permet d'identifier des transcrits chimériques ou antisens impliquant les L1 et d'annoter ces isoformes en fonction des différents événements d’épissage alternatif subits. Au cours de ce travail, il est apparu que la compréhension du lien entre polymorphisme des insertions et phénotype nécessite une vue complète des différentes copies L1HS présentes chez un individu donné. Afin de disposer d'un catalogue aussi complet que possible de ces polymorphismes identifiés dans des échantillons humains sains ou pathologiques et publiés dans des journaux scientifiques, nous avons développé euL1db, la base de données des insertions de rétrotransposon L1HS chez l’Homme. En conclusion, ce travail aidera à comprendre l’impact des L1 sur l’expression des gènes, à l'échelle du génome
Retrotransposons are mobile genetics elements, which form almost half of our genome. Only the L1HS subfamily of the Long Interspersed Element-1 class (LINE-1 or L1) has retained the ability to jump autonomously in humans. Their mobilization in the germline – but also in some somatic tissues – contributes to human genetic diversity and to diseases, such as cancer. L1 reactivation can be directly mutagenic by disrupting genes or regulatory sequences. In addition, L1 sequences themselves contain many regulatory cis-elements. Thus, L1 insertions near a gene or within intronic sequences can also produce more subtle genic alterations. To explore L1-mediated genic alterations in a genome-wide manner, we have developed a dedicated RNA-seq analysis software able to identify L1 chimeric or antisense transcripts and to annotate these novel isoforms with their associated alternative splicing events. During the course of this work, it appeared that understanding the link between L1HS insertion polymorphisms and phenotype or disease requires a comprehensive view of the different L1HS copies present in a given individual or sample. To provide a comprehensive summary of L1HS insertion polymorphisms identified in healthy or pathological human samples and published in peer-reviewed journals, we developed euL1db, the European database of L1HS retrotransposon insertions in humans. This work will help understanding the overall impact of L1 insertions on gene expression, at a genome-wide scale

Les rétrotransposons sont des éléments génétiques mobiles qui constituent presque la moitié de notre génome. Seule la sous-famille L1HS appartenant à la classe des Long Interspersed Element-1(LINE-1 ou L1) a gardé une capacité de mobilité autonome chez l’Homme. Leur mobilisation dans la lignée germinale, mais Aussi dans certains tissus somatiques, contribue à la diversité du génome humain ainsi qu’à certaines maladies comme le cancer. Ainsi, de nouvelles copies de L1 peuvent directement s'intégrer dans des séquences codantes ou régulatrices, et altérer leur fonction. De plus, les séquences L1 contiennent elles-mêmes plusieurs éléments cis-régulateurs et leur insertion à proximité ou dans un gène peut produire des altérations génétiques plus subtiles. Afin d'explorer l'ensemble de ces altérations à l'échelle du génome, nous avons développé un logiciel dédié à l’analyse des données de séquençage d'ARN qui permet d'identifier des transcrits chimériques ou antisens impliquant les L1 et d'annoter ces isoformes en fonction des différents événements d’épissage alternatif subits. Au cours de ce travail, il est apparu que la compréhension du lien entre polymorphisme des insertions et phénotype nécessite une vue complète des différentes copies L1HS présentes chez un individu donné. Afin de disposer d'un catalogue aussi complet que possible de ces polymorphismes identifiés dans des échantillons humains sains ou pathologiques et publiés dans des journaux scientifiques, nous avons développé euL1db, la base de données des insertions de rétrotransposon L1HS chez l’Homme. En conclusion, ce travail aidera à comprendre l’impact des L1 sur l’expression des gènes, à l'échelle du génome
Retrotransposons are mobile genetics elements, which form almost half of our genome. Only the L1HS subfamily of the Long Interspersed Element-1 class (LINE-1 or L1) has retained the ability to jump autonomously in humans. Their mobilization in the germline – but also in some somatic tissues – contributes to human genetic diversity and to diseases, such as cancer. L1 reactivation can be directly mutagenic by disrupting genes or regulatory sequences. In addition, L1 sequences themselves contain many regulatory cis-elements. Thus, L1 insertions near a gene or within intronic sequences can also produce more subtle genic alterations. To explore L1-mediated genic alterations in a genome-wide manner, we have developed a dedicated RNA-seq analysis software able to identify L1 chimeric or antisense transcripts and to annotate these novel isoforms with their associated alternative splicing events. During the course of this work, it appeared that understanding the link between L1HS insertion polymorphisms and phenotype or disease requires a comprehensive view of the different L1HS copies present in a given individual or sample. To provide a comprehensive summary of L1HS insertion polymorphisms identified in healthy or pathological human samples and published in peer-reviewed journals, we developed euL1db, the European database of L1HS retrotransposon insertions in humans. This work will help understanding the overall impact of L1 insertions on gene expression, at a genome-wide scale

A large proportion of the variation in traits between individuals can be attributed to variation in the nucleotide sequence of the genome. The most commonly studied traits in human genetics are related to disease and disease susceptibility. Although scientists have identified genetic causes for over 4,000 monogenic diseases, the underlying mechanisms of many highly prevalent multifactorial inheritance disorders such as diabetes, obesity, and cardiovascular disease remain largely unknown. Identifying genetic mechanisms for complex traits has been challenging because most of the variants are located outside of protein-coding regions, and determining the effects of such non-coding variants remains difficult. In this dissertation, I evaluate the hypothesis that such non-coding variants contribute to human traits and diseases by altering the regulation of genes rather than the sequence of those genes. I will specifically focus on studies to determine the functional impacts of genetic variation associated with two related complex traits: gestational hyperglycemia and fetal adiposity. At the genomic locus associated with maternal hyperglycemia, we found that genetic variation in regulatory elements altered the expression of the HKDC1 gene. Furthermore, we demonstrated that HKDC1 phosphorylates glucose in vitro and in vivo, thus demonstrating that HKDC1 is a fifth human hexokinase gene. At the fetal-adiposity associated locus, we identified variants that likely alter VEPH1 expression in preadipocytes during differentiation. To make such studies of regulatory variation high-throughput and routine, we developed POP-STARR, a novel high throughput reporter assay that can empirically measure the effects of regulatory variants directly from patient DNA. By combining targeted genome capture technologies with STARR-seq, we assayed thousands of haplotypes from 760 individuals in a single experiment. We subsequently used POP-STARR to identify three key features of regulatory variants: that regulatory variants typically have weak effects on gene expression; that the effects of regulatory variants are often coordinated with respect to disease-risk, suggesting a general mechanism by which the weak effects can together have phenotypic impact; and that nucleotide transversions have larger impacts on enhancer activity than transitions. Together, the findings presented here demonstrate successful strategies for determining the regulatory mechanisms underlying genetic associations with human traits and diseases, and value of doing so for driving novel biological discovery.

Dissertation

Genome-wide neutral diversity levels are shaped by both positive and purifying selection on linked sites. In humans like most species, the relative importance of these types of selection in shaping patterns of neutral diversity remains an open question. We can infer their relative contribution from observed patterns of neutral diversity by using information about recombination rates and targets of natural selection. To this end, I fit a joint model of the effects of positive selection (selective sweeps) and purifying selection (background selection) to genetic polymorphism data from the 1000 Genomes Project. I show that a model of the effects of background selection provides a good fit to patterns in diversity data and that incorporating the effects of selective sweeps does not improve the fit. Using my approach, the effects of background selection explain up to 60% of the variation in neutral diversity levels on the 1Mb scale and account for patterns in the data for which positive selection via selective sweeps had been invoked as explanations. I find that over 80% of the selected regions affecting neutral diversity levels are located outside of exons and that phylogenetic conservation is the best predictor of the source of selection in these regions. My results show that the genome-wide effects of background selection are pervasive, with measurable reductions in neutral diversity throughout almost the entirety of the autosomes. I provide maps of the effects of background selection and software for making similar inferences, which should provide important tools for future research that relies on interpreting patterns in neutral diversity levels.

The extent of genetic structuring of a population results from a balance of forces producing local genetic differentiation, and counter-forces producing genetic homogeneity. An understanding of these forces is essential when investigating evolutionary processes in a species. It has been predicted that, when populations experience severe reductions in size, they lose genetic variability. Small population size may occur as a consequence of founder events, such as with the introduction and spread of a pest species. The predictions as described were examined by studying the molecular evolution and population genetics of European rabbit (Oryctolagus cuniculus (L)) populations in Australia, using variation in mitochondrial DNA control region sequences. The results suggest that there is a higher rate of gene flow within populations in semi arid/arid areas, which is attributable to the extreme environmental heterogeneity found in these areas and may be best explained by a process whereby local populations becoming extinct and are recolonised by individuals drawn from other populations.
Master of Science (Hons)

The extent of genetic structuring of a population results from a balance of forces producing local genetic differentiation, and counter-forces producing genetic homogeneity. An understanding of these forces is essential when investigating evolutionary processes in a species. It has been predicted that, when populations experience severe reductions in size, they lose genetic variability. Small population size may occur as a consequence of founder events, such as with the introduction and spread of a pest species. The predictions as described were examined by studying the molecular evolution and population genetics of European rabbit (Oryctolagus cuniculus (L)) populations in Australia, using variation in mitochondrial DNA control region sequences. The results suggest that there is a higher rate of gene flow within populations in semi arid/arid areas, which is attributable to the extreme environmental heterogeneity found in these areas and may be best explained by a process whereby local populations becoming extinct and are recolonised by individuals drawn from other populations.

Codeine-containing medication is commonly used for pain after c-section. In most people, 10% of codeine is biotransformed into morphine by the Cytochrome P450 enzyme 2D6 (CYP2D6). Individuals who convert up to 50 fold more codeine into morphine, ultrarapid metaboizers, are at a greater risk for adverse effects. Conversely poor metabolizers, individuals who convert almost no codeine into morphine, are at risk for untreated pain. The pharmacodynamic relationship between codeine-analgesia and CYP2D6 genotype is studied for possible development of a titration model. To minimize these treatment risks, alternatives to opioids are sought. Reiki, an ancient Japanese form of healing used to treat pain and depression, has not been systematically reviewed for its efficacy in treating pain. My systematic review of Reiki literature (n=12) showed that while most trials yielded a positive result on primary outcomes, all existing Reiki studies lacked in one of the three key areas of proper patient allocation concealment, randomization or blinding which can lead to the introduction of bias. We designed a randomized controlled trial using distant Reiki for postpartum pain, taking careful steps to control for each of those three key areas. Eighty pregnant women scheduled for an elective c-section where recruited and randomly allocated to one of the two arms (n=40 Reiki and n=40 control). Women were monitored in hospital for up to three days. Visual Analogue Scores (VAS) for pain were recorded 4 times per day; and all pain medication, adverse effects and milestone recovery rates after c-section were recorded. Blood samples were taken to determine CYP2D6 genotype. We determined that distant Reiki did not reduce women’s pain; neither the measured pain nor the cumulative dose of pain medication differed between groups. Moreover, rates of recovery after c-section were also not different between the two groups. This led to the conclusion that distant Reiki was not suitable as a primary method of controlling pain after c-section. Our second study (n=45) looked for correlation between CYP2D6 genotype and effectiveness of codeine analgesia. Only a small sample of the women were genetic extremes (n=2 poor metabolizers and n=3 ultrarapid metabolizers), while most were, as expected, extensive or intermediate metabolizers. An individual examination of each of these cases provided valuable insight into patients where CYP2D6 polymorphism is clinically relevant. Two of the three ultrarapid metabolizers stopped opioid analgesia due to adverse effects, while both poor metabolizers complained that the codeine-containing medication was not providing analgesia (i.e. ineffective pain treatment). Healthcare providers need to be aware of patient response to pharmacotherapy and use this information to individualize postpartum opioid analgesia.