Gene transcription rates are influenced by proteins, known as Transcription Factors (TFs), that interact with DNA. The locations of TFs on the genome directly influence gene expression and the functional characteristics of a cell. TF binding locations can be estimated for entire genomes using high throughput chromatin immunoprecipitation sequencing (ChIP-Seq). While the analysis of ChIP-Seq binding locations is standardized for a single experiment, complications arise when data sets, taken from different labs and experimental conditions, are combined. In this thesis, I present my method for the simultaneous comparison of multiple ChIP-Seq data sets. My method of comparing multiple ChIP-Seq data sets extends the analysis of a single data set through the addition of two stages, a combination stage, and an extraction stage. Typically, one of two approaches are used to combine information from multiple datasets. Either estimated binding sites are extracted from each dataset and then combined (e.g. by various intersections or unions) or the "raw" genomic signals are analyzed by clustering or dimensionality reduction methods. Both approaches have strengths, but also substantial drawbacks. The method presented here relies both on estimating the binding sites and comparing the “raw” genomic signals between data sets. Once the binding locations have been found, the first step in the combination stage is to define an alternate feature space (AFS). The AFS is the union of all binding locations determined for all data sets. The AFS represents a subset of the genome that is likely to have TF binding in any condition where the protein is active. Once the AFS is defined, the read density is determined from the “raw” genomic signal of each of the data sets. The density is determined for all locations in the AFS resulting in a unified density matrix (UDM). The UDM is the final product of the combination stage of the analysis. After the data sets are homogenized into the UDM, the extraction stage is applied to the matrix. The extraction stage consists of applying machine learning techniques and other methods used to analyze the “raw” genomic signal, to help elucidate underlying similarities and differences between the data sets. I applied this method to the binding locations of the TF TAL1 across 22 ChIP-Seq data sets from the hematopoietic and endothelial lineages. Once the UDM had been generated and normalized, using quantile normalization, hierarchical clustering and principle component analysis (PCA) were applied. Clusters, formed by hematopoietic stem cells (HSCs), Erythroid, and T-cell acute lymphoblastic leukemia (T-ALL), were found using hierarchical clustering. The principle components (PCs) of the UDM provided weights for each peak. Using those weights I could separate groups of cellular conditions including T-ALL, Erythroid, HSC, and Endothelial Colony Forming Cells (ECFCs.) The weights also provided a quantitative measure of importance for each peak in the AFS based on how much weight they provided towards the group of interest. Functional analysis techniques, including de novo motif search and Gene Ontology, were applied to the peak partitions defined using the PCs. Motifs that were enriched in the T-ALL TAL1 partition, and not the Erythroid, were annotated and found to be similar to those that had previously been published, including Runx1 motif and a preference for the CC Ebox (CACCTG). In addition to finding the CC Ebox in T-ALL, I also show that it does not form a composite motif with GATA, indicating an alternative mechanism for the binding of TAL1 in T-ALL. This thesis establishes that heterogeneous collections of ChIP-Seq datasets, from multiple labs and experimental conditions, can be meaningfully combined, and provides an algorithmic template for doing so.

ETS1 est un facteur de transcription (FT) spécifique transposé dans les leucémies aigües. Le rôle essentiel d'ETS1 a été décrit au cours de l'hématopoïèse, plus particulièrement dans la différenciation lymphocytaire T. Son expression temporelle coordonnée participe au contrôle des transitions du stade double négatif (DN) CD4-/CD8- au stade double positif (DP) CD4+/CD8+jusqu'au stade simple positif (SP) CD4+ ou CD8+. Au cours de l'ontogenèse T, ETS1 transactive notamment l'expression des chaînes β et α du récepteur des cellules T (TCR). Nous avons criblé à grande échelle les cibles d'ETS1 aux stades DN et DP en ChIP-Seq, ainsi que desmarques histone et de l'ARN polymérase II (Pol II). Afin de faciliter nos analyses bioinformatiques, nous avons développé deux logiciels, CoCAS et AmaMineReg, qui permettent d'identifier plus facilement les cibles à partir de données brutes et de discriminer les vrais des faux positifs. Nous avons trouvé 5900 cibles en DN et 3400 en DP, principalement intergéniques dont 2000 sont communes, non caractérisées et correspondent aux gènes induits par la réponse immédiate à la signalisation TCR. Parmi les cibles différentiellement exprimées entre les deux stades, ETS1 active les gènes thymus-spécifiques et réprime les gènes hématopoïétiques non T spécifiques,en fonction de la co-occurrence avec le motif RUNX1. Nous avons également caractérisé très clairement le site de fixation en conditions natives, qui se révèle être CTTCCT.De plus, ETS1 co-localise avec des marques chromatines permissives aux régions inter- et intragéniques,caractérisées par un contenu GC, densité de motifs de fixation de FT (SFFT) et conservation inter-espèces accrus. / ETS1 is a specific transcription factor (TF) transposed in acute leukemias. key role of ETS1 wasdescribed during hematopoiesis, especially in T lymphocyte differentiation. Its temporal expression participates in the coordinated control of phase transitions from the CD4-/CD8-double negative (DN) stage to CD4+/CD8+ double positive (DP) up to CD4 or CD8 single positivestage (SP). During ontogenesis T ETS1 notably transactivates the expression of the alpha and beta chains of the T-Cell receptor (TCR). We performed genome-wide screening of ETS1 at both DN and DP stages via ChIP-Seq, as well as histone hallmarks and RNA polymerase II (PolII). To facilitate computational analysis we developed two new software suites, and COCASAmaMineReg, which allow easier identification of targets from raw data and to discriminate between true and false positives. We found 5900 targets in 3400 in DN and DP, mostly intergenic, out of which 2000 are common, and correspond to uncharacterized genes induced bythe immediate response to TCR signaling. Among targets differentially expressed between thetwo stages, Ets1 activates thymus-specific genes and represses non T-specific haematopoietic genes depending on the co-occurrence with the RUNX1 motif. We also very clearly characterized the binding site in native conditions, which proved to be CTTCCT. Furthermore, Ets1 colocalizes with permissive chromatin marks in inter-and intra-genic regions, characterized byincreased GC content, TF binding motifs (TFBS) density as well as inter-species conservation

En 2010, Haurie et al. ont identifié deux isoformes différentes de la Pol III humaine : Pol IIIα, quin’est présente que dans les cellules souches embryonnaires et dans celles tumorales, et Pol IIIβ,exprimée constitutivement.L’expression ectopique de Pol IIIα affecte profondément l’équilibre de cellules différentiées, jusqu’àen induire la transformation oncogénique. Ceci n’est pas observé dans le cas de l’expression ectopiquede Pol IIIβ.A fin de définir les causes moléculaires de la transformation cellulaire induite par Pol IIIα, nousavons décidé d’étudier son transcriptome en comparaison avec celui de Pol IIIβ, à la recherche desgènes qui pourraient soutenir l’oncogenèse observée. Dans notre étude, nous avons adopté latechnique du ChIP-Seq, une approche innovatrice et puissante qui permet de cartographier tous lessites de liaison d’une protéine sur le génome. Elle comporte la purification de la protéine d’intérêtcomplexe avec ces cibles génomiques et le séquençage de ces dernières de manière massive.Le premier but de cette thèse a été représenté par la mise a point d’un protocole de ChIP-Seqoptimisé spécifiquement pour Pol IIIα et Pol IIIβ. Une fois obtenue une préparation de chromatine dequalité adéquate aux standards du ChIP-Seq, nous avons effectué les séquençages massifs etcartographié sur le génome tous les loci contactés par les deux isoformes de polymérase.De cette manière, nous avons identifié 1287 loci occupés par Pol IIIα et 1281 par Pol IIIβ. Les deuxpolymérases se co-localisent sur la plupart de ces sites, mais montrent aussi une occupationpréférentielle et spécifique sur une fraction de loci Pol III. Cette disproportion pourrait contribuer auphénomène cellulaire observé par Haurie et al.Le manuscrit de thèse détaille les résultats de cette analyse et les conclusions qui en dérivent.Nous y avons ajouté une synthèse des travaux réalisés antérieurement et concertants la caractérisationdes promoteurs de gène qui codent pour les snoARN chez la levure S.cerevisiae, positionnée en toutefin de document. / In eukaryotes, transcription is carried out by DNA-dependent RNA polymerases I, II and III (or I-V inplants). These RNA polymerases are specialized in the transcription of specific groups of genes.Human RNA polymerase III (Pol III) transcribes small noncoding RNAs involved in the regulation oftranscription (7SK RNA), RNA processing (U6 RNA, RNAse P, RNAse MRP), translation (tRNAs,5S RNA) or other cellular processes (vault RNAs [multidrug resistance], adenoviral RNAs [VA-I,VA-II], Epstein-Barr virus RNAs [EBER1, EBER2]). It has furthermore been reported that somemicroRNAs of viral or cellular origin may also be transcribed by Pol III.Interestingly, increased Pol III transcription levels accompany or cause cell transformation. Themechanisms underlying this phenomenon are still largely unknown. Recently, two distinct isoforms ofhuman Pol III have been discovered (Haurie et al., 2010). RPC32β-containing Pol IIIβ is ubiquitouslyexpressed and essential for growth and survival of human cells. In contrast, RPC32α-containing PolIIIα is dispensable for cell survival and its expression is restricted to undifferentiated embryonic stem(ES) cells and to tumor cells.The distinct effects of Pol IIIα and Pol III β on cell growth and transformation may be explained bythe transcription of isoform-specific target genes. To identify such isoform-specific target genes, wespecifically targeted RPC32α and of RPC32β subunits in chromatin immunoprecipitation (ChIP)experiments and analyzed the co-precipitated DNA sequences by high throughput sequencing (ChIPseq.).Genome wide localization of RPC32α and RPC32β subunits of Pol III revealed the presence of bothsubunits on many of the known Pol III-transcribed genes, suggesting redundant activities of bothisoforms of Pol III in transcription of these genes. We also found that some of the genes known to betranscribed by Pol III are only occupied by either RPC32α or by RPC32β, suggesting that these genesare exclusively transcribed by Pol IIIα or by Pol IIIβ, respectively.RPC32α and RPC32β ChIP-seq. results furthermore led to the identification of novel Pol III candidategenes in HeLa cells. Moreover, we found high levels of Pol IIIα or Pol III β at some of the annotatedtRNA pseudogenes, implicating that these genes may be transcribed. The functions of RNAstranscribed from novel putative Pol III genes or from tRNA pseudogenes remain to be determined.

A sinalização celular desencadeada na presença do hormônio andrógeno em células da próstata é dependente da ativação do receptor de andrógeno (AR), que é um fator de transcrição codificado pelo gene NR3C4. O AR quando não ligado ao hormônio andrógeno é encontrado no citoplasma, e a ligação do hormônio ao AR promove seu deslocamento para o núcleo. O AR se liga a motivos de DNA, promovendo a transcrição de determinados genes por meio de um complexo mecanismo de regulação ainda não totalmente conhecido. Neste trabalho exploramos a capacidade dos RNAs longos intergênicos não-codificadores (lincRNAs) se ligarem ao AR e contribuírem para a regulação transcricional de genes. Utilizamos a imunoprecipitação de RNA, seguida de sequenciamento em larga escala (RIP-Seq) para a identificação e quantificação de lincRNAs associados fisicamente ao AR na linhagem celular de próstata LNCaP. Em paralelo utilizamos dados de transcriptoma e de marcas epigenéticas para verificar se a interação física dos lincRNAs com o AR influenciaria a composição da cromatina e a expressão dos genes vizinhos desses ARA-lincRNAs (Androgen Receptor Associated LincRNAs). Como resultado deste estudo descrevemos pela primeira vez centenas de LincRNAs associados fisicamente ao receptor de andrógeno após o tratamento com o hormônio. Observamos que uma parte desses ARA-lincRNAs pode modificar in cis a expressão de genes codificadores de proteínas vizinhos e essa capacidade de regulação é reforçada pela presença de marcas epigenéticas que são características de reguladores in cis. Além disso, mostramos que as regiões da cromatina contendo domínios topologicamente associativos (TADs) que possuem ARA-lincRNAs apresentam fatores de transcrição, marcas epigenéticas e nível de transcrição gênica diferenciadas. Os resultados apresentados neste trabalho estendem a importância dos lincRNAs durante eventos regulatórios complexos e mostra pela primeira a atuação dessas moléculas em sinergia com um fator de transcrição modificando a cromatina e alterando a regulação gênica. / The cell signaling events triggered in the presence of the androgen hormone in prostate cells is dependent on the activation of the androgen receptor (AR), which is a transcription factor encoded by the NR3C4 gene. AR when not bound to the androgen hormone is found in the cytoplasm, and binding of the hormone to AR promotes its displacement to the nucleus. AR binds to DNA motifs, promoting the transcription of certain genes through a complex regulatory mechanism not yet fully undestood. In this work we explore the ability of long non-coding intergenic RNAs (lincRNAs) to bind to AR and contribute to the transcriptional regulation of genes. We used immunoprecipitation of RNA, followed by large-scale sequencing (RIP-Seq) for the identification and quantification of lincRNAs physically associated with AR in the LNCaP prostate cell line. In parallel we used transcriptome data and data on epigenetic marks to verify whether the physical interaction of lincRNAs with AR would influence the chromatin composition and the expression of genes neighboring these ARA-lincRNAs (Androgen Receptor Associated LincRNAs). As a result of this study we described for the first time in the literature hundreds of LincRNAs physically associated with the androgen receptor after treatment with the hormone. We have observed that part of these ARA-lincRNAs can modify in cis the expression of neighboring protein coding genes and this regulatory ability is enhanced by the presence of epigenetic marks that are characteristic of in cis regulators. In addition, we have shown that chromatin regions containing topologically associating domains (TADs) that possess ARA-lincRNAs have different transcription factors, epigenetic marks and gene transcription levels. The results presented in this work extend the importance of the lincRNAs during complex regulatory events and shows for the first time the performance of these molecules in synergy with a transcription factor modifying the chromatin and altering gene regulation.

Com o desenvolvimento do sequenciamento em larga escala, novas tecnologias surgiram para auxiliar o estudo de sequências de ácidos nucleicos (DNA e cDNA); como consequência, o desenvolvimento de novas ferramentas para analisar o grande volume de dados gerados fez-se necessário. Entre essas novas tecnologias, uma, em particular, chamada Imunoprecipitação de Cromatina seguida de sequenciamento de DNA em larga escala ou CHIP-Seq, tem recebido muita atenção nos últimos anos. Esta tecnologia tornou-se um método usado amplamente para mapear sítios de ligação de proteínas de interesse no genoma. A análise de dados resultantes de experimentos de ChIP-Seq é desaadora porque o mapeamento das sequências no genoma apresenta diferentes formas de viés. Os métodos existentes usados para encontrar picos em dados de ChIP-Seq apresentam limitações relacionadas ao número de amostras de controle e tratamento usadas, e em relação à forma como essas amostras são combinadas. Nessa tese, mostramos que métodos baseados em testes estatísticos de hipótese tendem a encontrar um número muito maior de picos à medida que aumentamos o tamanho da amostra, o que os torna pouco conáveis para análise de um grande volume de dados. O presente estudo descreve um método estatístico Bayesiano, que utiliza meta-análise para encontrar sítios de ligação de proteínas de interesse no genoma resultante de experimentos de ChIPSeq. Esse métodos foi chamado Meta-Analysis Bayesian Approach ou MABayApp. Nós mostramos que o nosso método é robusto e pode ser utilizado com diferentes números de amostras de controle e tratamentos, assim como quando comparando amostras provenientes de diferentes tratamentos. / With the development of high-throughput sequencing, new technologies emerged for the study of nucleic acid sequences (DNA and cDNA) and as a consequence, the necessity for tools to analyse a great volume of data was made necessary. Among these new technologies, one in special Chromatin Immunoprecipitation followed by massive parallel DNA Sequencing, or ChIP-Seq, has been evidenced during the last years. This technology has become a widely used method to map locations of binding sites for a given protein in the genome. The analysis of data resulting from ChIP-Seq experiments is challenging since it can have dierent sources of bias during the sequencing and mapping of reads to the genome. Current methods used to nd peaks in this ChIP-Seq have limitations regarding the number of treatment and control samples used and on how these samples should be used together. In this thesis we show that since most of these methods are based on traditional statistical hypothesis tests, by increasing the sample size the number of peaks considered signicant changes considerably. This study describes a Bayesian statistical method using meta-analysis to discover binding sites of a protein of interest based on peaks of reads found in ChIP-Seq data. We call it Meta- Analysis Bayesian Approach or MABayApp. We show that our method is robust and can be used for dierent number of control and treatment samples, as well as when comparing samples under dierent treatments.

La compréhension des mécanismes qui contrôlent l'expression des gènes est un enjeu majeur pour la recherche médicale. Elle nécessite un ensemble d'approches pangénomiques telles que les puces à ADN et plus récemment le séquençage à très haut débit qui génèrent une masse toujours plus grande de données numériques à traiter. Au cours de ma thèse, j'ai développé plusieurs outils informatiques innovants pour faciliter leur exploitation. Ainsi, j'ai créé une librairie R (AgiND) qui vérifie la qualité des données de puces à ADN Agilent et permet de les normaliser. Le nombre croissant d'expériences stockées dans Gene Expression Omnibus a motivé la mise en place du projet TBrowser. Une méthode originale DBF-MCL a été créée pour extraire des signatures transcriptionnelles annotées par l'intégration de diverses sources d'information. Stockées dans une base de données, elles sont accessibles à travers une interface Java, un service web SOAP et une librairie R/Bioconductor (RTools4TB). Enfin, un pipeline d'analyse dédié au ChIP-seq a été implémenté. Tous ces outils ont servi pour l'étude de diverses maladies dans le cadre de collaborations. / Understanding the mechanisms that control gene expression is a major challenge for medical research. This requires using a large set of pangenomic approaches such as those using DNA microarrays and high-throughput sequencing that generate an ever growing mass of digital data. During my thesis, I have developed several computer-based tools to facilitate their processing and analysis. I have created a R library (AgiND) that controls the quality of Agilent DNA microarray data and allows their statistical normalization. The growing number of experiences stored in Gene Expression Omnibus has motivated the development of the TBrowser project. An original method, DBF-MCL, was created to extract annotated transcriptional signatures by integrating various sources of information. Stored in a database, these signatures are accessible using a Java interface, a SOAP web service and a R/Bioconductor library (RTools4TB). Finally, a pipeline dedicated to the ChIP-seq analyses has been implemented. All these tools were used to study various diseases in collaborations.

Pour comprendre comment les gènes du développement exercent de multiples fonctions temporelles, nous prenons comme modèle le facteur de transcription Otx2. Celui-ci est impliqué dans la gastrulation, le développement de l’œil, du système olfactif, de la glande pinéale, du thalamus et de la région cranio-faciale. Dans la rétine adulte, deux tissus distincts expriment Otx2 : l’épithélium pigmenté (RPE) et la rétine neurale, contenant les photorécepteurs. L’ablation globale du gène Otx2 entraîne la dégénérescence exclusive des photorécepteurs alors qu’elle modifie l’expression de gènes surtout dans le RPE. Ces faits suggèrent un mécanisme non autonome, confirmé par des expériences de gain et perte de fonction restreintes au RPE. Pour approcher les fonctions de la protéine Otx2 dans la rétine neurale et le RPE, une étude à grande échelle de ses cibles génomiques a été menée. Les profils distincts d’occupation du génome du RPE et de la rétine neurale suggèrent des fonctions différentes d’Otx2. Dans la rétine neurale, ce profil est très proche de celui du facteur paralogue Crx, indiquant une redondance fonctionnelle entre Otx2 et Crx. Nous avons émis l’hypothèse qu’une combinatoire de partenaires protéiques différents permet de moduler l’action d’Otx2 en sélectionnant des cibles génomiques distinctes. Pour identifier cette combinatoire in vivo et la corréler aux fonctions exercées par Otx2, nous avons créé une lignée de souris exprimant une protéine de fusion Otx2-TAP-tag à un niveau physiologique. Cet outil permettra la purification des complexes protéiques Otx2 in vivo et leur identification par analyse protéomique. / In the present work, we study the Otx2 transcription factor as a model to understand how developmental genes achieve multiple functions throughout time. Otx2 is first implied in gastrulation, and then participates to the development of the eye, the olfactory system, the pineal gland, the thalamus and the craniofacial region. Otx2 is expressed in two distinct tissues: retinal pigmented epithelium (RPE) and neural retina including photoreceptors. Global Otx2 gene ablation leads to exclusive photoreceptor degeneration although most of the affected genes are RPE specific. These elements suggest a non-cell-autonomous mechanism, confirmed by RPE restricted gain and loss of function. To understand Otx2 functions in the neural retina and in the RPE, a large scale study of its genomic targets has been yielded. Genome occupancy profiles in RPE and neural retina suggest different Otx2 functions. In the neural retina, Otx2 genome occupancy profile is very close to the one of its paralogue Crx, indicating functional redundancy between both transcription factors. We hypothesized that a different combination of protein partners allows modulating Otx2 action by selecting distinct target genes. To identify Otx2 combinatory in vivo and correlate it to Otx2 functions, we produced a mouse line expressing an Otx2-TAP-tag fusion protein at physiological level. This tool will allow purification of Otx2 protein complexes in vivo and their identification by proteomic analysis.

Cette thèse s’intéresse aux mécanismes permettant d’expliquer plusieurs des fonctions de l’homéogène Otx2 au cours du développement. Une première partie étudie l’importance de la régulation de son expression dans la régionalisation du système nerveux central. A la fin de la gastrulation la frontière d’expression postérieure d’Otx2 déterminera la position de l’organiseur isthmique responsable de l’induction du mésencéphale et du métencéphale. Un modèle murin a été mis au point dans lequel cette frontière est abolie au profit d’une présence uniforme du gène. A l’encontre du modèle actuel, l’isthme est alors correctement induit, et est de plus déplacé antérieurement, signe qu’un seuil net de concentration d’Otx2 est nécessaire à sa fonction régionalisante. Une seconde partie étudie l’importance du contexte cellulaire dans les modalités d’action d’Otx2 au niveau de la rétine adulte. Otx2 est exprimé dans les deux tissus qui composent cet organe, la neurorétine et le RPE. Une étude par ChIP-seq dans ces deux tissus a pu montrer que l’homéogène y occupait des sites de fixation très différents, suggérant des fonctions distinctes. L’écrasante majorité des sites occupés par Otx2 dans la neurorétine l’était également par son paralogue Crx, indice d’une redondance fonctionnelle. Une nouvelle lignée de souris a permis l’analyse des partenaires protéiques d’Otx2 dans la neurorétine, et pu démontrer qu’Otx2 ne formait pas d’interactions avec les autres facteurs de ce tissu, faisant en fait de Crx l’acteur principal de la famille Otx. Cette analyse a également dévoilé une série de partenaires jusque-là inconnus d’Otx2, potentiellement associée à de nouvelles fonctions de la protéine. / The molecular mechanisms explaining several functions of the homeogene Otx2 during embryonic development are the focus of this work. In a first part the importance of the regulation of its expression in the regionalisation of the central nervous system is studied. At the end of gastrulation the posterior border of Otx2 expression will position the isthmic organizer responsible for the induction of the midbrain and hindbrain. A mouse model was developed where this border is replaced by an ubiquitous expression of the gene. Contrary to the predictions of the current model, the organizer then correctly arises, and is shifted anteriorly. A concentration threshold of Otx2 thus appears necessary to its regionalising function. In a second part the importance of the cellular context in Otx2 function in the adult retina is examined. Otx2 is expressed in both tissues of this organ, the neural retina and RPE. A ChIP-seq analysis performed on both tissues revealed that this homeogene occupies very different sets of binding sites, which suggests distinct functions of the transcription factor. Most Otx2-bound sites in the neural retina were also bound by its paralogue Crx, with which a functional redundancy may therefore exist. A new mouse line finally allowed the study of the complete Otx2 interactome in the neural retina; this analysis showed that Otx2 does not interact with other important transcription factors of this tissue, and that Crx may therefore be the main actor of the Otx family in neural retina function. It also led to the discovery of a series of previously unknown partners of Otx2, which could be associated to new functions of this homeogene.

La ségrégation active des molécules d'ADN chez les bactéries compte sur l'assemblage d'une structure nucléoprotéique nucléée sur des sites centromerique localisés près de l'origine de réplication. Pour les systèmes de partition de type I, les seuls présent sur les chromosomes et le plus fréquents sur les plasmides a bas nombre de copie, la protéine qui se lie au centromère, ParB, montre la capacité de propagation sur l'ADN à partir de la nucléation au niveau du site centromerique pars. Ainsi, le complèxe de partition contient plus que 90% des ParB présent dans la cellule et interagit avec ParA, une ATPase dont l'activité dépend de ParB et de l'ADN. Deux modèles concurrents qui sont actuellement proposés pour expliquer le mécanisme d'assemblage du complèxe de partition : les modèles de 'spreading and bridging' (Graham et al., 2014), et le 'nucleation and caging' (Sanchez et al., 2015) . Nous avons utilisé le système de partition du plasmide F et pour déterminer si la mode d'assemblage était aussi conservée sur les chromosomes, nous avons étudié le système natif de Vibrio cholerae du chromosome 1. D'abord, nous avons exploré les mécanismes d'incompatibilité basé sur les sites centromériques pour comprendre l'assemblage des complèxes de partition dans les conditions de titrage ParB par le site parS. Ensuite, nous avons décrit un protocole optimisé de ChIP-sequencing à haut-débit, de la paillasse jusqu'à l'analyse des données. Puis, en utilisant le ChIP-sequencing, la microscopie à épifluorescence et la modélisation physico-mathématique, nous avons révélé que l'assemblage du complèxe de partition est robuste et cohérent avec le modèle de 'nucleation and caging' à la fois sur les chromosomes et les systèmes plasmidiques. / The active partition of low copy number plasmids and most bacterial chromosomes relies on the assembly of a nucleoprotein superstructure nucleated at centromere-like sites near the origin of replication. In the case of type I partition systems, the most widespread on plasmids and the only ones present on chromosomes, centromere binding proteins, ParB, display the capability to propagate along DNA from their nucleation point at the centromere-like site, parS. This structure, termed the partition complex, contains over 90% of the available ParB in the cell and interacts with ParA, a ParB- and DNA-dependent ATPase, to segregate and position replicons within the cell. Two concurrent models exist to explain the assembly mechanism of the partition complex: the spreading and bridging (Graham et al., 2014), and the nucleation and caging (Sanchez et al., 2015) models. We used the partition system of the archetypal plasmid F and the naturally occurring partition system of Vibrio cholerae's chromosome 1. First, we explored the mechanisms of centromere-based incompatibilities to gain insight into partition complex assembly in low levels of ParB through parS titration of ParB. Next, we describe an optimized a high- resolution ChIP-sequencing protocol from the lab bench to data analysis. Then, through ChIP-sequencing, epifluorescence microscopy and physico-mathematical modeling, we revealed that the partition complex assembly mechanism is robust and consistent with the nucleation and caging model on both chromosomal and plasmid systems.

Les gliomes sont les tumeurs primitives les plus fréquentes du cerveau et restent de mauvais pronostic en raison de l’inefficacité des traitements actuels. Des cellules souches cancéreuses ont été isolées à partir de gliomes de haut grade de l’adulte. Ces cellules souches de gliomes (GSC) peuvent fournir tous les sous-types cellulaires qui composent la tumeur. De nombreuses données indiquent que la résistance aux traitements est due en grande partie aux GSC. Cibler les GSC et leurs propriétés souches constitue donc un enjeu thérapeutique important. [...] Une solution pertinente de ciblage thérapeutique est de forcer les GSC à quitter leur état souche. Dans ce cadre, mes principaux travaux ont eu pour but de caractériser les changements épigénétiques des marques d’histones qui accompagnent la répression des propriétés des GSC par un groupe de micro-ARN, miR-302-367. [...] L’étude de cette plasticité par notre équipe a abouti à l’identification de miR-302-367. Son expression forcée, à l’aide de lentivirus, bloque de façon irréversible les propriétés souches et initiatrices de tumeur des GSC. L’effet suppresseur de tumeur exercé par miR offre la possibilité d’identifier les mécanismes qui régulent le maintien ou la perte des propriétés des GSC. A l’aide d’un modèle formé par une lignée de GSC et de sa contrepartie dépourvue des propriétés souches et tumorigènes GSC-miR-302-367, je me suis attachée à caractériser les méthylations de l’histone H3, qui font parties du code d’histone associé à une transcription génique respectivement active ou réprimée. Je me suis axée sur la triméthylation de la lysine 4 (H3K4me3) et de la lysine 27 (H3K27me3), respectivement permissive et répressive de la transcription. Une analyse par ChIP-seq (Immunoprécipitation de la chromatine-séquençage) des gènes associés à ces marques a été associée à la caractérisation des transcriptomes des cellules par exon-array. Nos résultats montrent que l’expression du groupe de miR-302-367 ne modifie pas de façon globale les taux des marques H3K4me3 et H3K27me3. Par contre, des changements dans des groupes de gènes circonscrits ont pu être identifiés. La corrélation positive observée entre les marques d’histones et les taux d’expression des gènes montre une conservation du code d’histone dans les cellules cancéreuses, au moins pour les marques étudiées. L’analyse des termes GO (Gene Ontology) indique que la perte des propriétés induites par miR-302-367 s’accompagne d’un engagement de GSC dans une voie de différenciation. Les gènes portant la marque répressive dans les GSC-miR-302-367 participent notamment à des catégories fonctionnelles associées à l’expression de propriétés souches et tumorigènes. L’analyse du groupe de gènes portant une marque permissive dans les GSC et répressive dans les GSC-miR-302-367, a révélé un réseau de facteurs de transcription susceptible de participer au contrôle des propriétés souches des GSC. La répression à l’aide de siRNA d’un des membres de ce réseau, le facteur de transcription ARNT2, nous a permis de révéler son rôle dans le maintien des capacités prolifératives des GSC issues de gliomes distincts et dans l’expression du facteur de transcription Nanog, connu pour son rôle central dans le contrôle des propriétés souches des GSC. Nos résultats montrent que l’analyse des changements de marques d’histone offre donc non seulement une vue d’ensemble des différents réseaux moléculaires associés au maintien ou au contraire à la répression des propriétés des GSC, mais permet d’identifier de nouveaux acteurs. L’effet stimulateur d’ARNT2 sur la croissance cellulaire et l’expression de Nanog, dans des GSC dérivées de gliomes différents aux altérations génomiques distinctes, indique que ce facteur de transcription tient une place centrale, insoupçonnée jusqu’à présent, dans la hiérarchie des gènes qui gouvernent les propriétés des GSC. / Gliomas, the most frequent primary brain tumors, are resistant to current therapies and the survival rate of patients is very low. Within high-grade gliomas, a cell sub-population bearing stem-like properties has been isolated. These cells, called glioma stem cell (GSC), are capable of generating all glioma cellular sub-types. Recent data indicates that resistance of these aggressive tumors to therapies is mostly due to GSCs. Thus, targeting the GSCs and their stem-like properties is imperative in order to improve current therapies. [...] Another effective solution to treat GSCs is to force them to lose their stem-like properties. In this context, the aims of my major project were to characterize the epigenetic modifications of histone marks accompanying the loss of GSC stem-like properties under the influence of a cluster of micro-RNA, miR-302-367. GSCs are endowed with an exceptional plasticity, allowing them to gain or lose their stem-like state in response to modifications in their micro-environment. Our results identified the implication of miR-302-367 in the regulation of GSC plasticity. Its stable expression using lentivirus inhibits in an irreversible manner the stem-like and tumorigenic properties of GSC. The tumor-suppressor effect of this miR offers the possibility to decipher the mechanisms responsible for the maintenance or the loss of GSC stem-like properties. Using the model of GSC and their counterparts, GSC-miR-302-367, who lost their stem-like and tumorigenic properties, my aim was to identify the methylation status of histone H3 of the histone code which is known to be associated either to an active or to a repressive gene transcription. I focused on the trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3), which are associated with an activation or repression of gene transcription, respectively. We performed a ChIP-seq (Chromatin-immunoprecipitation-sequencing) analysis of the respective associated genes followed by a transcriptomic (exon-array) analysis of both cell lines. Our results show that miR-302-367 expression does not alter in a global manner the expression levels of H3K4me3 and H3K27me3. On the contrary, we were able to detect modifications in a discrete group of genes. At least for the studied marks, the positive correlation between the identified histone marks and the gene expression levels indicates that the histone code is well preserved in cancer. GO (Gene Ontology) analysis indicates that miR-302-367-induced loss of stem-like properties is accompanied with activation of the differentiation process in GSC. Genes implicated in the regulation of stem-like and tumorigenic properties were found to bear the repressive histone mark in GSC-miR-302-367. From our analysis of the group of genes bearing the active histone mark in GSC and the repressive one in GSC-miR-302-367, emerged a network of transcription factors that could possibly participate in the regulation of GSC stem-like properties. Down-regulation using siRNA of a member of this network, namely ARNT2, highlighted its role in the maintenance of the proliferative dynamic, as well as the expression of the transcription factor Nanog (a major regulator of GSC stem-like properties), in GSC derived from distinct gliomas. Our histone mark modification analysis, not only elucidated the molecular pathways implicated in the maintenance or, on the contrary, in the loss of GSC stem-like properties, but also, highlighted the implication of new actors in these processes. The activator effect of ARNT2 on GSC proliferation, as well as on the expression of Nanog, observed in GSC bearing distinct genetic alterations and derived from different glioma, indicates that this transcription factor plays a major role, not documented thus far, in the regulation of GSC stem-like properties.