Current approaches integrating large scale data and information from a variety of sources to reveal molecular basis of cellular events do not adequately benefit from pathway information. Here, we portray a network structure based pathway enrichment system that fuses and exploits model and data: signalling pathways are taken as the biological models while microarray and ChIP-seq data are the sample input data sources among many other alternatives. Our model- and data-driven hybrid system allows to quantitatively assessing the biological activity of a cyclic pathway and simultaneous enrichment of the significant paths leading to the ultimate cellular response. Signal Transduction Score Flow (SiTSFlow) algorithm is the fundamental constituent of proposed network structure based pathway enrichment system. SiTSFlow algorithm converts each pathway into a cascaded graph and then gene scores are mapped onto the protein nodes. Gene scores are transferred to en route of the pathway to form a final activity score describing behaviour of a specific process in the pathway while enriching of the gene node scores. Because of cyclic pathways, the algorithm runs in an iterative manner and it terminates when the node scores converge. The converged final activity score provides a quantitative measure to assess the biological significance of a process under the given experimental conditions. The conversion of cyclic pathways into cascaded graphs is performed by using a linear time multiple source Breadth First Search Algorithm. Furthermore, proposed network structure based pathway enrichment system works in linear time in terms of nodes and edges of given pathways. In order to explore various biological responses of several processes in a global signalling network, the selected small pathways have been unified based on their common gene and process nodes. The merge algorithm for pathways also runs in linear time in terms of nodes and edges of given pathways. In the experiments, SiTSFlow algorithm proved the convergence behaviour of activity scores for several cyclic pathways and for a global signalling network. The biological results obtained by assessing of experimental data by described network structure based pathway enrichment system were in correlation with the expected cellular behaviour under the given experimental conditions.

The regulation of gene expression at the right time, place, and degree is crucial for many cellular processes such as proliferation and development. In addition, in order to maintain cellular life, cells must rapidly and appropriately respond to various environmental stimuli. Sequence-specific transcription factors (TFs) can recognize functional regulatory DNA elements in a sequence-specific manner so that they can regulate only a specific group of genes, a process which enables cells to cope with diverse internal and external stimuli. Human has approximately 1,400 sequence-specific TFs whose aberrant expression causes a wide range of detrimental consequences including developmental disorders, diseases, and cancers; therefore, it is pivotal to identify the binding sites of each sequence-specific TF in order to unravel its roles in and mechanisms of gene regulation. Even though some TFs have been intensively studied, the majority of TFs still remain to be studied, particularly the tasks of identifying their genome-wide target genes and deciphering their biological roles in specific cellular contexts. Many questions remain unanswered: how many sites on the human genome a sequence-specific TF can bind; whether all TF-bound sites are functional; how a TF achieves binding specificity onto its targets; how and to what extent a TF is involved in gene regulation. Comprehensive identification of the binding sites of sequence-specific TFs and follow-up molecular studies including gene expression microarrays will provide close answers to these questions. Chromatin Immunoprecipitation coupled with recently developed high-throughput sequencing (ChIP-seq) allows us to perform genome-scale unbiased identification of the binding sites of sequence-specific TFs. Here, to gain insight into gene regulatory functions of TFs as well as their influences on gene expression, we conducted, in diverse cell lines, genome-wide identification of the binding sites of several sequence-specific TFs (CTCF, E2F4, MYC, Pol II) that are involved in a wide range of biological functions, including cell proliferation, development, apoptosis, genome stability, and DNA repair. Analysis of ChIP-seq data provided not only comprehensive binding profiles of those TF across the genome in diverse cell lines, but also revealed tissue-specific binding of CTCF, MYC, and Pol II as well as combinatorial usage among these three factors. Analyses also showed that some CTCF binding sites were inherited from parents to children and regulated in an individual-specific as well as allele-specific manner. Finally, genome-wide target identification of several TFs will broaden our understanding of the gene regulatory roles of these sequence-specific TFs. / text

L'axe hypothalamo-hypophyso-surrénalien (HPA) permet de maintenir l'homéostasie de l'organisme face à divers stress. Qu'ils soient de nature psychologique, physique ou inflammatoire/infectieux, les stress provoquent la synthèse et la libération de CRH par l'hypothalamus. Les cellules corticotropes hypophysaires perçoivent ce signal et en réaction, produisent et sécrètent l'ACTH. Ceci induit la synthèse des glucocorticoïdes (Gc) par le cortex surrénalien; ces stéroïdes mettent le système métabolique en état d’alerte pour la réponse au stress et à l’agression. Les Gc ont le rôle essentiel de contrôler les défenses de l'organisme, en plus d'exercer une rétro-inhibition sur l'axe HPA. L'ACTH est une petite hormone peptidique produite par le clivage d'un précurseur: la pro-opiomélanocortine (POMC). À cause de sa position critique dans la normalisation de l'homéostasie, le contrôle transcriptionnel du gène Pomc a fait l'objet d'études approfondies au cours des dernières décennies. Nous savons maintenant que la région promotrice du gène Pomc permet une expression ciblée dans les cellules POMC hypophysaires. L'étude du locus Pomc par des technologies génomiques m'a permis de découvrir un nouvel élément de régulation qui est conservé à travers l'évolution des mammifères. La caractérisation de cet enhancer a démontré qu'il dirige une expression restreinte à l'hypophyse, et plus particulièrement dans les cellules corticotropes. De façon intéressante, l'activité de cet élément dépend d'un nouveau site de liaison recrutant un homodimère du facteur de transcription Tpit, dont l'expression est également limitée aux cellules POMC de l'hypophyse. La découverte de cet enhancer ajoute une toute nouvelle dimension à la régulation de l'expression de POMC. Les cytokines pro-inflammatoires IL6/LIF et les Gc sont connus pour leur antagonisme sur la réaction inflammatoire et sur le promoteur Pomc via l'action des facteurs de transcription Stat3 et GR respectivement. L'analyse génomique des sites liés ii par ces deux facteurs nous a révélé une interrelation complexe et a permis de définir un code transcriptionnel entre ces voies de signalisation. En plus de leur action par interaction directe avec l’ADN au niveau des séquences régulatrices, ces facteurs interagissent directement entre eux avec des résultats transcriptionnels différents. Ainsi, le recrutement de GR par contact protéine:protéine (tethering) sur Stat3 étant lié à l'ADN provoque un antagonisme transcriptionnel. Inversement, le tethering de Stat3 sur GR supporte une action synergique, tout comme leur co-recrutement à l'ADN sur des sites contigus ou composites. Lors d'une activation soutenue, ce synergisme entre les voies IL6/LIF et Gc induit une réponse innée de défense cellulaire. Ainsi lors d'un stress majeur, ce mécanisme de défense est mis en branle dans toutes les cellules et tissus. En somme, les travaux présentés dans cette thèse définissent les mécanismes transcriptionnels engagés dans le combat de l'organisme contre les stress. Plus particulièrement, ces mécanismes ont été décrits au niveau de la réponse globale des corticotropes et du gène Pomc. Il est essentiel pour l'organisme d'induire adéquatement ces mécanismes afin de faire face aux stress et d'éviter des dérèglements comme les maladies inflammatoires et métaboliques. / The hypothalamo-pituitary-adrenal (HPA) axis regulates homeostasis in various conditions of stress contributing to both the stress response and its termination. Psychological, physical or inflammatory/infectious stresses all prompt the synthesis and secretion of hypothalamic CRH. The pituitary corticotrope cells receive this signal and in turn, secrete ACTH which triggers the synthesis of glucocorticoids (Gc) by the adrenal cortex; these steroids induce a general state of alertness in order to fight or flight aggressions and stresses. Glucocorticoids have the critical role to restrict the stress response by exerting a negative feedback on the HPA axis. ACTH is a small peptidic hormone produced after cleavage of a precursor protein: pro-opiomelanocortin (POMC). Due to its critical role in homeostasis, transcriptional control of the Pomc gene has been intensely studied during the last decades. Previous investigations identified a promoter region that is sufficient for expression of Pomc in the appropriate pituitary cells. Genome-wide studies of the Pomc locus led me to discover a novel regulatory element that is conserved throughout mammalian evolution. The activity of this enhancer is restricted to the pituitary, and more precisely to the corticotrope lineage. Interestingly, its activity depends on a novel transcription factor binding motif that binds homodimers of Tpit, a transcription factor that is only found in pituitary POMC cells. The discovery of this enhancer adds a new dimension in the control of pituitary Pomc expression. The IL6/LIF pro-inflammatory cytokines and the glucocorticoids are well known for their antagonism in control of the inflammatory response; at the Pomc promoter, their action is mediated by the transcription factors Stat3 and GR, respectively. The analysis of genomic sites bound by these two factors revealed a complex relationship and led us to define a transcription regulatory code linking these signalling pathways. In addition to their direct DNA interaction with cognate regulatory sequences, these factors iv interact with each other with different outcomes. Thus, the recruitment of GR on DNAbound Stat3 through protein:protein contacts (tethering) results in transcriptional antagonism. Conversely, Stat3 tethering to GR produces synergism; this is also the case when the two factors are co-recruited to DNA on contiguous or composite binding sites. Prolonged activation of the IL6/LIF and Gc pathways elicits a synergistic innate cell defense response in all cells and tissues. In summary, this doctoral work has defined transcriptional mechanisms that mediate and control the stress response. In particular, pituitary components of the stress response were defined at the level of the Pomc gene and as a global response of corticotrope cells. This response is critical for appropriate organism defense during stresses such as those produced in inflammatory and metabolic diseases.

Chez les eucaryotes, l'ARN polymérase (RNAP) III transcrit les ARN de transferts, l'ARN ribosomique 5S, et plusieurs douzaines d'autres ARNs non traduits. Le génome des mammifères contient plusieurs milliers d'éléments répétés, les SINEs. In vitro, leur transcription dépend de la RNAP III. Le taux de transcription de la RNAP III détermine la croissance et la prolifération cellulaires, sa dérégulation a été associée à de nombreux cancers. Afin de caractériser la distribution sur l'ensemble du génome de la RNAP III et de ses facteurs de transcription TFIIIB et TFIIIC, nous avons développé un protocole très spécifique de ChIP-seq en tandem. Nous avons déterminé l'ensemble des gènes liés par la RNAP III dans les cellules souches embryonnaires de souris. Cet ensemble est bien inférieur au nombre de gènes prédits dans le génome. Nous avons également observé la RNAP III et ses facteurs de transcription liés à 30 régions non annotées, seule une d'entre elles est conservée chez l'humain. Un très faible nombre de SINEs sur un demi-million prédits est associé à la RNAP III. Notre étude révèle de nombreux sites liés uniquement par TFIIIC, nommés " extra-TFIIIC loci ", ETC chez la levure. Ces sites sont associés à la protéine CTCF, et à la cohésine. La cohésine occupe les sites liés par CTCF, et contribue à la formation de boucles ADN, associées à la répression ou à l'activation de l'expression des gènes. Ces données suggèrent que TFIIIC peut jouer un rôle dans l'organisation de l'architecture chromosomique chez les souris. Nous avons également démontré que TCEA1, l'isoforme ubiquitaire de TFIIS, le facteur d'élongation de la RNAP II, est associée aux gènes actifs de classe III. Ceci suggére que TFIIS est un facteur de transcription de classe III. Finalement, la distribution de TFIIS aux gènes de classe II indique que le recrutement de TFIIS n'est pas suffisant pour contrôler la transition de la RNAP II pausée en 5' des gènes en élongation.

The recent advent of High-Throughput Sequencing (HTS) methods has triggered a revolution in gene regulation studies. Demand has never been higher to process the immense amount of emerging data to gain insight into the regulatory mechanisms of the cell. We address this issue by describing methods to analyze, integrate and interpret HTS data from different sources. In particular, we developed and benchmarked Pyicos, a powerful toolkit that offers flexibility, versatility and efficient memory usage. We applied it to data from ChIP-Seq on progesterone receptor in breast cancer cells to gain insight into regulatory mechanisms of hormones. Moreover, we embedded Pyicos into a pipeline to integrate HTS data from different sources. In order to do so, we used data sets from ENCODE to systematically calculate signal changes between two cell lines. We thus created a model that accurately predicts the regulatory outcome of gene expression, based on epigenetic changes in a gene locus. Finally, we provide the processed data in a Biomart database to the scientific community. / La llegada reciente de nuevos métodos de High-Throughput Sequencing (HTS) ha provocado una revolución en el estudio de la regulación génica. La necesidad de procesar la inmensa cantidad de datos generados, con el objectivo de estudiar los mecanismos regulatorios en la celula, nunca ha sido mayor. En esta tesis abordamos este tema presentando métodos para analizar, integrar e interpretar datos HTS de diferentes fuentes. En particular, hemos desarollado Pyicos, un potente conjunto de herramientas que ofrece flexibilidad, versatilidad y un uso eficiente de la memoria. Lo hemos aplicado a datos de ChIP-Seq del receptor de progesterona en células de cáncer de mama con el fin de investigar los mecanismos de la regulación por hormonas. Además, hemos incorporado Pyicos en una pipeline para integrar los datos HTS de diferentes fuentes. Hemos usado los conjuntos de datos de ENCODE para calcular de forma sistemática los cambios de señal entre dos líneas celulares. De esta manera hemos logrado crear un modelo que predice con bastante precisión los cambios de la expresión génica, basándose en los cambios epigenéticos en el locus de un gen. Por último, hemos puesto los datos procesados a disposición de la comunidad científica en una base de datos Biomart.

Over the last decade there has been a great effort to annotate noncoding regions of the genome, particularly those that regulate gene expression. These regulatory elements contain binding sites for transcription factors (TF), which interact with one another and transcriptional machinery to initiate, enhance, or repress gene expression. The Encyclopedia of DNA Elements (ENCODE) consortium has generated thousands of epigenomic datasets, such as DNase-seq and ChIP-seq experiments, with the goal of defining such regions. By integrating these assays, we developed the Registry of candidate Regulatory Elements (cREs), a collection of putative regulatory regions across human and mouse. In total, we identified over 1.3M human and 400k mouse cREs each annotated with cell-type specific signatures (e.g. promoter-like, enhancer-like) in over 400 human and 100 mouse biosamples. We then demonstrated the biological utility of these regions by analyzing cell type enrichments for genetic variants reported by genome wide association studies (GWAS). To search and visualize these cREs, we developed the online database SCREEN (search candidate regulatory elements by ENCODE). After defining cREs, we next sought to determine their potential gene targets. To compare target gene prediction methods, we developed a comprehensive benchmark of enhancer-gene links by curating ChIA-PET, Hi-C and eQTL datasets. We then used this benchmark to evaluate unsupervised linking approaches such as the correlation of epigenomic signal. We determined that these methods have low overall performance and do not outperform simply selecting the closest gene. We then developed a supervised Random Forest model which had notably better performance than unsupervised methods. We demonstrated that this model can be applied across cell types and can be used to predict target genes for GWAS associated variants. Finally, we used the registry of cREs to annotate variants associated with psychiatric disorders. We found that these "psych SNPs" are enriched in cREs active in brain tissue and likely target genes involved in neural development pathways. We also demonstrated that psych SNPs overlap binding sites for TFs involved in neural and immune pathways. Finally, by identifying psych SNPs with allele imbalance in chromatin accessibility, we highlighted specific cases of psych SNPs altering TF binding motifs resulting in the disruption of TF binding. Overall, we demonstrated our collection of putative regulatory regions, the Registry of cREs, can be used to understand the potential biological function of noncoding variation and develop hypotheses for future testing.

No description available.

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed.

No description available.

No description available.