<p>Transcriptional regulation has been studied intensively in recent decades. One important aspect of this regulation is the interaction between regulatory proteins, such as transcription factors (TF) and nucleosomes, and the genome. Different high-throughput techniques have been invented to map these interactions genome-wide, including ChIP-based methods (ChIP-chip, ChIP-seq, etc.), nuclease digestion methods (DNase-seq, MNase-seq, etc.), and others. However, a single experimental technique often only provides partial and noisy information about the whole picture of protein-DNA interactions. Therefore, the overarching goal of this dissertation is to provide computational developments for jointly modeling different experimental datasets to achieve a holistic inference on the protein-DNA interaction landscape. </p><p>We first present a computational framework that can incorporate the protein binding information in MNase-seq data into a thermodynamic model of protein-DNA interaction. We use a correlation-based objective function to model the MNase-seq data and a Markov chain Monte Carlo method to maximize the function. Our results show that the inferred protein-DNA interaction landscape is concordant with the MNase-seq data and provides a mechanistic explanation for the experimentally collected MNase-seq fragments. Our framework is flexible and can easily incorporate other data sources. To demonstrate this flexibility, we use prior distributions to integrate experimentally measured protein concentrations. </p><p>We also study the ability of DNase-seq data to position nucleosomes. Traditionally, DNase-seq has only been widely used to identify DNase hypersensitive sites, which tend to be open chromatin regulatory regions devoid of nucleosomes. We reveal for the first time that DNase-seq datasets also contain substantial information about nucleosome translational positioning, and that existing DNase-seq data can be used to infer nucleosome positions with high accuracy. We develop a Bayes-factor-based nucleosome scoring method to position nucleosomes using DNase-seq data. Our approach utilizes several effective strategies to extract nucleosome positioning signals from the noisy DNase-seq data, including jointly modeling data points across the nucleosome body and explicitly modeling the quadratic and oscillatory DNase I digestion pattern on nucleosomes. We show that our DNase-seq-based nucleosome map is highly consistent with previous high-resolution maps. We also show that the oscillatory DNase I digestion pattern is useful in revealing the nucleosome rotational context around TF binding sites. </p><p>Finally, we present a state-space model (SSM) for jointly modeling different kinds of genomic data to provide an accurate view of the protein-DNA interaction landscape. We also provide an efficient expectation-maximization algorithm to learn model parameters from data. We first show in simulation studies that the SSM can effectively recover underlying true protein binding configurations. We then apply the SSM to model real genomic data (both DNase-seq and MNase-seq data). Through incrementally increasing the types of genomic data in the SSM, we show that different data types can contribute complementary information for the inference of protein binding landscape and that the most accurate inference comes from modeling all available datasets. </p><p>This dissertation provides a foundation for future research by taking a step toward the genome-wide inference of protein-DNA interaction landscape through data integration.</p> / Dissertation

Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources.

The main topic of my PhD studies was to investigate the role of sumoylation in the function of Ikaros transcription factor, that regulates the lymphocyte differentiation and function. Sumoylation is a posttranslational modification that can change the properties and regulate the function of a given protein. Up to now, one study addressed the question of how sumoylationmodulates Ikaros function. It shows that Ikaros is sumoylated in total primary thymocytes, and that this dynamic event modulates Ikaros' repressive function. It also describes two consensus sumoylation sites on Ikaros (K58 and K240), the sumoylation of which leads to loss of Ikaros repressive function in ectopic reporter gene assays. The final conclusion of the study is that sumoylation does not alter the nuclear localization of Ikaros but acts as a mechanism disrupting its participation in both histone deacetylase (HDAC) dependent and independent repression. My work shows the presence of additional sumoylation site on Ikaros and demonstrates that sumoylation does not significantly alter its interaction with the nucleosome remodelling and histone deacetylase (NURD) complex in T-cell lines. The functional analysis of sumo-deficientmutants indicates a complex role of this modification in regulating Ikaros' transcriptional properties. The identification of this new sumoylation site contributes to a better understanding of Ikaros' dual repressive - activating function and suggests the existence of conditional Ikaros' interacting partners. Moreover, the different Ikaros splicing isoforms would have differentsumoylation profiles, which would complete the knowledge of their functional diversity.

Le contrôle du cycle cellulaire, processus fondamental pour la prolifération cellulaire, est souvent altéré au cours de la tumorigenèse. Les facteurs de transcription E2Fs sont des régulateurs majeurs de l'expression de gènes impliqués dans le cycle cellulaire, la réplication de l'ADN, la mort cellulaire programmée ou encore la différenciation cellulaire. La famille des facteurs E2Fs contient des membres qui agissent comme activateurs ou répresseurs de la transcription et dont l'activité est régulée par un grand nombre de corégulateurs transcriptionnels, incluant notamment les protéines à poche pRb, p107, p130. RIP140 (Receptor Interacting Protein of 140kDa) a été identifié comme un corépresseur de nombreux récepteurs nucléaires, une autre grande famille de facteurs de transcription qui, pour certains, régulent positivement l'expression du gène RIP140.Ce travail de thèse a permis d'identifier RIP140 comme un nouveau répresseur de l'activité transcriptionnelle du facteur E2F1, dans des expériences de transfection transitoire ainsi que sur l'expression de gènes endogènes. Nous avons également montré que l'expression ectopique de RIP140 bloque la progression des cellules dans le cycle cellulaire. Dans les cancers du sein, le niveau d'expression de RIP140 présente une corrélation inverse avec celui de différents gènes cibles des facteurs E2Fs et semble discriminer les tumeurs luminales des tumeurs basales. Nous avons également démontré que le niveau d'ARNm RIP140 est régulé au cours du cycle cellulaire et que le promoteur du gène RIP140 est une cible directe des facteurs E2Fs. Cette régulation implique des sites de liaison des facteurs E2Fs et Sp1 de la région proximale du promoteur. La régulation de ce gène par E2F1 a également été observée au cours du processus de différenciation adipocytaire en utilisant un modèle murin E2F1-/-.En conclusion, ce travail a permis d'identifier RIP140 comme un nouvel acteur de la voie de signalisation par les facteurs E2Fs. / Cell cycle control, a fundamental process which controls cell proliferation, is frequently altered during tumorigenesis. The E2F transcription factors are central regulators of target gene expression involved in cell cycle regulation, DNA replication, apoptosis and differentiation. The E2F transcription factors family encompasses members which act as activators or repressors. Their activities are regulated by a large number of transcriptional coregulators, including in particular the pocket proteins pRb, p107, p130.The transcription coregulator RIP140 (Receptor Interacting Protein of 140kDa) has been identified as a partner of numerous nuclear receptors, another important transcription factor family. Some of these nuclear receptors positively regulate RIP140 gene expression.This work identified RIP140 as a new repressor of E2F1 transcriptional activity, both in transient transfection experiments and on the expression of endogenous target genes. We also showed that ectopic expression of RIP140 blocks cell cycle progression. In breast cancers, the level of RIP140 expression is inversely correlated with various target genes of E2Fs factors and seems to discriminate luminal from basal tumors. We also demonstrated that the RIP140 mRNA expression is regulated during cell cycle and that the RIP140 promoter is a direct target of E2F transcription factors. This regulation involves both E2F and Sp1 binding sites in the proximal region of the RIP140 promoter. The regulation of the RIP140 gene by E2F1 was also observed during adipocyte differentiation using an E2F1-/- mouse model.In conclusion, this study identified RIP140 as a new regulator of the E2F signalling pathway and as a novel E2F1 target gene. These results open new perspectives concerning the roles that this transcriptional coregulator might play in the control of cell proliferation and tumorigenesis.

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.

La famille des gènes Hox code pour des facteurs de transcription connus pour leur contribution essentielle à l’élaboration de l’architecture du corps et ce, au sein de tout le règne animal. Au cours de l’évolution chez les vertébrés, les gènes Hox ont été redéfinis pour générer toute une variété de nouveaux tissus/organes. Souvent, cette diversification s’est effectuée via des changements quant au contrôle transcriptionnel des gènes Hox. Chez les mammifères, la fonction de Hoxa13 n’est pas restreinte qu’à l’embryon même, mais s’avère également essentielle pour le développement de la vascularisation fœtale au sein du labyrinthe placentaire, suggérant ainsi que sa fonction au sein de cette structure aurait accompagné l’émergence des espèces placentaires. Au chapitre 2, nous mettons en lumière le recrutement de deux autres gènes Hoxa, soient Hoxa10 et Hoxa11, au compartiment extra-embryonnaire. Nous démontrons que l’expression de Hoxa10, Hoxa11 et Hoxa13 est requise au sein de l’allantoïde, précurseur du cordon ombilical et du système vasculaire fœtal au sein du labyrinthe placentaire. De façon intéressante, nous avons découvert que l’expression des gènes Hoxa10-13 dans l’allantoïde n’est pas restreinte qu’aux mammifères placentaires, mais est également présente chez un vertébré non-placentaire, indiquant que le recrutement des ces gènes dans l’allantoïde précède fort probablement l’émergence des espèces placentaires. Nous avons généré des réarrangements génétiques et utilisé des essais transgéniques pour étudier les mécanismes régulant l’expression des gènes Hoxa dans l’allantoïde. Nous avons identifié un fragment intergénique de 50 kb capable d’induire l’expression d’un gène rapporteur dans l’allantoïde. Cependant, nous avons trouvé que le mécanisme de régulation contrôlant l’expression du gène Hoxa au sein du compartiment extra-embryonnaire est fort complexe et repose sur plus qu’un seul élément cis-régulateur. Au chapitre 3, nous avons utilisé la cartographie génétique du destin cellulaire pour évaluer la contribution globale des cellules exprimant Hoxa13 aux différentes structures embryonnaires. Plus particulièrement, nous avons examiné plus en détail l’analyse de la cartographie du destin cellulaire de Hoxa13 dans les pattes antérieures en développement. Nous avons pu déterminer que, dans le squelette du membre, tous les éléments squelettiques de l’autopode (main), à l’exception de quelques cellules dans les éléments carpiens les plus proximaux, proviennent des cellules exprimant Hoxa13. En contraste, nous avons découvert que, au sein du compartiment musculaire, les cellules exprimant Hoxa13 et leurs descendantes (Hoxa13lin+) s’étendent à des domaines plus proximaux du membre, où ils contribuent à générer la plupart des masses musculaires de l’avant-bras et, en partie, du triceps. De façon intéressante, nous avons découvert que les cellules exprimant Hoxa13 et leurs descendantes ne sont pas distribuées uniformément parmi les différents muscles. Au sein d’une même masse musculaire, les fibres avec une contribution Hoxa13lin+ différente peuvent être identifiées et les fibres avec une contribution semblable sont souvent regroupées ensemble. Ce résultat évoque la possibilité que Hoxa13 soit impliqué dans la mise en place de caractéristiques spécifiques des groupes musculaires, ou la mise en place de connections nerf-muscle. Prises dans leur ensemble, les données ici présentées permettent de mieux comprendre le rôle de Hoxa13 au sein des compartiments embryonnaires et extra-embryonnaires. Par ailleurs, nos résultats seront d’une importance primordiale pour soutenir les futures études visant à expliquer les mécanismes transcriptionnels soutenant la régulation des gènes Hoxa dans les tissus extra-embryonnaires. / The Hox family of transcription factors is well known for its key contribution in the establishment of the body architecture in all the animal kingdom. During vertebrate evolution, Hox genes have been co-opted to pattern a variety of novel tissues/organs. Often, this diversification has been achieved by changes in Hox transcriptional control. In mammals, Hoxa13 function is not restricted to the embryo proper, but is also essential for the proper development of the fetal vasculature within the placental labyrinth, suggesting that its function in this structure accompanied the emergence of placental species. In chapter 2, we report on the recruitment of two other Hoxa genes, namely Hoxa10 and Hoxa11, in the extra embryonic compartment. We show that Hoxa10, Hoxa11 and Hoxa13 expression is required in the allantois, the precursor of the umbilical cord and fetal vasculature within the placental labyrinth. Interestingly, we found that Hoxa10-13 gene expression in the allantois is not restricted to placental mammals, but is also present in a non-placental vertebrate, indicating that the recruitment of these genes in the allantois most likely predates the emergence of placental species. We generated genetic rearrangements and used transgenic assays to investigate the regulatory mechanisms underlying Hoxa gene expression in the allantois. We identified a 50 kb intergenic fragment able to drive reporter gene expression in the allantois. However, we found that the regulatory mechanism controlling Hoxa gene expression in the extra-embryonic compartment is very complex and relies on more than one cis-regulatory element. In chapter 3, we used genetic fate mapping to assess the overall contribution of Hoxa13 expressing cells to the different embryonic structures. In particular, we focused on Hoxa13 fate-mapping analysis in the developing forelimbs. We could determine that, in the limb skeleton, all autopod (hand) skeletal elements, with the exception of a few cells in the most proximal carpal elements, originate from Hoxa13 expressing cells. In contrast, we found that, in the muscle compartment, Hoxa13 expressing cells and their descendants extend to more proximal limb domains, where they contribute to most of the muscle masses of the forearm and, in part, to the triceps. Interestingly we found that Hoxa13 expressing cells and their descendants are not identically distributed among different muscles. Within the same muscular mass, fibres with different Hoxa13lin+ contribution can be identified, and fibers with similar contribution are often clustered together. This result raises the possibility that Hoxa13 might be involved in establishing specific features of muscle groups, or in establishing nerve-muscle connectivity. Altogether, the data presented herein provide a better understanding of the role of Hoxa13 in both the embryonic and extra-embryonic compartment. Moreover, our results will be of key importance for further investigations aimed at unravelling transcriptional mechanisms underlying Hoxa gene regulation in extra embryonic tissues.

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

Network growing mechanisms are used to construct random networks that have structural behaviors similar to existing networks such as genetic networks, in efforts of understanding the evolution of complex topologies. Popular mechanisms, such as preferential attachment, are capable of preserving network features such as the degree distribution. However, little is known about such randomly grown structures regarding robustness to disturbances (e.g., edge deletions). Moreover, preferential attachment does not target optimizing the network's functionality, such as information flow. Here, we consider a network to be optimal if it's natural functionality is relatively high in addition to possessing some degree of robustness to disturbances. Specifically, a robust network would continue to (1) transmit information, (2) preserve it's connectivity and (3) preserve internal clusters post failures. In efforts to pinpoint features that would possibly replace or collaborate with the degree of a node as criteria for preferential attachment, we present a case study on both; undirected and directed networks. For undirected networks, we make a case study on wireless sensor networks in which we outline a strategy using Support Vector Regression. For Directed networks, we formulate an Integer Linear Program to gauge the exact transcriptional regulatory network optimal structures, from there on we can identify variations in structural features post optimization.

EIF4E, le facteur d’initiation de la traduction chez les eucaryotes est un oncogène puissant et qui se trouve induit dans plusieurs types de cancers, parmi lesquels les sous-types M4 et M5 de la leucémie aiguë myéloblastique (LAM). EIF4E est régulé à plusieurs niveaux cependant, la régulation transcriptionnelle de ce gène est peu connue. Mes résultats montrent que EIF4E est une cible transcriptionnelle directe du facteur nucléaire « kappa-light- chain- enhancer of activated B cells » (NF-κB).Dans les cellules hématopoïétiques primaires et les lignées cellulaires, les niveaux de EIF4E sont induits par des inducteurs de NF-κB. En effet, l’inactivation pharmaceutique ou génétique de NF-κB réprime l’activation de EIF4E. En effet, suite à l’activation de NF-κB chez l’humain, le promoteur endogène de EIF4E recrute p65 (RelA) et c-Rel aux sites évolutionnaires conservés κB in vitro et in vivo en même temps que p300 ainsi que la forme phosphorylée de Pol II. De plus, p65 est sélectivement associé au promoteur de EIF4E dans les sous-types LAM M4/M5 mais non pas dans les autres sous-types LAM ou dans les cellules hématopoïétiques primaires normales. Ceci indique que ce processus représente un facteur essentiel qui détermine l’expression différentielle de EIF4E dans la LAM. Les analyses de données d’expressions par séquençage de l’ARN provenant du « Cancer Genome Atlas » (TCGA) suggèrent que les niveaux d’ARNm de EIF4E et RELA se trouvent augmentés dans les cas LAM à pronostic intermédiaire ou faible mais non pas dans les groupes cytogénétiquement favorables. De plus, des niveaux élevés d’ARNm de EIF4E et RELA sont significativement associés avec un taux de survie relativement bas chez les patients. En effet, les sites uniques κB se trouvant dans le promoteur de EIF4E recrutent le régulateur de transcription NF-κB p65 dans 47 nouvelles cibles prévues. Finalement, 6 nouveaux facteurs de transcription potentiellement impliqués dans la régulation du gène EIF4E ont été prédits par des analyses de données ChIP-Seq provenant de l’encyclopédie des éléments d’ADN (ENCODE). Collectivement, ces résultats fournissent de nouveaux aperçus sur le control transcriptionnel de EIF4E et offrent une nouvelle base moléculaire pour sa dérégulation dans au moins un sous-groupe de spécimens de LAM. L’étude et la compréhension de ce niveau de régulation dans le contexte de spécimens de patients s’avère important pour le développement de nouvelles stratégies thérapeutiques ciblant l’expression du gène EIF4E moyennant des inhibiteurs de NF-κB en combinaison avec la ribavirine. / The eukaryotic translation initiation factor EIF4E is a powerful oncogene that is overexpressed in cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). EIF4E is regulated at multiple levels; however not much is known about the transcriptional regulation of this gene. My findings show that the nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) is a direct transcriptional regulator of EIF4E. EIF4E levels are induced in primary hematopoietic cells and in cell lines in response to NF-κB activating stimuli. Pharmacological and genetic inhibition of NF-κB suppresses EIF4E levels. NF-κB factors RelA (p65) and c-Rel are recruited to evolutionarily conserved κB sites in the EIF4E promoter in vitro and in vivo following NF-κB activation concurrent with the recruitment of p300 and phosphorylated Pol II. Furthermore, p65 is selectively associated with the EIF4E promoter in M4/M5 AML subtypes but not in other AML subtypes or normal primary hematopoietic cells and thus represents an underlying factor in determining the differential expression of EIF4E in AML. Analysis of gene expression RNA-Seq data from The Cancer Genome Atlas (TCGA) suggests that EIF4E and RELA mRNA levels are upregulated in intermediate and poor prognosis AML but not in the cytogenetically favorable group. Additionally, elevated EIF4E and RELA mRNA levels are significantly associated with worst patient survival outcome. Furthermore, 8 new putative NF-κB target genes that may be regulated with a pattern similar to EIF4E in poor prognosis AML were in silico predicted from Chip-Seq data. Finally, 6 new transcription factors that may be implicated in EIF4E gene regulation were predicted from the analysis of ChIP-Seq data from the encyclopedia of DNA elements (ENCODE). Collectively, these findings could offer novel insights into the transcriptional regulation of EIF4E and a novel molecular basis for its dysregulation in AML. Understanding this level of regulation within the context of patient specimens is important for the development of novel therapeutic strategies to target EIF4E gene expression with specific NF-κB inhibitors combined with ribavirin.

Genome-wide association studies in multifactorial diseases have contributed to our understanding of genetic risk and defined specific disease-associated loci in particular populations. However, risk cannot be fully explained by genetics and evidence points to both genetic and environmental factors being important in causation and pathophysiology. The role of vitamin D in calcium homeostasis is well established. Over the last 30 years it has become clear that vitamin D has a wider role in physiology and disease, notably in autoimmune, cancer and infectious conditions. However, the molecular mechanisms and possible causal role of these associations are poorly understood. Here I propose that the role of vitamin D in immune and inflammatory responses is significant, that genetic variation partly determines the response to vitamin D and that integrative analysis can yield important insights for disease mechanisms. For this I investigate the relationship between vitamin D and genetic risk involving the immune system by focusing on multiple sclerosis and sepsis, conditions classically defined as autoimmune and inflammatory respectively. I describe data resolving genetic variation associated with autoimmune diseases in vitamin D receptor binding sites; the association to multiple sclerosis of a genetic variant lying within a VDR binding site; the correlation of plasma vitamin D with genotype and cell specific transcriptomes in healthy volunteers; and the extent of vitamin D deficiency in severe sepsis and septic shock, its association with survival, correlation with gene expression and use in sub-classification to identify patients at higher risk of death. The limitations of each study and future work are discussed. Integrating epidemiological and clinical observations with genetic and functional genomics techniques has the potential to reveal interactions in population specific disease susceptibility that may lead to an improved understanding of disease mechanisms and clinical translation. The work I present here bridges molecular analysis, candidate and genome- wide, with phenotypic observations that are important in our understanding of disease.