Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. In particular, they have been implicated in the generation of in vitro cytokine-driven T cell polarization and therefore may determine the vigor, quality and/or longevity of such responses in vivo. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, such as 2,4-dinitrochlorobenzene (DNCB) that result in type 1/type 17 responses in mice, and chemicals that cause sensitization of the respiratory tract and occupational asthma, for example trimellitic anhydride (TMA) that induces preferential type 2 responses in mice. To explore the regulation and maintenance of these divergent responses generated by polarised T cell populations in vivo, BALB/c strain mice were exposed topically DNCB and TMA. DNA from draining lymph nodes (LN) was processed for methylated DNA (5mC) immunoprecipitation (MeDIP) followed by hybridization to a whole-genome DNA promoter array. A higher number of DNCB-associated differently methylated regions (DMR) were identified and there was significant crossover between allergen treatments. Promoter-associated DMR, unique to either DNCB or TMA, were generally hypomethylated. Pathway analyses highlighted a number of immune related pathways, including chemokine and cytokine signalling. A number of these DMR were hypothesised to be candidate biomarkers of chemical allergy. To confirm this, novel analysis of hydroxymethylated (5hmC) DNA in the in vivo allergen-activated LN was compared to analysis of 5mC to identify LN specific DMR. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses and the Nwc DMR was characteristic of TMA treatment, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but also provide a possible basis for differentiation between classes of chemical allergens. Finally, a combined population of effector/effector memory T cells (TEff/TEM) was isolated from the CD4+ and CD8+ populations of allergen-activated draining lymph nodes (LN). Levels of 5mC and 5hmC at T cell lineage cytokine prompters was determined and analysed by comparison with concurrently sorted naïve T cells. In CD8+ TEff/TEM from DNCB-stimulated LN, increased expression of Ifng and Gzmb correlated with a reduction 5mC at their respective promoters. There were also reduced levels of 5mC at an Ifng enhancer. In contrast, TMA-simulated CD4+ TEff/TEM were characterised by high levels of Il4 expression which were associated with a decrease in promoter 5mC and an increase in 5hmC, as well as increased 5hmC at an Il4 enhancer region. These data demonstrate that exposure to chemical allergens results in characteristic DNA methylation patterns indicative of epigenetic regulation of divergent T cell populations in vivo. Furthermore, it highlights a particularly important role for DNA hydroxymethylation at the Th2 locus. In conclusion, exposure to chemical allergens results in divergent patterns of 5mC and 5hmC. These provide possible biomarkers for the different classes of chemical allergens and represent an insight into the importance of 5mC and 5hmC in the control of polarised T cell responses in vivo.

Les dérivés de la cytosine sont d’importantes modifications épigénétiques dont le rôle dans l’évolution de la leucémie lymphoïde chronique (LLC) n’est pas totalement exploré. Dans ce contexte, notre première étude vise à examiner le niveau global de la 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) et 5-hydroxymethyluridine (5-hmU) dans des lymphocytes B purifiés de patients LLC (n=56) et d’individus sains (n=17). Les principaux acteurs de la régulation épigénétique (DNMT1/3A/3B, MBD2/4, TET1/2/3, SAT1) ont été évalués par PCR quantitative en temps réel. L’analyse a permis de mettre en exergue trois groupes de patients. En premier lieu, un groupe de patients stables (délai médian de progression [PFS] et délai au premier traitement [TFT] >120 mois), avec un profil épigénétique similaire au groupe contrôle. Deuxièmement, un groupe intermédiaire (PFS=84; TFT=120 mois) qui présente une augmentation de la déméthylation de l’ADN expliquée par l'induction SAT1 / TET2 pendant la progression de la maladie. Troisièmement, un groupe de patients avec une forme active de la maladie (PFS=52; TFT=112 mois) qui présentent une hyperlymphocytose, une réduction du temps de doublement des lymphocytes et des modifications épigénétiques majeures. Au sein de ce groupe, une réduction est observée pour la 5-mCyt, 5-hmCyt, 5-CaCyt et serait associée à une diminution des DNMTs, TETs et MBDs au cours de la progression de la maladie. Les profils épigénétiques mis en évidence sont indépendants du statut mutationnel IGHV mais sont associés avec les anomalies cytogénétiques. Nous nous sommes également intéressés à cette association et nous avons montré dans la deuxième étude que les modifications des dérivées de la cytosine peuvent affiner le pouvoir pronostic des anomalies cytogénétiques. En conclusion, nos résultats suggèrent que les variations de la méthylation ainsi que des intermédiaires de la déméthylation de l’ADN sont impliqués dans la progression de la LLC. / Cytosine derivatives are important epigenetic modifications whose role in the pathogenesis and evolution of chronic lymphocytic leukemia (CLL) is not fully explored. In this context, our first study aims to examine the global DNA level of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluridine (5-hmU) in purified B lymphocytes of CLL patients (n = 56) and healthy individuals (n = 17). The main actors in epigenetic regulation (DNMT1 / 3A / 3B, MBD2 / 4, TET1 / 2/3, SAT1) were evaluated by quantitative real time PCR. The analysis highlighted three groups of patients. First, a group of patients with stable disease (median time to progression [PFS] and time to first treatment [TFT]> 120 months), with an epigenetic profile similar to the control group. Secondly, an intermediate group (PFS = 84, TFT = 120 months) which shows an increase in DNA demethylation explained by SAT1 / TET2 induction during disease progression. Third, a group of patients with an active form of the disease (PFS = 52, TFT = 112 months) who have hyperlymphocytosis, a short lymphocyte doubling time, and major epigenetic changes. Within this group, a reduction is observed for 5-mCyt, 5-hmCyt, 5-CaCyt which is associated with a decrease in DNMTs, TETs and MBDs during disease progression. The identified epigenetic profiles are independent of IGHV mutational status but are associated with cytogenetic abnormalities. We were also interested in this association and we showed in the second study that modifications of cytosine derivatives levels can refine the prognostic power of cytogenetic abnormalities.In conclusion, our results suggest that methylation variations as well as DNA demethylation intermediates are involved in the progression of CLL.

Hematopoiesis is a highly orchestrated process, in which a single hematopoietic stem cell (HSC) gives a rise to all blood cellular components. For myeloid and lymphoid development precise controlled expression of the PU.1 transcription factor is needed. Deletion of PU.1 gene in mouse is lethal and its dysregulation during hematopoietic differentiation is associated with blood malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). MDS and AML are serious blood disorders characterized by expansion of immature blood cells and lack of differentiated functional cells. Not only genetic but also epigenetic aberrations represent a very important field for studying pathophysiology of leukemia genesis and dysregulation of the PU.1 gene represents intensively studied candidate mechanism. Modern therapy of selected MDS and subset of AML patients is based on treatment with DNA hypomethylating agent Azacytidine (AZA) interfering in PU.1 gene regulatory mechanism. However, poor response or resistance to this therapy often occurs. In this thesis we present data obtained from AZA-resistant clones of MDS/AML cell line OCI-M2. We analysed DNA methylation and DNA hydroxymethylation at the key regulatory element of the PU.1 gene (URE). We found that these epigenetic modifications at URE...