TET2 et P53, deux suppresseurs de tumeurs, jouent un rôle important dans l’homéostasie des cellules souches hématopoïétiques et sont trouvés mutés dans les hémopathies malignes. Ils sont aussi impliqués dans le contrôle du cycle cellulaire et les mécanismes de réparation des dommages de l’ADN, notamment la voie de réparation par excision de base (BER). Dans la première partie de ce travail, nous avons montré que la surexpression de TET2 et l’augmentation consécutive des 5hmC, ralentit la progression du cycle cellulaire et la transition G1/S et induit une instabilité centrosomique associée à une instabilité chromosomique dans un modèle cellulaire Ba/F3. De plus, la surexpression de TET2 induit l’augmentation de la mutagenèse particulièrement des transitions C->T dans les sites CpG dans un contexte déficient en thymidine DNA glycosylase (TDG), une protéine initiatrice du BER. Dans la seconde partie de ce travail, nous avons montré que l’activation de P53, par des antagonistes de MDM2, a un effet délétère sur tous les progéniteurs hématopoïétiques. Ces antagonistes induisent aussi une cytotoxicité non seulement dans les stades précoces de la mégacaryopoïèse mais surtout dans les stades tardifs. Cette cytotoxicité n’est pas réversible, contrairement à ce qui est observé en clinique, et ne peut pas être restaurée par des doses croissantes de thrombopoïétine. Au total, TET2 et P53 doivent être strictement régulés pour assurer l’homéostasie et la stabilité génétique des cellules hématopoïétiques. / Two tumor suppresors, TET2 and P53, play an important role in the homeostasis of hematopoietic stem cells and have been found mutated in hematological malignancies. They are also involved in cell cycle control and DNA repair mechanisms, including the base excision repair pathway (BER). In the first part of this work, we showed that TET2 overexpression and the consequent increase of 5hmC, inhibit cell cycle progression particularly G1/S transition and induces centrosome instability associated with chromosomal instability in Ba/F3 cellular model. In addition, overexpression of TET2 induces increased mutagenesis particularly transitions C->T at CpG sites in a context deficient in thymidine DNA glycosylase (TDG), a protein initiating BER. In the second part of this work, we have shown that p53 activation by MDM2 antagonists has deleterious effect on all haematopoietic progenitors. These antagonists also induce cytotoxicity not only in the early stages of megakaryopoiesis but also mainly in the late stages. This cytotoxicity is not reversible, in contrast to what is observed in clinic, and can not be restored by increasing doses thrombopoietin. To conclude, TET2 and P53 must be strictly controlled to ensure homeostasis and genetic stability of the hematopoietic cells.

Mammalian DNA methylathion is a chemical reaction catalyzed by DNA methyltransferases (DNMTs) and involves the addition of a methyl group from the methyl donor SAM to the carbon 5 position of cytosine (C) in a CpG dinucleotide. Specifically, DNA methylation is essential for normal development and is involved in numerous key mechanisms such as genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and may be involved in the regulation of single-copy gene expression. In the human genome the majority of CpGs are methylated whereas regions with high density of CpG sites, termed CpG islands and often co-localized within gene promoters, are typically free of this mark. Recently, a new modified cytosine, 5-hydroxymhetylcytosine (5-hmC), was identified and found at significant levels in mouse brain and both mouse and human embryonic stem (ES) cells. The conversion of 5-mC to 5-hmC is catalyzed by the ten-eleven translocation (TET) proteins of the 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase superfamily. Many studies were conducted since the identification of 5-hmC and significant levels of 5-mC hydroxylation were found in many other mouse and human tissues. Importantly, many of the techniques used for 5-mC detection, such as bisulphite sequencing and methyl-sensitive restriction digestion, are incapable of distinguishing between 5mC and 5hmC implying the necessity not only to develop techniques specific for 5-hmC characterization but also reevaluation of previously published 5mC data. The biological function of 5-hmC is unknown however many recent studies have suggested a role for 5-hmC as an intermediate of either passive or active demethylation. The majority of studies of 5- hmC and TETs have used mouse ES cells as model system. Therefore, very little is known about 5-hmC patterns and TET expression within and between normal tissues. During my PhD, I used the recently developed 5-hmC-specific antibody for tiling microarrays and 5hmC-qPCR to examine both global 5hmC content and locus-specific patterns of 5hmC in several normal human tissues and breast cancer. I found that global 5-hmC content is highly variable between tissues compared to global 5-mC content. Moreover, TETs genes are highly expressed in most of tissues tested. Importantly, both global 5-hmC content and TETs genes are rapidly and significantly reduced as consequence of adaptation of cells from normal human tissue to cell culture. Using the 5hmC-specific antibody for tiling microarrays and 5-hmC-qPCR to profile locus-specific patterns of 5hmC, I found that 5-hmC patterns are tissue-specific in human samples. In addition, comparing array data to RNA-seq data, 5- hmC was found to co-localize at gene bodies of active genes. Moreover, despite the global 5-hmC reduction in cell lines, 5-hmC content remains enriched in some specific loci. In summary, my results show that tissue type is a major modifier of both global and locus-specific 5hmC at genes in normal human tissues. Furthermore, I also show that both TET gene expression and 5hmC content are significantly reduced and 5-hmC profiles reprogrammed during the passage from tissues to cell culture.

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