Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development

Investigation of TET epigenetic enzyme role and mode of action in Drosophila

Chez les mammifères, la famille des enzymes TET a fait l’objet de nombreuses études qui ont mis en évidence leur rôle dans la régulation de l’expression des gènes notamment grâce à leur capacité d’oxydation des cytosines méthylées (5mC), initiant ainsi un processus actif de déméthylation de l’ADN, mais aussi via des fonctions indépendantes de leurs activités catalytiques qui sont moins bien décrites. Le génome de la Drosophile ne contenant pas ou peu de 5mC mais possédant un gène Tet, fait de cet organisme un modèle de choix pour étudier les fonctions non canoniques de ces enzymes. Par ailleurs, de récentes études, ont montré la présence de 6-méthylAdénine (6mA) chez différents métazoaires dont la souris, l’homme et la drosophile. Il a été montré que chez cette dernière, les 6mA seraient déméthylées par TET ce qui participerait au contrôle de l’expression du génome. Ainsi, au cours de ma thèse, j’ai principalement étudié les fonctions et les mécanismes moléculaires de TET chez la drosophile. Dans un premier temps, nous avons caractérisé le profil d’expression de Tet et de ces isoformes à différents stades développementaux et dans plusieurs tissus. Grâce à des analyses de transcriptome entre une lignée sauvage et une lignée mutante pour Tet, nous avons mis en évidence que Tet agit essentiellement comme un régulateur tissu-spécifique de l’expression des gènes et qu’il est requis pour le développement du cerveau, des disques d’ailes et de la glande lymphatique. L’étude de cet organe hématopoïétique larvaire montre que Tet est impliqué dans la régulation de la balance entre la maintenance et la différenciation des progéniteurs sanguins, mais aussi dans le contrôle du nombre de cellules du PSC (Posterior Signalling Center), une niche hématopoïétique. La combinaison d’approches de séquençage de troisième génération et de spectrométrie de masse nous a permis de montrer la présence d’un faible niveau de 6mA dans les cerveaux larvaires de drosophile, notamment au sein de motifs GAG, une caractéristique qui semble conservée chez les métazoaires. Cependant, contrairement à ce que l’on pouvait attendre, nos résultats indiquent que TET n’est pas impliquée dans la déméthylation des 6mA dans le cerveau larvaire. Par ailleurs, dans cet organe, la présence de 6mA sur les gènes n’est pas corrélée à leur niveau d’expression. En parallèle, j’ai participé à la caractérisation des cellules sanguines de la drosophile adulte, ce qui nous a notablement permis de mettre en évidence qu’une petite population de cellules originaires du PSC larvaire maintient un faible potentiel hématopoïétique chez l’imago, une partie de ces cellules étant capable de proliférer et de se différencier en réponse à une infection bactérienne.Enzymes of the TET family have been extensively studied in mammals and have been shown to play a role in regulation of gene expression through their ability to oxidize methylated cytosines (5mC) and thus initiate an active DNA demethylation process, but also through catalytic-independent functions that have been less described. The Drosophila genome contains little or no 5mC but encodes a Tet gene, which makes this organism a model of choice to study the non-canonical functions of this family. In addition, recent studies have reported the presence of 6-methyladenine (6mA) in different metazoans including mouse, human and drosophila in which this modification seems to be removed by TET and to influence gene expression. Thus, during my thesis, I mainly studied the functions and molecular mechanisms of action of TET in drosophila. First, we characterized the expression profile of Tet and its isoforms in different developmental stages and tissues. Our experiments highlighted the function of Tet as a tissue-specific regulator of gene expression and its involvement in the development of the brain, wing discs and lymph glands. The study of this larval hematopoietic organ showed that Tet is involved in the regulation of the balance between maintenance and differentiation of blood progenitors, but also in the control of cell number in the PSC, an hematopoietic niche. Using a combination of third generation sequencing and mass spectrometry, we also have shown that drosophila larval brain DNA contains low levels of 6mA, especially within the GAG motif, a characteristic that seems conserved in metazoans. However, in contrast with previous publications, our data show that TET is not involved in the demethylation of 6mA in the larval brain. Moreover, in this organ, the presence of 6mA on the genes does not correlate with their expression level. In parallel, I participated in the characterization of the blood cells of the adult drosophila. We notably discovered that a small population of cells originating from the larval PSC maintains a limited hematopoietic potential in the imago, as some of these cells are able to proliferate and to differentiate in response to bacterial infection

From Drosophila Blood Cells to Human Leukemia

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Prmt5 promotes vascular morphogenesis independently of its methyltransferase activity.

During development, the vertebrate vasculature undergoes major growth and remodeling. While the transcriptional cascade underlying blood vessel formation starts to be better characterized, little is known concerning the role and mode of action of epigenetic enzymes during this process. Here, we explored the role of the Protein Arginine Methyl Transferase Prmt5 in blood vessel formation as well as hematopoiesis using zebrafish as a model system. Through the combination of different prmt5 loss-of-function approaches we highlighted a key role of Prmt5 in both processes. Notably, we showed that Prmt5 promotes vascular morphogenesis through the transcriptional control of ETS transcription factors and adhesion proteins in endothelial cells. Interestingly, using a catalytic dead mutant of Prmt5 and a specific drug inhibitor, we found that while Prmt5 methyltransferase activity was required for blood cell formation, it was dispensable for vessel formation. Analyses of chromatin architecture impact on reporter genes expression and chromatin immunoprecipitation experiments led us to propose that Prmt5 regulates transcription by acting as a scaffold protein that facilitates chromatin looping to promote vascular morphogenesis

Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential

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Towards Excellence in Asthma Management: Final Report of an Eight-Year Program Aimed at Reducing Care Gaps in Asthma Management in Quebec

BACKGROUND AND OBJECTIVES: Asthma care in Canada and around the world persistently falls short of optimal treatment. To optimize care, a systematic approach to identifying such shortfalls or ‘care gaps’, in which all stakeholders of the health care system (including patients) are involved, was proposed

CBP and P300 regulate distinct gene networks required for human primary myoblast differentiation and muscle integrity

International audienceThe acetyltransferases CBP and P300 have been implicated in myogenesis in mouse immortalized cell lines but these studies focused only on the expression of a handful of myogenic factors. Hence, the respective role of these two related cofactors and their impact at global scale on gene expression rewiring during primary myoblast differentiation remain unknown. Here, we characterised the gene networks regulated by these two epigenetic enzymes during human primary myoblast differentiation (HPM). We found that CBP and p300 play a critical role in the activation of the myogenic program and mostly regulate distinct gene sets to control several aspects of HPM biology, even though they also exhibit some degree of redundancy. Moreover, CBP or P300 knockdown strongly impaired muscle cell adhesion and resulted in the activation of inflammation markers, two hallmarks of dystrophic disease. This was further validated in zebrafish where inhibition of CBP and P300 enzymatic activities led to cell adhesion defects and muscle fiber detachment. Our data highlight an unforeseen link between CBP/ P300 activity and the emergence of dystrophic phenotypes. They thereby identify CBP and P300 as mediators of adult muscle integrity and suggest a new lead for intervention in muscular dystrophy

Adenine methylation is very scarce in the drosophila genome and not erased by the Ten Eleven Translocation dioxygenase

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in drosophila, for which the erasure of this epigenetic mark has been ascribed to the Ten Eleven Translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on drosophila genome. Using axenic or conventional breeding conditions, we found only traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET. Further molecular and genetic analyses suggest that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans