Rôle des modifications post-traductionnelles de microphthalmia, un facteur de transcription nécessaire au développement des cellules pigmentées

PARIS-BIUSJ-Thèses (751052125) / SudocSudocFranceF

Implications des modifications post-transcriptionnelles dans la régulation de l activité de MITF in vivo (un facteur de transcription essentiel pour la lignée mélanocytaire)

Le facteur de transcription Microphthalmia (Mitf) et la voie de signalisation des Mitotic Activated Protein Kinase (MAPK) sont des éléments déterminants pour la différentiation, la prolifération et la survie des melanocytes. L altération des fonctions de l un ou l autre se manifeste par une perte totale ou partielle de ce type cellulaire. A l inverse, le mélanome est associé très majoritairement à une activation constitutive des MAPK, et parfois, à un gain d activité de MITF. Afin d étudier les interaction entre les MAPK et l activité de MITF, nous nous sommes intéressés aux modifications post transcriptionnelles qui permettent la génération d isoformes multiples dotées d activité différentes. MITF contient un site de phosphorylation, la serine 73 (S73), qui a été démontré par le passé comme jouant un rôle à la fois dans l augmentation de l activité et dans la réduction de la stabilité de MITF in vitro. Pour comprendre le rôle de cette serine in vivo, une tentative de mutation S73A de Mitf a été réalisée. Ce codon fait parti d un Exon splicing Enhencer et sa mutation réduit l affinité de fixation de la protéine SRp40 et l exclusion de l exon 2B dans lequel est situé ce site de phosphorylation. Pour dissocier l exclusion de l exon 2B et de sa phosphorylation, nous avons donc altéré la jonction de l exon 2A-2B afin qu elle ne soit plus reconnue par le spliceosome. En conséquence, l exon 2B ne peut plus être exclu des transcrits Mitf quelque soit le statut du codon 73. La comparaison de 3 nouveaux allèles Mitf S-S73A S-S73D et S-S73S, où l inclusion de l exon 2B est forcée, nous a permis d associer l absence de phosphorylation à un gain d activité de MITF.The Microphthalmia associated transcription factor MITF and the Mitotic Activated Protein Kinase signaling pathway (MAPK) are crucial elements for the differentiation, proliferation and survival of melanocytes. The alteration of one of these two elements results in the impairment of this cell type. In contrast, in most cases, melanoma is associated with a constitutive activation of the MAPK pathway and often with a gain-of-function of MITF. In order to study interactions between the MAPK pathway and MITF activity, we focused our insterest on post-translational modifications that give rise to multiple isoforms of different activities. In fact, MITF contains a phosphorylation site, Serine-73 (S73), which has been implicated both in the increase of activity and the degradation of MITF protein in vitro. To assess the role of this S73 in vivo, our group previously generated targeted mice harboring a S73A mutation in the Mitf gene. However, the S73 codon is part of an Exon Splicing Enhancer sequence, whose mutation leads to a decreased binding affinity of the splice regulatory protein SRp40 and exclusion of exon 2B coding for that serine. In order to dissociate potential effects of residue-73 mutation from the exclusion of exon 2B, we retargeted Mitf in a way that renders the 5 splice site of exon 2B non-functional and force the incorportation of exon 2B. The phenotypic analysis of 3 new alleles Mitf S-S73A, SS73D and SS73-S demonstrates a gain-of-function of the non-phosphorylatable MITF 2B+ isoform.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

The Discovery of the Antiviral Resistance Gene Mx: A Story of Great Ideas, Great Failures, and Some Success

The discovery of the Mx gene-dependent, innate resistance of mice against influenza virus was a matter of pure chance. Although the subsequent analysis of this antiviral resistance was guided by straightforward logic, it nevertheless led us into many blind alleys and was full of surprising turns and twists. Unexpectedly, this research resulted in the identification of one of the first interferon-stimulated genes and provided a new view of interferon action. It also showed that in many species, MX proteins have activities against a broad range of viruses. To this day, Mx research continues to flourish and to provide insights into the never-ending battle between viruses and their hosts

Endothelin signalling in the development of neural crest-derived melanocytes

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest- derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct+ even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early Dct+ cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+ melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6- dimethylpiperidinocarbonyl-L-α-methylleucyl-D-1-methoxycarbonyltryptophanyl- D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+ melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.SCOPUS: ar.jinfo:eu-repo/semantics/publishe