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Caractérisation Moléculaire Du Rôle De FOXL2 Et De Ses Partenaires Dans L’Ovaire Sain Et Pathologique Laetitia Herman

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Caractérisation Moléculaire Du Rôle De FOXL2 Et De Ses Partenaires Dans L’Ovaire Sain Et Pathologique Laetitia Herman Caractérisation moléculaire du rôle de FOXL2 et de ses partenaires dans l’ovaire sain et pathologique Laetitia Herman To cite this version: Laetitia Herman. Caractérisation moléculaire du rôle de FOXL2 et de ses partenaires dans l’ovaire sain et pathologique. Médecine humaine et pathologie. Université de Paris, 2020. Français. NNT : 2020UNIP7123. tel-03246869 HAL Id: tel-03246869 https://tel.archives-ouvertes.fr/tel-03246869 Submitted on 2 Jun 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Laëtitia HERMAN-Doctorat de Génétique-2020 Université de Paris ED 562 BioSPC UMR7592 Institut Jacques Monod Caractérisation moléculaire du rôle de FOXL2 et de ses partenaires dans l’ovaire sain et pathologique Par Laëtitia HERMAN Thèse de doctorat de Génétique Dirigée par Pr. Reiner Veitia Et par Dr. Anne-Laure Todeschini Présentée et soutenue publiquement le 22 septembre 2020 Devant un jury composé de : Virginie Rouiller-Fabre, PU, CEA, Présidente Corinne Cotinot, DR, INRAE, Rapportrice Eric Pailhoux, DR, INRAE, Rapporteur Francis Poulat, CR, IGH, Examinateur Daniel Vaiman, DR, Institut Cochin, Membre invité 1 Laëtitia HERMAN-Doctorat de Génétique-2020 2 Laëtitia HERMAN-Doctorat de Génétique-2020 Titre : Caractérisation moléculaire du rôle de FOXL2 et de ses partenaires dans l’ovaire sain et pathologique Résumé : FOXL2 est un facteur de transcription majeur des cellules de la granulosa, participant à leur différenciation, leur maintien et leurs fonctions. Une mutation spécifique (p.C134W) est retrouvée dans la majorité des formes adultes des tumeurs des cellules de la granulosa. FOXL2 joue donc un rôle à la fois dans l’ovaire sain mais aussi dans l’ovaire pathologique. Pour comprendre son action moléculaire nous avons identifié par des analyses haut-débit ses cibles transcriptomiques directes et indirectes ainsi que ses sites de fixation sur le génome. Nous avons identifié 1001 gènes cibles et de nouveaux rôles putatifs pour FOXL2. De plus, AKT inhiberait, probablement de façon indirecte, l’activité de FOXL2 sur des gènes impliqués principalement dans la migration cellulaire. FOXL2 possède de nombreux partenaires parmi lesquels ESR2, SMAD3 et TRIM28, semblent jouer un rôle dans le développement et le maintien des cellules de la granulosa. Nous avons réalisé une analyse des cibles transcriptomiques de ces partenaires et de la fixation de FOXL2, en leurs présences et en leurs absences. Pour ESR2, particulièrement, nous avons identifié de nouvelles cibles impliquées dans le développement, la régulation de la migration cellulaire et l’organisation de la matrice extracellulaire. Nous avons montré que la présence d’ESR2 est nécessaire à une bonne fixation de FOXL2 sur la chromatine et qu’ensemble ils régulent l’expression de plus de 150 gènes. De plus, leur action conjointe permet l’expression de Phactr1 assurant une migration, une invasion et une adhésion cellulaire correctes. Dans un second projet, toujours en cours, nous souhaitons caractériser les cibles de FOXL2 p.C134W, au niveau transcriptomique. Des cellules, portant la mutation à l’état hémizygote ainsi que des lignées contrôles WT pour FOXL2, ont été obtenues par CRISPR/Cas9 et analysées par RNAseq. Les résultats obtenus sont prometteurs et permettront de comprendre le rôle de FOXL2 dans ces tumeurs. Mots clefs : FOXL2, forkhead, ESR2, Facteur de transcription, régulation transcriptionnelle, ChIP-seq, RNA-seq, ovaire, cellules de la granulosa, cancer des cellules de la granulosa, cancer de l’ovaire. 3 Laëtitia HERMAN-Doctorat de Génétique-2020 Title : Molecular characterization of the role of FOXL2 and its partners in the healthy and pathological ovary Abstract : FOXL2 is a major transcription factor in ovarian granulosa cells, involved in their differentiation, maintenance and function. A specific mutation (p.C134W) is found in the majority of adult forms of granulosa cell tumors. Therefore FOXL2 has been described to play a role both in the healthy or pathological ovary. In order to understand its molecular action, we have identified its direct and indirect transcriptomic targets as well as its binding sites on the genome using high-throughput approaches. We have identified more than 1000 target genes and new putative roles for FOXL2. In addition, AKT is proposed to inhibit, probably indirectly, the activity of FOXL2 on genes mainly involved in cell migration. FOXL2 has many partners including ESR2, SMAD3 and TRIM28, which are thought to co-regulated FOXL2 targets in granulosa cells. We performed an analysis of the targets of these partners and of the binding of FOXL2 in their presence or absence. For ESR2, in particular, we identified more than 350 targets involved in development, in the regulation of cell migration and the organization of the extracellular matrix. We have shown that the presence of ESR2 is required for FOXL2 binding to chromatin and that they co-regulate the expression of more than 150 genes. Moreover, their joint action controls the expression of Phactr1 modulating cell migration, invasion and adhesion. In a second project, still in progress, I wish to characterize the targets of FOXL2 p.C134W, at the transcriptomic level. Cells carrying the mutation at the hemizygous state as well as WT control lines for FOXL2 were obtained by CRISPR/Cas9 and analyzed by RNAseq. The results obtained are promising and will help understand the role of FOXL2 in these tumors. Keywords : FOXL2, forkhead factor, ESR2, transcription factor, transcriptional regulation, ChIP-seq, RNA-seq, ovary, granulosa cells, granulosa cell tumors, ovarian cancer. 4 Laëtitia HERMAN-Doctorat de Génétique-2020 « Ce qui est incompréhensible, c’est que le monde soit compréhensible. » Albert Einstein (Comment je vois le monde, 1934) 5 Laëtitia HERMAN-Doctorat de Génétique-2020 6 Laëtitia HERMAN-Doctorat de Génétique-2020 Remerciements 7 Laëtitia HERMAN-Doctorat de Génétique-2020 8 Laëtitia HERMAN-Doctorat de Génétique-2020 Table des matières REMERCIEMENTS ...................................................................................................... 7 TABLE DES MATIERES ............................................................................................. 9 GLOSSAIRE ............................................................................................................... 13 I.SYNTHESE BIBLIOGRAPHIQUE ....................................................................... 19 I. Introduction générale......................................................................................... 19 II. La transcription et sa régulation ........................................................................ 21 A. Mécanismes généraux de la régulation transcriptionnelle ........................................ 21 1) Aspect général de la transcription ................................................................................ 21 a) Mise en évidence de la régulation de l’expression génique et de sa régulation .................. 21 b) Initiation, élongation, terminaison ....................................................................................... 23 c) Devenir de l’ARN messager ................................................................................................... 24 2) La régulation transcriptionnelle : de l’architecture du noyau à la reconnaissance d’une séquence d’ADN ................................................................................................................ 25 a) Les facteurs de transcriptions : effecteurs majeurs de la régulation transcriptionnelle ...... 26 b) De la régulation proximale à la régulation distale : promoteurs et enhancers .................... 30 c) Paysage de régulation et domaines topologiques associés .................................................. 32 B. Les facteurs de transcription de la famille des forkheads .......................................... 38 1. Aspect généraux sur les forkheads ............................................................................... 38 a) Organisation génomique et conservation des facteurs à domaine forkhead ....................... 38 b) Structure des facteurs à domaine forkhead .......................................................................... 41 2. Reconnaissance de l’ADN et mécanismes d’actions ..................................................... 43 a) Reconnaissance et fixation à l’ADN ....................................................................................... 43 b) Dimérisation et interaction avec des protéines partenaires ................................................ 43 c) Implications dans les voies de signalisation et régulations par modifications post- traductionnelles......................................................................................................................... 44 d) Fonctions des gènes FOX ....................................................................................................... 49 9 Laëtitia HERMAN-Doctorat de Génétique-2020 C. Cancer et régulation de la transcription par les forkheads .......................................
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