Genome-wide analysis of ATP-dependent chromatin remodeling functions in embryonic stem cells Daria Bou Dargham To cite this version: Daria Bou Dargham. Genome-wide analysis of ATP-dependent chromatin remodeling functions in embryonic stem cells. Biomolecules [q-bio.BM]. Université Paris-Saclay, 2015. English. NNT : 2015SACLS033. tel-01552176 HAL Id: tel-01552176 https://tel.archives-ouvertes.fr/tel-01552176 Submitted on 1 Jul 2017 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. THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY, préparée à L’Université Paris Sud NNT : 2015SACLS033 ÉCOLE DOCTORALE 577 Structure et Dynamique des Systèmes Vivants Spécialité de doctorat : Sciences de la Vie et de la Santé Par Mme Daria Bou Dargham Analyse de la fonction des facteurs de remodelage de chromatine ATP-dépendants dans le contrôle de l’expression du génome des cellules souches embryonnaires Thèse présentée et soutenue à Saclay, le 13/10/2015 : Composition du Jury : Mme Fabienne Malagnac Professeur, Université Paris Sud Présidente M Saadi Khochbin Directeur de Recherche, CNRS Rapporteur M Slimane Ait-Si-Ali Directeur de Recherche, CNRS Rapporteur M Daan Noordermeer Chargé de Recherche, CNRS Examinateur M Eric Soler Chargé de Recherche, INSERM Examinateur M Matthieu Gérard Chercheur CEA, CEA Saclay Directeur de thèse Titre : Analyse de la fonction des facteurs de remodelage de chromatine ATP-dépendants dans le contrôle de l’expression du génome des cellules souches embryonnaires Mots clés : Cellules Souches Embryonnaires, remodeleurs de la chromatine, ChIP-seq Résumé: Des expériences d’immunoprécipitation de la Les cellules souches embryonnaires (cellules ES) chromatine suivi par un séquençage à haute-débit constituent un excellent système modèle pour étudier (ChIP-seq) sur des cellules ES étiquetées pour les les mécanismes épigénétiques contrôlant la différents remodeleurs pour étudier leur distribution transcription du génome mammifère. Un nombre sur le génome, et un approche transcriptomique sur important de membres de la famille des facteurs de des cellules déplétées de chaque remodeleur par remodelage de chromatine ATP-dépendants ont une traitement avec des vecteurs shRNA (knockdown). fonction essentielle pour l’auto-renouvellement des Nous avons établi les profils de liaison des cellules ES, ou au cours de la différentiation. On remodeleurs sur des éléments régulateurs pense que ces facteurs exercent ces rôles essentiels (promoteurs, enhancers et sites CTCF) sur le en régulant l’accessibilité de la chromatine au niveau génome, et montré que ces facteurs occupent toutes des éléments régulateurs de la transcription, en les catégories d’éléments régulateurs du génome. La modulant la stabilité et le positionnement des corrélation entre les données ChIP-seq et les données nucléosomes. Dans ce projet, nous avons conduit une transcriptomiques nous a permis d’analyser le rôle étude génomique à grande échelle du rôle d’une des remodeleurs dans les réseaux de transcription dizaine des remodeleurs (Chd1, Chd2, Chd4, Chd6, essentiels des cellules ES. Nous avons notamment Chd8, Chd9, Ep400, Brg1, Smarca3, Smarcad1, démontré l’importance particulière de certains Smarca5, ATRX et Chd1l) dans les cellules ES. Une remodeleurs comme Brg1, Chd4, Ep400 et double stratégie expérimentale a été utilisée. Smarcad1 dans la régulation de la transcription chez les cellules ES. Title : Genome-wide analysis of ATP-dependent chromatin remodeling factors functions in embryonic stem cells Keywords: Embryonic Stem Cells, chromatin remodeling factors, ChIP-seq Abstract: This was done using a double experimental strategy. The characteristics of embryonic stem cells (ES First, a ChIP-seq (Chromatin Immunoprecipitation cells) make them one of the best models to study the followed by deep sequencing) strategy was done on epigenetic regulation exerted by different actors in ES cells tagged for each factor in the goal of order to control the transcription of the mammalian revealing the genomic binding profiles of the genome. Members of the Snf2 family of ATP- remodeling factors. Second, loss-of-function studies dependent chromatin remodeling factors were followed by transcriptome analysis in ES cells were shown to be of specific importance for ES cell self- performed in order to understand the functional role renewal and during differentiation. These factors are of remodelers. Data from both studies were believed to play essential roles in modifying the correlated to acquire a better understanding of the chromatin landscape through their capacity to role of remodelers in the transcriptional network of position nucleosomes and determine their ES cells. occupancy throughout the genome, making the Specific binding profiles of remodelers on chromatin more or less accessible to DNA binding promoters, enhancers and CTCF binding sites were factors. revealed by our study. Transcriptomic data analysis In this project, a genome-wide analysis of the of the deregulated genes upon remodeler factor function of a number of ATP-dependent chromatin knockdown, revealed the essential role of Chd4, remodelers (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Ep400, Smarcad1 and Brg1 in the control of Brg1, Ep400, ATRX, Smarca3, Smarca5, Smarcad1 transcription of ES cell genes. Altogether, our data and Alc1) in mouse embryonic stem (ES) cells was highlight how the distinct chromatin remodeling conducted. factors cooperate to control the ES cell state. Acknowledgements Initially I would like to thank my jury members for accepting to examine my thesis work. Thanks to Dr. Saadi Khochbin and Dr. Slimane Ait-Si-Ali for accepting to be my thesis reporters and carefully reading and commenting my thesis manuscript. Equally, I thank the examiners Dr. Eric Soler, Dr. Daan Noordermeer and Dr. Fabienne Malagnac for accepting to be a part of my thesis defense jury. I would like to deeply thank my thesis supervisor Dr. Matthieu Gerard for giving me the opportunity to conduct this interesting project in his lab. Thank you for your guidance, advice and great trust during these three years. I would like to equally thank Dr. Jean-Christophe Andrau for being my thesis tutor and participating in my thesis committees. In addition, I thank Dr. Ute Rogner for being a member of my thesis committees. Thank you for your advices and comments. I give special thanks to my collegues in our team. I thank Dr. Michel De Chaldée for his numerous advices and help especially at the beginning of my project, thank you as well for all the interesting discussions we had. I would like to thank Hélène Picaud for her everlasting humor and eventual technical help, keep on laughing! I equally thank Sylvie Jounier for her initial guidance and help, thank you for your patience and professionalism. I give my thanks as well to my collegues in the animal house for their company and help. Thank you Anne-Sophie Chaplault, Sylvain Thessier, Jean-Charles Robillard, Patrick Héry. Thank you for picking me up at occasional work weekends… I also would like to thank Dr.Sophie Chantalat and Florence Ribierre from CNG for their initial help and implication in my project. I would like to thank all my collegues in the SBiGeM and the CEA IRTELIS program for giving me the opportunity to conduct this project. Moreover, I thank my doctoral school especially Dr. Pierre Capy for his guidance. I thank my friends, in particular Dr. Elma El Khouri for her support and huge implication in reading and commenting my thesis manuscript. Finally, I would like to send my sincere and deep thanks to each of my parents, my two brothers and my husband Ghazi for their continuous encouragement, support and love. Without you I would have never been where I am now. Only he who attempts the absurd can achieve the impossible (A. Einstein) Table of contents Table of Contents Introduction Chapter I. Embryonic Stem Cells: Definition and Regulatory Pathways .................................................................................................................................................... 9 A. What are Embryonic Stem Cells? ................................................................................ 9 1. The Major Characteristics of Embryonic Stem Cells .................................................. 9 2. Important signaling pathways that control the mouse ES cells state ......................... 11 B. Naïve and primed pluripotency ................................................................................. 17 1. Mouse ES cell in serum versus 2i medium ................................................................ 17 2. EpiS cells versus ES cells .......................................................................................... 18 C. Human ES cells ......................................................................................................... 20 Chapter II. The Transcriptional and Epigenetic Control of the Embryonic Stem Cell State .................................................................................................................................................