Genetic and epigenetic mechanisms of the regulation of mouse embryonic stem cells self-renewal by the pluripotency transcription factor Nanog Victor HEURTIER Thèse de doctorat de Biologie Dirigée par Pablo NAVARRO GIL Laboratoire Epigénétique des cellules souches – Institut Pasteur Sorbonne Université Ecole doctorale Complexité du Vivant (ED 515) Présentée et soutenue publiquement le 18/09/2018 Devant un jury composé de : Président : Pr MOUCHEL-VIELH Emmanuèle, Professeur Rapporteur : Dr SAVATIER Pierre, Directeur de Recherche Rapporteur : Dr RADA-IGLESIAS Alvaro, Chef d’équipe Examinateur : Dr AZUARA Veronique, Maître de conférences Examinateur : Dr PINSKAYA Marina, Maître de conférences Directeur de thèse : Dr NAVARRO GIL Pablo, Chef d’équipe Abstract Mouse embryonic stem (ES) cells are derived from the pre-implantation blastocyst and are able to maintain their pluripotent state through virtually limitless cell divisions in vitro. The study of the mechanisms regulating the specific features of ES cells led to the discovery of the transcription factors (TFs) governing their unique transcriptome. Among those TFs, Nanog plays a central role in the gene regulatory network that supports ES cells self-renewal. However, the molecular mechanisms by which Nanog exerts its functions are not fully elucidated. We adapted an inducible CRISPR activation (CRISPRa) system in mouse ES cells and demonstrated its functionality to stimulate transcription at endogenous loci. We then accurately determined the list of Nanog responsive genes using gain (CRISPRa cell line) and loss of function experiments and total RNA sequencing. Moreover, the DNA binding profiles of distinct pluripotency TFs were assessed genome-wide by chromatin immunoprecipitation (ChIP) and sequencing upon Nanog depletion and revealed the importance of the latter in the regulation of the pluripotency network activity at thousands of loci. We additionally showed that Nanog employs distinct mechanisms to activate and repress its targets through binding rather distal or proximal DNA regulatory elements respectively. We further investigated the action of Nanog overexpression in the absence of the LIF cytokine (otherwise mandatory to support ES cells self-renewal) and showed that Nanog both activates pluripotency sustaining factors and, to a greater extent, represses differentiation determinants. Furthermore, we found that Nanog-dependent gene repression is strongly correlated with the enrichment of the repressive histone mark H3K27me3 thus proposing a new mechanism through which Nanog could indirectly repress its targets such as Otx2, a major early differentiation factor. We further explored the regulation of long non-coding RNAs (lncRNAs) expression by Nanog and selected non-coding candidates that could potentially be involved in the maintenance of mouse ES cell self-renewal. So far, only one of them seems to give promising results and will be further investigated. Finally, a fortuitously observed off-target effect of a specific gRNA, together with CRISPRa, led to the induction of the 2-cell-like state transcriptomic profile spontaneously found in a small subset of ES cells. CRISPRa off-target binding sites were determined genome- wide by ChIP sequencing and candidate genes potentially responsible for the induction of the observed effect are now under investigation. 2 À mon père, Qui m’a toujours encouragé et soutenu, Sans qui cette thèse ne serait sans doute pas. 3 Acknowledgements I would like to express my deep gratitude to my supervisor Pablo Navarro Gil for giving me the opportunity to work first as a Master and then a PhD student in his laboratory. Thank you for the great liberty you gave me in my work and the faith you had in me which allowed me to feed my curiosity and develop my scientific autonomy. Thank you for useful critiques and accurate analyses which pushed me to do always better and helped me building my scientific critical spirit. I want to thank all the past and present members of the laboratory with whom I had a real pleasure to work, discuss, exchange and laugh every day during these five years and made my time in the lab enjoyable. Thank you Agnès for your kindness, advice and support as well as all you do for us every day and that we often don’t even realize. Thank you Philippe for all our discussions, from science to politics, from music to fishing, without forgetting our silly jokes. I wish to thank Nicola for his excellent advice that I didn’t follow enough. It was also a pleasure to work with Inma, always enthusiastic and encouraging, thank you for your help and kindness. I am particularly grateful for the help of Nick in analysing the massive amount of data we generate, for great discussions and with whom it was a pleasure to work with. Thank you Thaleia for your help, humour and rational scientific point of view. Thank you Laure for your patience and for the personal engagement you put in the project. Finally, thank you Laurence for your help with all our administrative work. I wish you all best of luck and success in your future professional and personal lives. I wish to acknowledge the help provided by Didier Montarras by introducing me to the world of research and of Institut Pasteur from the beginning. I am very grateful for your tutoring and help. Un grand merci Didier. I would like to thank Damien for his help on the gRNA design and our long discussions. I want to thank my PhD committee (Alice Jouneau, Marina Pinskaya, Cosmin Saveanu) for following my work all along my thesis and for their useful critiques and suggestions. I would like to thank the whole Department of Stem Cells and Developmental Biology, and more specifically Aurélien, Brendan, Sylvia, Sabela, Mathieu, Coralie, Aurélie, Clémire, Catherine, Edith, Marc, Sylvain, Glenda. I want to thank Sara for welcoming me in the Department during my Master and for her support. I would like to thank the organizers of the MD-PhD programs from the Sorbonne Université and the Ecole de l’INSERM for allowing me to do this PhD. 4 I want to thank Alexandra for her everyday life support, kindness and care as well as her constant and precious help in the lab. Je voudrais finalement remercier ma famille pour leur soutien tout au long de mes études, leur affection et leur intérêt dans mon travail. Merci à mes deux frères Lucien et Sylvain, à ma grand-mère Mimine et à mes parents, Stéphanie et Jean-François. 5 Table of content Introduction .................................................................................................................... 8 I. Early development & embryonic stem cells ...................................................................... 9 A. Early mouse embryo development .............................................................................. 9 B. The establishment of pluripotency in vitro ............................................................... 11 C. Origin and properties ................................................................................................. 12 D. The spectrum of pluripotent cells .............................................................................. 13 E. The maintenance of the pluripotent state .................................................................. 14 II. Signalling pathways regulating pluripotency................................................................... 15 A. LIF signalling ............................................................................................................ 15 B. FGF signalling ........................................................................................................... 18 C. Wnt signalling ........................................................................................................... 19 III. Transcription Factors-based regulation of pluripotency ............................................... 21 A. Pluripotency factors................................................................................................... 21 B. Oct4 (Pou5f1) ............................................................................................................ 21 C. Sox2 ........................................................................................................................... 22 D. Nanog ........................................................................................................................ 23 E. LIF independent self-renewal.................................................................................... 28 F. Otx2............................................................................................................................... 30 IV. Polycomb regulation and bivalent domains in pluripotency ......................................... 32 A. Polycomb complexes and functions .......................................................................... 32 B. Bivalent domains and Polycomb regulation in pluripotent cells............................... 33 V. Long non-coding RNAs & mouse ES cells ..................................................................... 36 A. Description and characteristics.................................................................................. 36 B. LncRNAs function in mouse ES cells ....................................................................... 37 VI. From CRISPR discovery to CRISPR activators ........................................................... 40 A. Historical background ............................................................................................... 40