Molecular mechanisms underlying heterochromatin formation in the mouse embryo Joanna Weronika Jachowicz To cite this version: Joanna Weronika Jachowicz. Molecular mechanisms underlying heterochromatin formation in the mouse embryo. Embryology and Organogenesis. Université de Strasbourg, 2015. English. NNT : 2015STRAJ094. tel-01681610v2 HAL Id: tel-01681610 https://tel.archives-ouvertes.fr/tel-01681610v2 Submitted on 11 Jan 2018 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. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE des SCIENCES de la VIE et de la SANTÉ THÈSE présentée par : Joanna Weronika JACHOWICZ soutenue le : 17 Décembre 2015 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Aspects moléculaires et cellulaire de la biologie du développement Molecular mechanisms underlying heterochromatin formation in the mouse embryo THÈSE dirigée par : Dr Maria Elena TORRES-PADILLA , Université de Strasbourg RAPPORTEURS : Prof Didier TRONO EPFL, Lausanne Dr Andrew BANNISTER The Gurdon Institute, Cambridge AUTRES MEMBRES DU JURY : Dr Michael WEBER ESBS, Illkirch Table of Contents Acknowledgements ............................................................................................................................. ! 4 ! Abstract ............................................................................................................................................... ! 5 ! Avant propos ....................................................................................................................................... ! 7 ! I. Introduction ............................................................................................................................. ! 11 ! 1. Characteristics of (hetero)chromatin in mammalian cells ...................................................... ! 11 ! 1.1. Brief introduction to chromatin .................................................................................... ! 11 ! 1.2. Euchromatin versus heterochromatin .......................................................................... ! 12 ! 1.3. Maintenance and establishment of heterochromatin in somatic and stem cells ......... ! 14 ! 1.3.1. DNA methylation ................................................................................................... ! 15 ! 1.3.2. Histone modifications and related pathways ....................................................... ! 16 ! 1.3.3. Histone variants .................................................................................................... ! 18 ! 1.3.4. DNA binding factors .............................................................................................. ! 19 ! 1.3.5. Small RNAs and RNAi pathway .............................................................................. ! 21 ! 1.3.6. Nuclear organization ............................................................................................. ! 22 ! 1.4. Concluding remarks ....................................................................................................... ! 24 ! 2. Characteristics of heterochromatin during mouse development ....................................... ! 24 ! 2.1. Mouse development at a glance ................................................................................... ! 24 ! 2.2. Formation of gametes ................................................................................................... ! 25 ! 2.2.1. Primordial germ cells specification ....................................................................... ! 25 ! 2.2.2. Formation of sex specific gametes ........................................................................ ! 27 ! 2.3. Preimplanation development ........................................................................................ ! 32 ! 2.3.1. Most important events in preimplantation embryos ........................................... ! 32 ! 2.3.2. DNA methylation ................................................................................................... ! 34 ! 2.3.3. Histone variants .................................................................................................... ! 35 ! 2.3.4. Pathways responsible for establishment of repressive and active histone marks ! 37 ! 2.3.5. Nuclear organization in preimplantation embryos ............................................... ! 41 ! 3. Heterochromatin during mouse development – specific regions and open questions about their establishment ....................................................................................................................... ! 45 ! 3.1. Pericentric repeats ........................................................................................................ ! 45 ! 3.2. L1 transposable elements ............................................................................................. ! 47 ! 3.2.1. Publication 1 – LINEs in mice ................................................................................. ! 49 ! II. Main questions and objectives ..................................................................................... ! 50 ! Part I – Pericentric repeats ............................................................................................................ ! 50 ! Part II – L1 transposable elements ................................................................................................ ! 50 ! ! 2 ! III. Results & Discussions ....................................................................................................... ! 52 ! Part I ! Pericentric repeats ............................................................................................................ ! 52 ! 1.1. Summary of Publication 2 ............................................................................................. ! 52 ! 1.2. Publication 2 .................................................................................................................. ! 53 ! 1.3. Discussion ...................................................................................................................... ! 54 ! Part II ! L1 transposable elements ................................................................................................ ! 59 ! 2.1. L1 biology during early mouse development ................................................................ ! 59 ! 2.1.1. Description of the transcription pattern of L1spa elements from fertilization until morula stage .......................................................................................................................... ! 59 ! 2.1.2. Protein expression ................................................................................................. ! 60 ! 2.2. Establishment of tools and experimental conditions .................................................... ! 61 ! 2.2.1. TALEs design and verification in human and mouse cells ..................................... ! 61 ! 2.2.2. Establishment of experimental conditions for TALE expression in mouse embryos ... ! 62 ! 2.3. Role of the repression of L1 elements during preimplantation development of mouse embryos ..................................................................................................................................... ! 64 ! 2.3.1. Artificial tethering of the L1 TALE !VP64 activators and their effect on the level of L1 transcript at 4! and 8!cell stage mouse embryos .................................................................. ! 64 ! 2.3.2. The consequences of the L1 activation on the preimplantation development of mouse embryos ..................................................................................................................... ! 66 ! 2.3.3. Dissecting the plausible cause of the developmental arrest ................................ ! 67 ! 2.4. Figures ........................................................................................................................... ! 69 ! 2.5. Materials & Methods..................................................................................................... ! 80 ! 2.6. Discussion ...................................................................................................................... ! 82 ! IV. Concluding remarks .......................................................................................................... ! 88 ! V. Bibliography ........................................................................................................................... ! 90 ! ! 3 ! Acknowledgements Firstly, I would like to express my sincere gratitude to my advisor Dr. Maria Elena Torres ! Padilla for the continuous support during my studies, for her patience, motivation, and immense knowledge. Her guidance has helped me throughout the research and the thesis writing and without it I would have been probably lost. Thank you for showing me how amazing science is and for proving that one can be a great researcher but still stay a human being. I could not have imagined having a better advisor and