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Dual Role of Med12 in PRC1-Dependent Gene Repression & Ncrna-Mediated Transcriptional Activation

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Dual Role of Med12 in PRC1-Dependent Gene Repression & Ncrna-Mediated Transcriptional Activation Dual Role of Med12 in PRC1-dependent Gene Repression & ncRNA-mediated Transcriptional Activation Dissertation zur Erlangung des Grades "Doktor der Naturwissenschaften" am Fachbereich Biologie der Johannes Gutenberg-Universität in Mainz Thaleia Papadopoulou geb. am 25.07.1986 in Athen, Griechenland Mainz, 2016 1 Tag der mündlichen Prüfung: 28.10.2016 2 Σα βγεις στον πηγαιμό για την Ιθάκη, να εύχεσαι νάναι μακρύς ο δρόμος, γεμάτος περιπέτειες, γεμάτος γνώσεις. Τους Λαιστρυγόνας και τους Κύκλωπας, τον θυμωμένο Ποσειδώνα μη φοβάσαι, τέτοια στον δρόμο σου ποτέ σου δεν θα βρεις, αν μέν’ η σκέψις σου υψηλή, αν εκλεκτή συγκίνησις το πνεύμα και το σώμα σου αγγίζει. Τους Λαιστρυγόνας και τους Κύκλωπας, τον άγριο Ποσειδώνα δεν θα συναντήσεις, αν δεν τους κουβανείς μες στην ψυχή σου, αν η ψυχή σου δεν τους στήνει εμπρός σου. As you set out for Ithaka hope the voyage is a long one, full of adventure, full of discovery. Laistrygonians and Cyclops, angry Poseidon—don’t be afraid of them: you’ll never find things like that on your way as long as you keep your thoughts raised high, as long as a rare excitement stirs your spirit and your body. Laistrygonians and Cyclops, wild Poseidon—you won’t encounter them unless you bring them along inside your soul, unless your soul sets them up in front of you. (Translation by Edmund Keeley/Philip Sherrard) Brichst du auf gen Ithaka, wünsch dir eine lange Fahrt, voller Abenteuer und Erkenntnisse. Die Lästrygonen und Zyklopen, den zornigen Poseidon fürchte nicht, solcherlei wirst du auf deiner Fahrt nie finden, wenn dein Denken hochgespannt, wenn edle Regung deinen Geist und Körper anrührt. Den Lästrygonen und Zyklopen, dem wütenden Poseidon wirst du nicht begegnen, falls du sie nicht in deiner Seele mit dir trägst, falls deine Seele sie nicht vor dir aufbaut. (Tranlation by lyrikwelt.de/) The poem Ithaka by Constantinos P. Cavafy very much describes the journey towards a PhD. I dedicate this thesis to all those who contributed into “keeping my thoughts raised high” during this “voyage” 3 Table of Contents Dual Role of Med12.......................................................................................................................... 1 in PRC1-dependent Gene Repression & ......................................................................................... 1 ncRNA-mediated Transcriptional Activation .................................................................................... 1 INDEX OF ABBREVIATIONS .......................................................................................................... 8 SUMMARY ....................................................................................................................................... 9 ZUSAMMENFASSUNG ................................................................................................................. 10 INTRODUCTION ............................................................................................................................ 12 Stem cell origin ........................................................................................................................... 12 Transcriptional regulation in pluripotency and differentiation .................................................... 14 Embryonic Stem Cell Transcription Factors ........................................................................... 14 Chromatin Regulators ............................................................................................................. 15 ncRNAs ................................................................................................................................... 20 The General Transcriptional Machinery, Co-Activators & cis-Regulatory Elements ............. 24 Genome organization.............................................................................................................. 35 AIM OF THE STUDY ..................................................................................................................... 37 MATERIALS & METHODS ............................................................................................................ 38 Cell culture .................................................................................................................................. 38 mESCs .................................................................................................................................... 38 Freezing/thawing cells ............................................................................................................ 39 HEK293T ................................................................................................................................. 39 Transfections .............................................................................................................................. 40 siRNA Transfections in mESCs .............................................................................................. 40 Calcium phosphate Transfections in HEK293T cells ............................................................. 40 PEI Transfections in HEK293T cells....................................................................................... 41 Generation of stable ESC Knockdown Lines ............................................................................. 41 Alkaline Phospatase Staining ..................................................................................................... 42 Site directed mutagenesis in Zrf1 SANT domain ....................................................................... 42 Chromatin Association Assays ................................................................................................... 43 Chromatin Associations after RNaseA treatment ...................................................................... 44 Cell Fractionation Assays ........................................................................................................... 45 RNA extraction, cDNA synthesis and RTqPCR ......................................................................... 45 Immunoprecipitations (IPs) ......................................................................................................... 49 Preparing mESCs for IPs ........................................................................................................ 49 4 Preparing HEK293Ts for IPs .................................................................................................. 49 Preparation of cell extracts for IP ........................................................................................... 50 Chromatin Immunoprecipitations (ChIP) .................................................................................... 51 ChIP for all Abs ....................................................................................................................... 51 ChIP for FLAG-tagged lines .................................................................................................... 53 RNA- Chromatin Immunoprecipitation (RNA- ChIP) .................................................................. 55 RNA-Immunoprecipitations ........................................................................................................ 57 Immunofluorescent staining ....................................................................................................... 59 Genome-wide approaches ......................................................................................................... 60 RNA-sequencing ..................................................................................................................... 60 ChIP-sequencing .................................................................................................................... 60 Mass Spectrometry ..................................................................................................................... 60 Results ............................................................................................................................................ 61 PART 1: Common functions of PRC1 and Med12-Mediator in pluripotent mESCs ..................... 61 PRC1 and Mediator occupy similar chromatin regions in mESCs ............................................ 61 Ring1b-dependent recruitment of Med12 at chromatin ............................................................. 66 Ring1b and Med12 repress key developmental genes in pluripotency ..................................... 69 Med12 and Ring1b depleted mESCs fail to properly differentiate............................................. 74 PART 2: Remodeling of Med12-Mediator enhances gene activation in early stem cell differentiation in a ncRNA-dependent manner .............................................................................. 78 Med12 acts in concert with ncRNAs as a transcriptional activator of developmental genes in RA-mediated differentiation ........................................................................................................ 78 Zrf1 associates with activating ncRNAs during differentiation ................................................... 82 Zrf1 is implicated in the Med12-Dlx1as-dependent activation of Hoxd11 ................................. 84 Zrf1 assembles the Kinase Module of Mediator during differentiation ...................................... 86 Epigenetic regulation by Zrf1 on the Hoxd11 locus ................................................................... 89 ZRF1 interacts with transcription
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