Genetic determinants of inter-individual variations in DNA methylation Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie und Vorklinische Medizin der Universität Regensburg vorgelegt von Julia Wimmer (geb. Wegner) aus Neubrandenburg im Jahr 2014 The present work was carried out at the Clinic and Polyclininc of Internal Medicine III at the University Hospital Regensburg from March 2010 to August 2014. Die vorliegende Arbeit entstand im Zeitraum von März 2010 bis August 2014 an der Klinik und Poliklinik für Innere Medizin III des Universitätsklinikums Regensburg. Das Promotionsgesuch wurde eingereicht am: 01. September 2014 Die Arbeit wurde angeleitet von: Prof. Dr. Michael Rehli Prüfungsausschuss: Vorsitzender: Prof. Dr. Herbert Tschochner Erstgutachter: Prof. Dr. Michael Rehli Zweitgutachter: Prof. Dr. Axel Imhof Drittprüfer: Prof. Dr. Gernot Längst Ersatzprüfer: PD Dr. Joachim Griesenbeck Unterschrift: ____________________________ Who seeks shall find. (Sophocles) Table of Contents TABLE OF CONTENTS .......................................................................................................................... I LIST OF FIGURES ................................................................................................................................ IV LIST OF TABLES ................................................................................................................................... V 1 INTRODUCTION ........................................................................................................................... 1 1.1 THE CONCEPT OF EPIGENETICS ........................................................................................................ 1 1.1.1 DNA methylation ................................................................................................................... 2 1.1.1.1 Setting and erasing methylation at CpG dinucleotides ......................................................... 2 1.1.1.2 Methyl-CpG binding proteins, the readers of DNA methylation ........................................... 6 1.1.2 The histone code ................................................................................................................... 8 1.1.2.1 Histone acetylation ................................................................................................................... 10 1.1.2.2 Histone methylation .................................................................................................................. 10 1.1.2.3 Translation of the histone code .............................................................................................. 11 1.1.3 Non-coding RNAs ............................................................................................................... 12 1.2 EPIGENETIC REPROGRAMMING DURING EMBRYOGENESIS ............................................................. 13 1.3 REGULATORS OF INTER-INDIVIDUAL EPIPHENOTYPES .................................................................... 16 2 RESEARCH OBJECTIVES ........................................................................................................ 17 3 MATERIALS & EQUIPMENT ..................................................................................................... 18 3.1 EQUIPMENT ..................................................................................................................................... 18 3.2 CONSUMABLES ................................................................................................................................ 20 3.3 CHEMICALS ..................................................................................................................................... 21 3.4 ENZYMES, KITS, AND PRODUCTS FOR MOLECULAR BIOLOGY ........................................................ 21 3.5 ANTIBIOTICS .................................................................................................................................... 22 3.6 MOLECULAR WEIGHT STANDARDS .................................................................................................. 23 3.7 CELL LINES ...................................................................................................................................... 23 3.8 E. COLI STRAINS .............................................................................................................................. 23 3.9 DEOXYRIBONUCLEIC ACIDS ............................................................................................................. 23 3.9.1 Murine genomic DNA ......................................................................................................... 23 3.9.2 Circular DNA ........................................................................................................................ 24 3.9.2.1 Plasmids .................................................................................................................................... 24 3.9.2.2 Bacterial Artificial Chromosomes ........................................................................................... 24 3.10 OLIGONUCLEOTIDES ................................................................................................................... 25 3.10.1 PCR primers .................................................................................................................... 25 3.10.2 RT-qPCR primers ........................................................................................................... 25 3.10.3 qPCR primers ................................................................................................................. 26 3.10.4 Primers for the MassARRAY system (Sequenom) ................................................. 27 3.10.4.1 EpiTYPER primers ................................................................................................................... 27 3.10.4.2 iPLEX primers ........................................................................................................................... 33 3.10.5 Primers for hybridization probes .................................................................................. 34 3.11 ANTIBODIES FOR IMMUNOPRECIPITATION EXPERIMENTS .......................................................... 40 3.12 DATABASES AND SOFTWARE ...................................................................................................... 40 4 METHODS ................................................................................................................................... 42 4.1 GENERAL CELL AND BACTERIA CULTURE METHODS ....................................................................... 42 4.1.1 Cell line cultures .................................................................................................................. 42 4.1.1.1 Murine embryonic stem cells .................................................................................................. 42 4.1.1.2 Assessing cell number and vitality ......................................................................................... 46 4.1.2 Bacterial culture ................................................................................................................... 47 I 4.1.2.1 Bacterial growth medium ......................................................................................................... 47 4.1.2.2 Transformation of E. coli ......................................................................................................... 48 4.1.2.3 Glycerol stock ........................................................................................................................... 50 4.2 LABORATORY ANIMALS .................................................................................................................... 50 4.3 GENERAL MOLECULAR BIOLOGICAL METHODS ............................................................................... 51 4.3.1 Preparation and analysis of DNA ..................................................................................... 51 4.3.1.1 Isolation of circular DNA from E. Coli .................................................................................... 51 4.3.1.2 Isolation of genomic DNA from mammalian cells ................................................................ 54 4.3.1.3 Fragmentation of genomic DNA and chromatin ................................................................... 55 4.3.1.4 Agarose gel electrophoresis ................................................................................................... 58 4.3.1.5 Pulsed-field gel electrophoresis (PFGE) ............................................................................... 60 4.3.1.6 Purification of DNA fragments by gel extraction .................................................................. 63 4.3.1.7 Purification of DNA by phenol/chloroform extraction ........................................................... 63 4.3.1.8 Polyethylene glycol 8000 (PEG8000) precipitation of DNA................................................ 64 4.3.1.9 Alcohol precipitation of DNA ................................................................................................... 65 4.3.1.10 Polymerase chain reaction ...................................................................................................... 67 4.3.1.11 In vitro methylation and demethylation of genomic DNA ...................................................