Analysis of tissue-specific & allele- specific DNA methylation Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät IV – Chemie und Pharmazie der Universität Regensburg vorgelegt von Elmar Schilling aus Schwenningen 2009 The present work was carried out in the Department of Hematology and Oncology at the University Hospital Regensburg from June 2005 to June 2009 and was supervised by PD. Dr. Michael Rehli. Die vorliegende Arbeit entstand in der Zeit von Juni 2005 bis Juni 2009 in der Abteilung für Hämatologie und internistische Onkologie des Klinikums der Universität Regensburg unter der Anleitung von PD. Dr. Michael Rehli. Promotionsgesuch eingereicht am: 30. Juli 2009 Die Arbeit wurde angeleitet von PD. Dr. Michael Rehli. Prüfungsausschuss: Vorsitzender: Prof. Dr. Sigurd Elz 1. Gutachter: Prof. Dr. Roland Seifert 2. Gutachter: PD. Dr. Michael Rehli 3. Prüfer: Prof. Dr. Gernot Längst Lob und Tadel bringen den Weisen nicht aus dem Gleichgewicht. (Budha) TABLE OF CONTENTS 1 INTRODUCTION .............................................................................................. 1 1.1 THE CONCEPT OF EPIGENETICS ............................................................................................. 1 1.2 DNA METHYLATION .............................................................................................................. 2 1.2.1 DNA methyltransferases ................................................................................................ 3 1.2.2 Methyl-CpG-binding proteins.......................................................................................... 4 1.3 FUNCTIONS AND MOLECULAR CONSEQUENCES OF DNA METHYLATION IN HEALTHY CELLS ....... 5 1.3.1 Global methylation landscapes ...................................................................................... 6 1.3.2 Genomic immunity: De novo methylation of integrated foreign DNA ............................. 6 1.3.3 Development: Tissue specific DNA methylation ............................................................ 7 1.3.4 Imprinting ........................................................................................................................ 8 1.3.5 Resetting of imprints ....................................................................................................... 8 1.3.6 X-chromosome inactivation ............................................................................................ 9 1.3.7 Interindividual phenotypical differences and inheritance of DNA methylation ............. 11 1.4 MAPPING DNA METHYLATION ............................................................................................. 11 2 AIMS............................................................................................................... 17 3 MATERIAL ..................................................................................................... 18 3.1 EQUIPMENT ........................................................................................................................ 18 3.2 CONSUMABLES .................................................................................................................. 19 3.3 CHEMICALS ........................................................................................................................ 20 3.4 DNA OLIGONUCLEOTIDES ................................................................................................... 20 3.4.1 Human .......................................................................................................................... 20 3.4.1.1 Real-time primer for MCIp ................................................................................................. 20 3.4.1.2 Nested amplification of bisulfite-treated primer .................................................................. 22 3.4.2 Murine ........................................................................................................................... 23 3.4.2.1 MassARRAY Primer .......................................................................................................... 23 3.4.2.2 Real-time PCR primer for MCIp ......................................................................................... 26 3.4.2.3 Real-time PCR primer for validation of CNV ...................................................................... 26 3.4.2.4 Real-time PCR primer for RT-PCR .................................................................................... 27 3.4.2.5 Primer for amplification of genomic DNA for sequencing ................................................... 28 3.4.2.6 Additional internal sequencing primer ................................................................................ 29 3.5 ENZYMES, KITS AND REAGENTS ........................................................................................... 30 3.6 MOLECULAR WEIGHT STANDARDS ....................................................................................... 31 3.7 BACTERIAL STRAINS AND PLASMIDS .................................................................................... 31 3.8 DATABASES AND SOFTWARE ............................................................................................... 32 i 4 METHODS ...................................................................................................... 33 4.1 GENERAL MOLECULAR BIOLOGY ......................................................................................... 33 4.1.1 Bacterial culture ............................................................................................................ 33 4.1.1.1 Bacterial growth medium ................................................................................................... 33 4.1.1.2 Preparation of chemically competent E. coli ...................................................................... 34 4.1.1.3 Transformation of chemically competent E. coli................................................................. 35 4.1.1.4 Glycerol stocks .................................................................................................................. 35 4.1.1.5 Plasmid isolation from E. coli ............................................................................................. 35 4.2 MOLECULAR TECHNOLOGIES .............................................................................................. 36 4.2.1 Polymerase chain reaction (PCR) ................................................................................ 36 4.2.1.1 Primer design..................................................................................................................... 36 4.2.1.2 Standard PCR for cloning or sequencing of gDNA ............................................................ 37 4.2.1.3 Reverse transcription (RT-PCR) ........................................................................................ 38 4.2.1.4 Real-time quantitative PCR analysis .................................................................................. 38 4.2.2 Creation of 0% to 100% methylated DNA as a control ................................................ 41 4.2.3 Molecular cloning ......................................................................................................... 41 4.2.4 Restriction digest .......................................................................................................... 42 4.2.5 CIAP treatment ............................................................................................................. 42 4.2.6 Ligation reaction ........................................................................................................... 42 4.2.7 PEG-precipitation ......................................................................................................... 42 4.2.8 Agarose gel electrophoresis ......................................................................................... 43 4.2.9 Purification of DNA fragment by gel extraction ............................................................ 44 4.2.10 Sequencing of genomic DNA ....................................................................................... 44 4.3 METHYL-CPG-IMMUNOPRECIPITATION (MCIP) ..................................................................... 45 4.3.1 Preparation of MBD2-Fc Fusionprotein ........................................................................ 46 4.3.1.1 Protein production using MBD2-Fc expressing Drosophila S2 cells .................................. 46 4.3.1.2 MBD2-Fc protein purification ............................................................................................. 46 4.3.1.3 MBD2-Fc quality and quantity assessment ........................................................................ 47 4.3.2 Binding MBD2-Fc to beads .......................................................................................... 49 4.3.3 DNA fragmentation ....................................................................................................... 49 4.3.4 Enrichment of highly methylated DNA ......................................................................... 50 4.4 MICROARRAY HANDLING AND ANALYSIS .............................................................................. 51 4.4.1 Gene Expression analysis ............................................................................................ 51 4.4.1.1 Labelling reaction ..............................................................................................................