DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR BIOLOGIE DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN CHROMATIN REMODELING IN EPSTEIN-BARR VIRUS AFTER INDUCTION OF THE LYTIC PHASE: MOLECULAR CHARACTERIZATION OF THE ROLE OF BZLF1 AND ITS INTERACTIONS MARISA SCHÄFFNER Dissertation eingereicht am 30. April 2015 Erstgutachter: Prof. Dr. Dirk Eick Zweitgutachter: Prof. Dr. Heinrich Leonhardt Tag der mündlichen Prüfung: 26.11.2015 ERKLÄRUNG Hiermit erkläre ich, dass die vorliegende Arbeit mit dem Titel „CHROMATIN REMODELING IN EPSTEIN-BARR VIRUS AFTER INDUCTION OF THE LYTIC PHASE: MOLECULAR CHARACTERIZATION OF THE ROLE OF BZLF1 AND ITS INTERACTIONS“ von mir selbstständig und ohne unerlaubte Hilfsmittel angefertigt wurde, und ich mich dabei nur der ausdrücklich bezeichneten Quellen und Hilfsmittel bedient habe. Die Arbeit wurde weder in der jetzigen noch in einer abgewandelten Form einer anderen Prüfungskommission vorgelegt. München, 30. April 2015 Marisa Schäffner CONTENT 1. Introduction....................................................................................................................... 7 1.1 The architecture of chromatin.......................................................................................... 7 1.1.1 Nucleosomes are histone octamers ........................................................................ 7 1.1.1.1 Nucleosome components.................................................................................. 7 1.1.1.2 Histone modifications and histone variants...................................................... 8 1.1.1.3 Nucleosome positioning ................................................................................. 10 1.1.2 Enhancer-promoter contacts................................................................................. 12 1.2 Chromatin regulators and their way of functions........................................................... 14 1.2.1 Chromatin remodelers.......................................................................................... 14 1.2.2 Pioneer factors...................................................................................................... 20 1.3 Epigenetic regulation in EBV ........................................................................................ 22 1.3.1 EBV and its life cycle........................................................................................... 22 1.3.2 EBV and its lytic switch transactivator BZLF1 ................................................... 24 1.3.3 Epigenetic regulation mechanisms upon lytic reactivation.................................. 26 1.3.4 Interactions of BZLF1 with viral and host cell proteins ...................................... 28 1.4 Scope of my thesis work............................................................................................. 30 2. Material............................................................................................................................ 31 2.1 Oligonucleotides ............................................................................................................ 31 2.2 Plasmids ......................................................................................................................... 31 2.3 Antibodies...................................................................................................................... 32 2.4 Bacterial strains.............................................................................................................. 32 2.5 Eukaryotic cell lines....................................................................................................... 33 CONTENT II 2.6 Cell culture media and additives.................................................................................... 33 2.6.1 Media for the cultivation of bacteria....................................................................... 33 2.6.2 Media for the cultivation of eukaryotic cells .......................................................... 33 2.7 Chemicals and enzymes................................................................................................. 34 2.8 Buffers and solutions ..................................................................................................... 35 2.9 Commercial kits............................................................................................................. 36 2.10 Software ....................................................................................................................... 37 2.11 Devices and consumables ............................................................................................ 37 3. Methods............................................................................................................................ 38 3.1 Bacterial culture............................................................................................................. 38 3.1.1 Growth and storage of bacterial cultures ................................................................ 38 3.1.2 Transformation of bacteria...................................................................................... 38 3.2 Eukaryotic cell culture ................................................................................................... 39 3.2.1 Cell culture conditions ............................................................................................ 39 3.2.2 Storage of eukaryotic cells...................................................................................... 39 3.2.3 Transient transfection of HEK293 cells.................................................................. 39 3.2.4 Electroporation of eukaryotic cells ......................................................................... 40 3.2.5 Establishment of stable cell lines............................................................................ 40 3.2.6 Flow cytometry ....................................................................................................... 40 3.3 Nucleic acid techniques ................................................................................................. 41 3.3.1 DNA purification from E.coli................................................................................. 41 3.3.2 DNA purification from eukaryotic cells ................................................................. 41 3.3.3 Purification of DNA from PCR products and agarose gels .................................... 41 3.3.4 Electroelution of DNA from native gels................................................................. 42 3.3.5 Dephosphorylation and ligation.............................................................................. 42 3.3.6 Polymerase chain reaction (PCR) ........................................................................... 42 3.3.7 Quantitative real time PCR (qPCR)........................................................................ 42 3.3.8 Mutagenesis PCR.................................................................................................... 43 3.3.9 Isolation of RNA from cells.................................................................................... 43 3.3.10 Reverse transcription of RNA............................................................................... 44 3.4 Protein analysis techniques............................................................................................ 44 3.4.1 Preparation of whole cell extracts........................................................................... 44 3.4.2 Preparation of nuclear cell extracts......................................................................... 44 3.4.3 Purification of Strep-tag fusion proteins................................................................. 45 3.4.4 Electromobility shift assay (EMSA) for detecting protein-DNA interactions ....... 45 3.4.5 Determination of the equilibrium dissociation constant (Kd value) ....................... 46 3.4.6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)........... 47 3.4.7 Western blot............................................................................................................ 47 3.4.8 Silver staining ......................................................................................................... 47 CONTENT III 3.5 Chromatin Immunoprecipitation (ChIP)........................................................................ 48 3.5.1 Chromatin preparation ............................................................................................ 48 3.5.2 Chromatin immunoprecipitation and purification of ChIP DNA ........................... 48 3.5.3 Quantitation of ChIP DNA by qPCR...................................................................... 49 3.6 Sequential ChIP (ReChIP) ............................................................................................. 49 3.6.1 Chromatin preparation for BZLF1-directed ChIP .................................................. 49 3.6.2 ReChIP starting with a BLZF1-directed antibody.................................................. 50 3.6.3 ReChIP starting with an H3K4me1-directed antibody ........................................... 52 3.7 Co-Immunoprecipitation (CoIP).................................................................................... 53 3.7.1 CoIPs of GFP-tagged bait proteins ......................................................................... 53 3.7.2 CoIPs of Strep-tagged bait proteins .......................................................................