DNA-encoded signals regulate genomic binding of transcription factors Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Jonas Telorac aus Berlin Juni 2014 Diese Arbeit wurde unter der Leitung von Dr. Sebastiaan H. Meijsing im Zeitraum von Juni 2010 bis Juni 2014 am Max-Planck-Institut für molekulare Genetik angefertigt. 1. Gutachter: Dr. Sebastiaan H. Meijsing Max-Planck-Institut für molekulare Genetik 2. Gutachter: Prof. Dr. Markus Wahl Freie Universität Berlin Disputation am: 13.10.2014 Acknowledgements I am very grateful to my advisor Dr. Sebastiaan H. Meijsing for always letting me follow own research ideas and being open for scientific discussion as well as his valuable advice. I also want to thank Prof. Dr. Markus Wahl for reviewing this work. For the great atmosphere and all the fruitful discussions I want to thank everybody who accompanied me during my time at the MPI-MG, especially all my fellow lab members. I really enjoyed all those fun and controversial discussions, especially with Stephan Starick. In addition, I also want to thank Dr. Sergey Prykhozhij for all his help with the Zebrafish experiments and for a nice time during the late hour microscopy evenings, but most importantly for his patience in trying to teach me injections. I also want to express my gratitude to Dr. Morgane Thomas-Chollier. Large parts of this work would not have been possible without her help and sophisticated bioinformatical analyses. I also want to thank Dr. Saskia Hutten and the rest the Lamond Lab at the University of Dundee in Scotland for teaching me a lot about subnuclear structures and for a great time during my stay in Dundee. For their commendable technical assistance I am very grateful to Edda Einfeldt and Katja Borzym. Table of Content 1 Introduction ................................................................................................................... 1 1.1 Transcription ........................................................................................................... 1 1.1.1 Initiation .......................................................................................................... 1 1.1.2 Elongation ........................................................................................................ 2 1.1.3 Termination ..................................................................................................... 2 1.2 Regulation of transcription ..................................................................................... 3 1.3 Chromatin influences accessibility of binding sites ............................................... 4 1.3.1 Nucleosome positioning .................................................................................. 5 1.3.2 Histone modifications and histone variants ..................................................... 5 1.3.3 DNA methylation and CpG content ................................................................ 6 1.3.4 Spatial genomic organization .......................................................................... 6 1.4 The nuclear receptor superfamily as a tool to study regulation of TF binding ....... 7 1.5 The glucocorticoid receptor as model transcription factor ..................................... 9 1.6 DNA-encoded signals that specify GR binding .................................................... 11 1.7 Background of my research: Analysis of ChIP-Seq data ..................................... 11 1.8 Nalm-6 as a model to study cell type specific signals .......................................... 13 1.9 Aim of this thesis .................................................................................................. 15 2 Materials ...................................................................................................................... 17 2.1 Chemicals .............................................................................................................. 17 2.2 Enzymes, Proteins, DNA Kits, Plasmids .............................................................. 18 2.3 Oligonucleotides ................................................................................................... 19 2.4 Plasmids ................................................................................................................ 21 2.4.1 pGL3-Promoter .............................................................................................. 21 2.5 pcDNA3.1PA-D57 (kind gift by Dr. Ulrich Stelzl) .............................................. 21 2.5.1 pFireV5-DM (kind gift by Dr. Ulrich Stelzl) ................................................ 22 2.6 Antibodies ............................................................................................................. 23 2.6.1 Antibodies for Lumier assay.......................................................................... 23 2.6.2 Antibody for ChIP ......................................................................................... 23 2.6.3 Antibodies for western blot ........................................................................... 23 2.6.4 Antibodies for Immunofluorescence ............................................................. 23 Table of Content 2.7 Lab Ware ............................................................................................................... 23 2.8 Organisms ............................................................................................................. 24 2.8.1 Bacterial strains ............................................................................................. 24 2.8.2 Mammalian cell lines..................................................................................... 24 2.9 Media .................................................................................................................... 24 2.9.1 LB Medium ................................................................................................... 24 2.9.2 SOB Medium ................................................................................................. 24 2.10 Buffers .................................................................................................................. 24 2.11 General buffers ..................................................................................................... 24 2.11.1 TE-Buffer....................................................................................................... 24 2.11.2 PBS buffer ..................................................................................................... 25 2.12 Lumier Buffers ...................................................................................................... 25 2.12.1 Lumier lysis buffer ........................................................................................ 25 2.12.2 TBST II .......................................................................................................... 25 2.12.3 Carbonate buffer ............................................................................................ 25 2.13 ChIP buffers .......................................................................................................... 25 2.13.1 IP Lysis Buffer .............................................................................................. 25 2.13.2 RIPA buffer ................................................................................................... 25 2.13.3 RIPA wash buffer .......................................................................................... 25 2.13.4 LiCl wash buffer ............................................................................................ 25 2.13.5 Crosslink reversal solution ............................................................................ 25 2.14 DNase Buffers ....................................................................................................... 25 2.14.1 DNase I reaction buffer ................................................................................. 25 2.15 MNase assay buffers ............................................................................................. 25 2.15.1 Lysis buffer .................................................................................................... 25 2.15.2 Storage buffer ................................................................................................ 26 2.15.3 Reaction buffer .............................................................................................. 26 2.15.4 Stopping buffer .............................................................................................. 26 2.16 Nuclear extract ...................................................................................................... 26 Table of Content 2.16.1 PBSI buffer .................................................................................................... 26 2.16.2 Buffer A ......................................................................................................... 26 2.16.3 Buffer B ......................................................................................................... 26 2.16.4 Buffer D ......................................................................................................... 26 2.17 DNA pull-down buffers ........................................................................................ 26 2.17.1 DW buffer .....................................................................................................