Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften an der Fakultät für Biologie der Ludwig-Maximilians-Universität München Dynamics of histone modifications Annette N. D. Scharf aus Offenburg Eingereicht am 23. Juli 2009 Mündliche Prüfung am 24. November 2009 1. Gutachter: Prof. Dr. Peter Becker 2. Gutachter: Prof. Dr. Dirk Eick 3. Gutachter: Prof. Dr. Thomas Cremer 4. Gutachter: Prof. Dr. Michael Boshart 5. Gutachter: PD Dr. Stefan Müller 6. Gutachter: Prof. Dr. Charles David 7. SV: Prof. Dr. Axel Imhof Ehrenwörtliche Versicherung Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt ist. München, den .......................... .................................................................................. (Unterschrift) Dedicated to my father Table of contents Contents Acknowledgments ........................................................... 13 Summary ................................................................... 14 Zusammenfassung ........................................................... 15 1. Introduction ............................................................. 17 1.1 Epigenetics .............................................................. 18 1.2 Chromatin .............................................................. 18 1.3 Histone modifications..................................................... 21 1.3.1 Acetylation ........................................................... 23 1.3.2 Methylation .......................................................... 24 1.3.2.1 H3K36 ........................................................... 25 1.3.2.2 H3K9 ............................................................ 26 1.3.2.3 H3K27 ........................................................... 26 1.3.2.4 H4K20 ........................................................... 27 1.3.3 Demethylation ........................................................ 28 1.4 Histone code ............................................................ 28 1.5 Replicating chromatin .................................................... 30 1.5.1 Disassembly of parental nucleosomes .................................... 30 1.5.2 Relocation of parental histones .......................................... 31 1.5.3 Deposition of newly synthesized histones ................................. 32 1.5.4 Chromatin maturation ................................................. 33 1.6 Objective ................................................................ 35 2. Materials and Methods .................................................... 37 2.1 Materials ................................................................ 38 2.1.1 Technical devices ...................................................... 38 2.1.2 Chemicals and consumables ............................................ 39 2.1.3 Kits and enzymes ..................................................... 41 2.1.4 Plasmids ............................................................. 41 2.1.5 Media ............................................................... 42 2.1.5.1 Media for E. coli ................................................... 42 2.1.5.2 Media for HeLa cells ................................................ 42 9 Table of contents 2.1.6 Antibiotics ........................................................... 43 2.1.7 Antibodies ........................................................... 43 2.1.7.1 Primary antibodies ................................................. 43 2.1.7.2 Secondary antibodies ............................................... 43 2.1.8 E. coli strains ......................................................... 43 2.1.9 DNA and protein markers .............................................. 44 2.1.10 Protease inhibitors ................................................... 44 2.1.11 Mass spectrometry material ........................................... 44 2.1.12 Bioinformatic tool .................................................... 45 2.2 Methods ................................................................ 45 2.2.1 Microbiology methods ................................................. 45 2.2.1.1 Preparation of competent cells ....................................... 45 2.2.1.2 Plasmid transformation ............................................. 46 2.2.1.3 Isolation of Plasmid DNA from E. coli ................................ 46 2.2.2 Nucleic acid methods .................................................. 47 2.2.2.1 Storage of DNA .................................................... 47 2.2.2.2 DNA quantification................................................ 47 2.2.2.3 Agarose gel electrophoresis .......................................... 48 2.2.2.4 Restriction digest .................................................. 48 2.2.2.5 Polymerase Chain Reaction ......................................... 48 2.2.2.6 RT PCR .......................................................... 49 2.2.3 Tissue culture methods ................................................ 49 2.2.3.1 Cultivation of HeLa cells ............................................ 49 2.2.3.2 Harvesting of HeLa cells ............................................ 50 2.2.3.3 Storage of HeLa cells ............................................... 50 2.2.3.4 Synchronization of HeLa cells ........................................ 50 2.2.3.5 SILAC labeling .................................................... 51 2.2.3.6 Flow cytometric analysis of the cell cycle .............................. 51 2.2.4 Protein methods ...................................................... 52 2.2.4.1 Protein quantification .............................................. 52 2.2.4.2 SDS-Polyacrylamid-Gelelectrophoresis ............................... 52 10 Table of contents 2.2.4.3 Coomassie staining ................................................. 53 2.2.4.4 Western blotting ................................................... 53 2.2.4.5 Histone extraction ................................................. 54 2.2.4.6 MALDI-TOF analysis .............................................. 55 2.2.4.7 Quantification of MALDI signals. .................................... 55 2.2.4.8 Tandem MS ....................................................... 56 2.2.4.9 AspN digest ....................................................... 56 2.2.5 Chromatin methods ................................................... 57 2.2.5.1 Preparation of chromatin assembly extract from Drosophila embryos ...... 57 2.2.5.2 Preparation of biotinylated DNA ..................................... 58 2.2.5.3 Chromatin assembly on immobilized DNA ............................ 59 2.2.5.4 Expression and purification of Drosophila histones ...................... 61 2.2.5.5 Octamer reconstitution ............................................. 63 2.2.5.6 Chromatin assembly by salt dialysis .................................. 63 2.2.5.7 HMT assay ........................................................ 64 3. Results and Discussion .................................................... 67 3.1 Results .................................................................. 68 3.1.1 H3K36 methylation during transcription ................................. 69 3.1.2 H4K20 monomethylation during chromatin assembly ...................... 81 3.1.3 Histone modifications during chromatin assembly in vivo ................... 93 3.2 Discussion .............................................................. 103 3.2.1 H3K36 methylation during transcription ................................. 103 3.2.2 H4K20 monomethylation during chromatin assembly ...................... 105 3.2.3 Histone modifications during chromatin assembly in vivo .................. 109 3.2.4 General outlook ....................................................... 113 References .................................................................. 118 Abbreviations ............................................................... 131 Curriculum vitae ............................................................. 135 11 Acknowledgments Acknowledgments First of all, I would like to thank Prof. Dr. Axel Imhof for giving me the opportunity to work on an exciting project and for his continual guidance, support and advice throughout the thesis. His inquisitive nature and extraordinary example as a scientific investigator and mentor are contagious and continuously boosted my motivation for science. I also want to express my gratitude to Prof. Dr. Peter Becker for always having time for me, his advice and constant support and for providing not only a scientifically motivating environment but also a perfect lab atmosphere. I would also like to thank Dr. Sandra Hake and Dr. Ana Villar-Garea for being on my internal thesis committee and for their helpful discussions and guidance of my research projects. I am grateful to all past and present members of the institute for creating a scientifically motivating environment, help whenever needed, discussions and criticism and of course for the fun. Special thanks go to the mass spec team Ana, Christa, Ignasi, Lars, Pierre and Tilman, the HPI team Andreas, Irene and Jochen, the HMT and SMARTER team Jörn, Julia and Simone, the chromatomics team Julian, Teresa and Viola. I wish to acknowledge my collaborators for their excellent experimental contribution and scientific interest, especially Oliver Bell, Dirk Schübeler, Karin Meier, Alexander Brehm, Elisabeth Kremmer, Volker Seitz and Teresa Barth. Heartfelt thanks go to Boehringer Ingelheim Fonds for their