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Molecular Mechanisms of Nucleosome Positioning and DNA Methylation in Chromatin

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Molecular Mechanisms of Nucleosome Positioning and DNA Methylation in Chromatin Molecular mechanisms of nucleosome positioning and DNA methylation in chromatin Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät III - Biologie und vorklinische Medizin der Universität Regensburg durchgeführt am Lehrstuhl für Biochemie III der Universität Regensburg vorgelegt von: ANNA SCHRADER SENSERSTR. 8 81371 MÜNCHEN Abgabedatum: 29. Juli 2009 Die vorliegende Arbeit wurde unter der Betreuung von Prof. Dr. Gernot Längst in der Zeit von Februar 2006 bis Juli 2009 am Institut für Biochemie III der Universität Regensburg erstellt. Prüfungskomitee: Vorsitzender: Prof. Dr. Reinhard Wirth 1. Gutachter: Prof. Dr. Gernot Längst 2. Gutachter: Prof. Dr. Alexander Brehm 3. Gutachter (Prüfer): Prof. Dr. Ralf Wagner Ersatzprüfer: Prof. Dr. Michael Thomm Table of Contents Abbreviations........................................................................................... 1 A. Zusammenfassung ............................................................................... 5 B. Introduction ........................................................................................ 7 I. The Chromatin structure ............................................................................................ 7 1. In General ......................................................................................................................... 7 1.1. The nucleosome - basic packaging unit of chromatin.................................................. 7 1.2. Chromatin higher order structures ............................................................................. 8 1.3. Compartmentation of chromatin within the nucleus................................................. 12 2. Nucleosome assembly ................................................................................................... 14 II. Modifications in chromatin ...................................................................................... 15 1. Histone modifications .................................................................................................... 15 1.1. Posttranslational modifications of histones ............................................................... 15 1.2. Histone variants ........................................................................................................ 17 2. Chromatin dynamics...................................................................................................... 18 2.1. Important chromatin remodeling subfamilies for this study...................................... 23 2.2. Mechanism of and influences on nucleosome mobility.............................................. 26 3. Positioning of nucleosomes on DNA.............................................................................. 29 3.1. Sequence-dependent nucleosome positioning ........................................................... 29 3.2. Nucleosome positioning by chromatin remodeling enzymes ...................................... 34 3.3. Additional factors influencing nucleosome positioning.............................................. 36 4. DNA methylation ............................................................................................................ 37 4.1. DNA methylation – Enzymes and mechanism ........................................................... 37 4.2. DNA methylation in mammals................................................................................... 41 4.3. Mammalian DNA methyltransferases ........................................................................ 46 III. Interplay between Chromatin remodeling and DNA methylation ........................... 57 1. In vitro studies on DNA methylation in chromatin ....................................................... 58 2. In vivo studies on DNA methylation in chromatin........................................................ 59 C. Objectives ......................................................................................... 62 1. Nucleosome positioning by chromatin remodeling enzymes....................................... 62 2. Maintenance methylation in the context of chromatin ................................................. 62 D. Material and methods........................................................................ 63 I. Material sources ........................................................................................................ 63 1. Laboratory chemicals and biochemicals....................................................................... 63 2. Enzymes ......................................................................................................................... 64 3. Buffers and solutions .................................................................................................... 64 4. Kits.................................................................................................................................. 65 5. Radioactive material ...................................................................................................... 66 6. Medias ............................................................................................................................ 66 7. Antibodies....................................................................................................................... 66 8. Eukaryotic tissue culture cell lines ............................................................................... 67 9. Bacteria........................................................................................................................... 67 10. DNA-constructs ............................................................................................................ 67 11. Oligonucleotides...........................................................................................................68 12. Fluorescence labeled Oligonucleotides ...................................................................... 71 13. Recombinant Baculoviruses for Sf9 or Sf21 cells...................................................... 72 14. Drosophila melanogaster: maintenance, embryo collection and extracts ................ 72 15. Chromatographic material........................................................................................... 72 16. Blotting material...........................................................................................................72 17. Instruments .................................................................................................................. 73 18. Free software and online tools.................................................................................... 75 II. Methods.................................................................................................................... 76 1. Biochemical methods (DNA-specific methods) ............................................................. 76 1.1. Standard procedures................................................................................................. 76 1.2. Determination of DNA concentration......................................................................... 76 1.3. Analysis of DNA quality and quantity........................................................................ 76 1.4. Hybridization of Oligonucleotides .............................................................................. 77 1.5. Radioactive and fluorescent labeling of DNA ............................................................. 77 1.6. Precipitation and isolation of radioactive DNA fragments .......................................... 77 1.7. Generation and analysis of hemimethylated and methylated DNA ............................ 78 1.8. Preparation of DNA fragments for the assembly of mononucleosomes ...................... 80 2. Molecularbiological methods (Protein-specific methods).............................................. 84 2.1. Standard procedures in protein analysis................................................................... 84 2.2. Protein quantification................................................................................................ 84 2.3. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ................................................ 84 2.4. Coomassie blue staining of protein gels .................................................................... 85 2.5. Semi-dry Western Blot .............................................................................................. 85 3. Isolation of chromatin remodeling complexes and the DNA methyltransferase Dnmt1............................................................................... 86 3.1. Expression of recombinant proteins with the baculovirus system............................. 86 3.2. Purification of recombinant proteins using affinity chromatography......................... 88 4. Chromatin – Assembly and analysis of arrays ........................................................... 90 4.1. Chromatin reconstitution using the salt gradient dialysis technique ........................ 90 4.2. Chromatin assembly using the Drosophila embryo extract (DREX) ........................... 92 4.3. Chromatin analysis by Micrococcal Nuclease (MNase) digest .................................... 92 5. Chromatin – Preparation of positioned mononucleosomes.......................................... 93 5.1. Assembly of mononucleosomes using HP-Mix ........................................................... 94 5.2. Isolation of positioned
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