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The Role of the Ubiquitin-Proteasome System in the Regulation of Nuclear Hormone Receptor-Dependent Transcription Dissertation

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The Role of the Ubiquitin-Proteasome System in the Regulation of Nuclear Hormone Receptor-Dependent Transcription Dissertation The Role of the Ubiquitin-Proteasome System in the Regulation of Nuclear Hormone Receptor-Dependent Transcription Dissertation for the award of the degree “Doctor rerum naturalium (Dr. rer. nat.)“ Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Tanja Prenzel born in Königs Wusterhausen Göttingen, 2010 Members of the Thesis Committee: Prof. Dr. Steven A. Johnsen (Reviewer) Molecular Oncology, University of Göttingen Medical School, Göttingen Prof. Dr. Holger Reichardt (Reviewer) Cellular and Molecular Immunology, University of Göttingen Medical School, Göttingen Dr. Tobias Pukrop Hematology/Oncology, University of Göttingen Medical School, Göttingen Date of the oral examination: 22th October 2010 Affidavit I hereby declare that the PhD thesis entitled “The role of the ubiquitin-proteasome system in the regulation of nuclear hormone receptor-dependent transcription” has been written independently and with no other sources and aids than quoted. _____________________________ Tanja Prenzel September, 2010 Göttingen Table of Contents Table of Contents Abbreviations ............................................................................................................................. I List of Figures .......................................................................................................................... V Summary ............................................................................................................................... VII Zusammenfassung ............................................................................................................... VIII 1 Introduction ...................................................................................................................... 1 1.1 Nuclear receptors ......................................................................................................... 1 1.2 Nuclear hormone receptor family ................................................................................ 1 1.3 Estrogen and estrogen receptor .................................................................................... 2 1.4 Glucocorticoid and glucocorticoid receptor................................................................. 4 1.5 Post-translational modifications of steroid receptors ................................................... 5 1.5.1 Post-translational modifications of ERα ............................................................... 5 1.5.2 Post-translational modifications of GR ................................................................ 7 1.6 Nuclear hormone receptors and disease ....................................................................... 8 1.6.1 The role of estrogen receptor in breast cancer ...................................................... 8 1.7 Mechanisms of NHR-mediated transcriptional regulation ........................................ 10 1.7.1 “Classical”, cyclic transcriptional regulation through estrogen responsive elements ............................................................................. 10 1.7.2 “Tethered”, indirect transactivation through protein-protein interactions.......... 11 1.7.3 “Non-genomic activity” of ERα ......................................................................... 11 1.7.4 Mechanisms of GR-regulated transcription ........................................................ 12 1.8 Three-dimensional structural organization of genomes ............................................. 13 1.8.1 Nuclear architecture ............................................................................................ 13 1.8.2 ERα-bound chromatin network .......................................................................... 15 1.9 Ubiquitin-proteasome system .................................................................................... 16 1.9.1 Ubiquitin ............................................................................................................. 16 1.9.2 Proteasome ......................................................................................................... 16 1.9.2.1 19S regulatory particle ................................................................................ 17 1.9.2.2 20S core particle .......................................................................................... 17 1.9.3 Proteasomal protein degradation ........................................................................ 18 1.9.4 The UPS as a potential chemotherapeutic target ................................................ 19 1.9.5 Proteasome inhibitors ......................................................................................... 20 1.9.6 Involvement of the UPS in NHR-regulated gene transcription .......................... 21 1.9.6.1 The UPS in ERα-mediated gene transcription ............................................ 22 1.9.6.2 The UPS in GR-mediated gene transcription .............................................. 23 1.10 Aims of study ............................................................................................................. 24 2 Material ........................................................................................................................... 25 2.1 Technical equipment .................................................................................................. 25 2.2 Consumable materials ................................................................................................ 26 2.3 Chemicals ................................................................................................................... 27 2.4 Kits and reagents ........................................................................................................ 29 2.5 Nucleic acids .............................................................................................................. 29 Table of Contents 2.5.1 Vectors and expression constructs ...................................................................... 29 2.5.2 Oligonucleotides ................................................................................................. 30 2.5.2.1 siRNA oligonucleotides .............................................................................. 30 2.5.2.2 RT-PCR primers .......................................................................................... 30 2.5.2.3 qPCR primers .............................................................................................. 30 2.5.2.4 ChIP primers ............................................................................................... 31 2.5.2.5 3C primers ................................................................................................... 32 2.5.2.6 TaqMan probes ............................................................................................ 32 2.6 Proteins ...................................................................................................................... 33 2.6.1 Molecular weight standards ................................................................................ 33 2.6.2 Enzymes ............................................................................................................. 33 2.6.3 Antibodies ........................................................................................................... 33 2.6.3.1 Primary antibodies....................................................................................... 33 2.6.3.2 Secondary antibodies................................................................................... 34 2.7 Cells ........................................................................................................................... 34 2.7.1 Bacteria cells....................................................................................................... 34 2.7.1.1 BAC clones ................................................................................................. 34 2.7.2 Eukaryotic cells .................................................................................................. 34 2.8 Buffers and media ...................................................................................................... 34 2.9 Software ..................................................................................................................... 36 3 Methods ........................................................................................................................... 37 3.1 Cell culture ................................................................................................................. 37 3.1.1 Cell culture of adherent cells .............................................................................. 37 3.1.2 Liposome-mediated plasmid transfection ........................................................... 37 3.1.3 Reverse-transfection with siRNA ....................................................................... 38 3.1.4 Colony formation assay ...................................................................................... 38 3.1.5 Measurement of DNA of single cells by flow cytometry ................................... 38 3.1.6 Apoptosis assay .................................................................................................. 39 3.1.7 Fluorescence recovery after photobleaching (FRAP) ........................................ 39 3.2 Molecular biology ...................................................................................................... 40 3.2.1
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