Aus der Klinik für Allgemein-, Viszeral-, Gefäß- und Transplantationschirurgie der Ludwig-Maximilians-Universität München (Direktor: Prof. Dr. med. Jens Werner) The Epigenetic Role of PHF20/MOF Histone Acetyltransferase Complex in Lung Cancer Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Xiaoyan Wang Aus Shandong, China 2017 Mit der Genehmigung der Medizinischen Fakultät der Universität München Berichterstatterin: Prof. Dr. med. Christiane J. Bruns Mitberichterstatter: PD Thomas Düll Prof. Wolfgang Zimmermann Mitbetreuung durch den promovierten Mitarbeiter: Dr. rer. nat Yue Zhao Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 07. 12. 2017 Something attempted, something done. Declaration I hereby declare that the thesis is my original work. The work and data presented in the thesis were performed independently. X-ray crystallography, NMR titration experiments, NMR structure determination, and fluorescence spectroscopy were performed by our collaborator Dr. Brianna J. Klein (Department of Pharmacology, University of Colorado School of Medicine, USA) and Dr. Gaofeng Cui (Department of Biochemistry and Molecular Biology, Mayo Clinic, USA). Parts of the results have been published in the following manuscripts: Klein BJ*, Wang X*, Cui G*, Yuan C, Botuyan MV, Lin K, Lu Y, Wang X, Zhao Y, Bruns CJ, Mer G, Shi X, Kutateladze TG. PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation. Cell Rep. 2016 Oct 18;17(4):1158-1170. No unauthorized data were included. Information from the literature was cited and listed in the reference. All the data presented in the thesis will not be used in any other thesis for scientific degree application. The work for the thesis began from Nov. 2014 with the supervision from Prof. Dr. Xiaobing Shi in UT M.D. Anderson Cancer Center, Houston, United States and Prof. Dr. med. Christiane J. Bruns and Dr Yue Zhao in Germany. 03.2017 Xiaoyan Wang Table of Contents 1 INTRODUCTION ............................................................. 5 1.1 Overview of lung cancer ................................................................ 5 1.1.1 Incidence and mortality of lung cancer .............................................................. 5 1.1.2 Types of lung cancer .......................................................................................... 6 1.1.3 Lung cancer risk factors ..................................................................................... 6 1.1.4 Lung cancer survival rate ................................................................................... 7 1.2 Cancer epigenetics ......................................................................... 7 1.2.1 Epigenetics ......................................................................................................... 7 1.2.2 Cancer epigenetics ............................................................................................. 8 1.3 PHF20 ............................................................................................ 9 1.3.1 PHF20 in human cancers ................................................................................... 9 1.3.2 Epigenetic role of PHF20 ................................................................................. 10 1.4 Aims of this study ......................................................................... 12 2 MATERIAL AND METHODS ...................................... 13 2.1 Materials ...................................................................................... 13 2.1.1 Eukaryotic cell lines ......................................................................................... 13 2.1.2 Bacterial strains ................................................................................................ 14 2.1.3 Plasmids constructs .......................................................................................... 14 2.1.4 shRNA ............................................................................................................. 15 2.1.5 Antibodies ........................................................................................................ 15 2.1.6 Oligos for plasmids cloning and point mutagenesis ........................................ 16 2.1.7 Common chemical reagents ............................................................................. 17 2.1.8 Materials for PCR and quantitative real-time PCR .......................................... 19 2.1.9 Reagents for Western blotting ......................................................................... 21 2.1.10 Materials for ChIP .......................................................................................... 21 2.1.11 Reagents for bacterial culture ........................................................................ 27 2.1.12 Reagents for mammalian cell culture ............................................................ 27 2.2 Methods in molecular biology and biochemistry ........................ 28 2.2.1 Solution stock preparation ............................................................................... 28 2.2.2 Buffer preparation ............................................................................................ 29 1 2.2.3 Plasmid cloning by PCR .................................................................................. 31 2.2.4 Plasmid cloning using Gateway technique ...................................................... 35 2.2.5 PCR mutagenesis ............................................................................................. 36 2.2.6 Protein purification .......................................................................................... 37 2.2.7 Peptide microarray assay ................................................................................. 39 2.2.8 Pull-down assay of biotin-labeled histone peptides .......................................... 39 2.2.9 Western blotting ................................................................................................ 40 2.2.10 Quantitative real-time PCR and RNA-Seq analysis ..................................... 42 2.2.11 Cross-linking chromatin immunoprecipitation (X-ChIP) ............................. 45 2.3 Methods in cell biology ................................................................ 46 2.3.1 Cell culture ....................................................................................................... 46 2.3.2 Protein extraction and concentration measurement ......................................... 46 2.3.3 Lenti-virus packaging ...................................................................................... 47 2.3.4 Virus transduction .............................................................................................. 48 2.4 Methods in structural biology ................................................... 51 2.4.1 X-ray crystallography ...................................................................................... 51 2.4.2 NMR titration experiments .............................................................................. 52 2.4.3 NMR structure determination .......................................................................... 52 2.4.4 Fluorescence spectroscopy ............................................................................... 54 2.5 Online databases and analysis .................................................. 54 2.5.1 GLOBOCAN project and online analysis ........................................................ 54 2.5.2 TCGA database and cBioPortal for Cancer Genomics .................................... 55 2.5.3 Kaplan-Meier plotter analysis .......................................................................... 55 3 RESULTS ......................................................................... 57 3.1 PHF20 is required for cancer cell growth and survival ............. 57 3.1.1 PHF20 gene is amplified in human cancers including lung cancer ................. 57 3.1.2 PHF20 is overexpressed in lung adenocarcinoma cell lines ............................ 59 3.1.3 Depletion of PHF20 inhibits cell proliferation and division in H1792 cells ... 59 3.1.4 Depletion of PHF20 inhibits colony formation in H1792 cells ....................... 61 3.2 PHF20 regulates the expression of genes involved in cancer and cell cycle ......................................................................................................... 62 3.2.1 RNA-seq in PHF20-depleted cells ................................................................... 62 2 3.2.2 Validation of RNA-seq results via qRT-PCR .................................................. 63 3.3 PHF20 recognizes histone H3K4me2 via its PHD finger .......... 64 3.3.1 PHF20 PHD finger binds to H3K4me2 in histone peptide microarray assay .. 64 3.3.2 PHF20 PHD finger specifically recognizes H3K4me2 in histone peptides pull- down assay .................................................................................................................... 65 3.3.3 HSQC titration experiments indicate direct binding between PHF20 PHD finger and H3K4me2 ............................................................................................................... 66 3.3.4 The PHF20 PHD finger binds to the H3K4me2 peptide with highest affinity in fluorescence spectroscopy assay ................................................................................... 67 3.4 Molecular basis for the recognition of H3K4me2 .....................