Mechanisms Directing Receptor-Specific Gene Regulation by the Androgen and Glucocorticoid Receptor Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry, Pharmacy of Freie Universität Berlin by Marina Kulik • 2021 The dissertation was prepared under the supervision of Dr. Sebastiaan H. Meijsing at the Max Planck Institute for Molecular Genetics in Berlin from September 2015 to February 2021. 1st Reviewer: Dr. Sebastiaan Meijsing 2nd Reviewer: Prof. Dr. Markus Wahl Date of defense: 21.05.2021 1 Selbstständigkeitserklärung Hiermit bestätige ich, dass ich die vorliegende Arbeit selbstständig und unter Zuhilfenahme der angegebenen Literatur erstellt habe. Acknowledgments First of all, I would like to express my gratitude to my supervisor Sebastiaan Meijsing for his support and guidance during my PhD. I would like to thank Martin Vingron for the opportunity to be part of his research group and for the great collaborations. Especially, I would like to thank Stefan Haas for introducing me to the world of RNA-seq and Gözde Kibar, who contributed with her bioinformatical analyses to this work. I would like to thank Sarah Kinkley for her support and for giving me the opportunity to finish my PhD in her group. I wish to thank Stefan Prekovic, Isabel Mayayo-Peralta and Wilbert Zwart from the NKI in Amsterdam for sharing their expertise in “nuclear signaling” and the great collaboration. I am particularly grateful to Melissa Bothe, her computational analyses and support in the lab contributed a lot to this work. My special gratitude goes to Laura Glaser for her continuous advice and for freezing my cells in the evening countless times. I wish to thank Edda Einfeldt for her great technical support and for always having an open ear. I would like to thank Verena Thormann, Nilofar Badra-Azar, Stefanie Schöne and all the members of the Vingron group for the discussions, life saving coffee breaks and the fun outside the institute. Finally, I would like to thank my parents, my friend Aline Behn, who accompanied me all the time since school years, and my husband Stanislav Kulik for his loving support and eternal optimism. 3 Contents Abstract ................................................................................................................................... 9 Zusammenfassung ............................................................................................................... 11 1. Introduction ................................................................................................................... 13 1.1 Transcriptional Regulation .............................................................................................................. 13 1.2 Influence of the Chromatin Landscape ........................................................................................ 15 1.3 Genomic Enhancers ......................................................................................................................... 17 1.4 Transcription Factors ....................................................................................................................... 21 1.5 Role of Cofactors .............................................................................................................................. 23 1.6 Steroid Hormone Receptors are Ligand-Inducible Transcription Factors .............................. 24 1.7 Steroid Hormone Receptors: Structure and Function ................................................................ 25 1.8 Paralogous Transcription Factors AR and GR ............................................................................ 26 1.9 Aim of the Thesis ............................................................................................................................. 31 2. Materials and Methods .................................................................................................. 33 2.1 General Materials .............................................................................................................................. 33 2.1.1 Cell Lines ........................................................................................................................................ 33 2.1.2 DNA Plasmids ............................................................................................................................... 33 2.1.3 Antibodies ...................................................................................................................................... 34 2.2 Bacteria Transformation, Bacterial Cell Culture and Plasmid Isolation ................................... 35 2.3 Cell Culture ........................................................................................................................................ 36 2.3.1 Transfection of U2OS Cells with Lipofectamine for Stable AR Integration ...................... 36 2.3.2 Transfection of U2OS Cells with Amaxa Nucleofector ......................................................... 37 2.4 Polymerase Chain Reaction (PCR) ................................................................................................ 37 2.5 DNA Sanger Sequencing ................................................................................................................. 38 2.6 mRNA Isolation and cDNA Synthesis ......................................................................................... 38 2.7 Quantitative Real-Time PCR .......................................................................................................... 38 2.8 Polyacrylamide Gel Electrophoresis and Western Blotting ....................................................... 39 2.9 Hormone Binding Assay ................................................................................................................. 40 2.10 Calculation to Determine the Bound Receptor Fraction ......................................................... 41 5 2.11 Chromatin Immunoprecipitation ................................................................................................. 42 2.12 Omni-ATAC-Seq ........................................................................................................................... 44 2.13 CRISPR/Cas9 Genomic Editing ................................................................................................. 48 2.14 STARR-Seq with FAIRE-Library ................................................................................................ 50 2.15 STARR-Seq Analysis for Individual Enhancers ........................................................................ 53 2.16 Rapid immunoprecipitation mass spectrometry of endogenous proteins ............................. 54 2.17 Computational Analysis ................................................................................................................. 55 2.17.1 RNA-Seq ...................................................................................................................................... 55 2.17.2 RNA-Seq Gene-Expression Categories of AR and GR (Venn Diagram) ......................... 55 2.17.3 Heatmaps for Top 50 AR, GR, Shared and Non-Regulated Genes ................................... 56 2.17.4 ChIP-Seq ...................................................................................................................................... 56 2.17.5 Omni-ATAC-Seq ........................................................................................................................ 57 2.17.6 STARR-Seq .................................................................................................................................. 57 2.17.7 Heatmaps and Profile Plots at AR and GR Peaks ................................................................. 57 2.17.8 Intersecting Genomic Binding and Gene Regulation ........................................................... 58 2.17.9 Shared FAIRE-STARR and H3K27ac Peaks near Different Gene Categories ................ 58 2.17.10 Motif Analysis and GC Content of AR and GR-specific Sites ......................................... 59 2.17.11 AR and GR Binding Sites in the AQP3 Enhancer .............................................................. 59 3. Results ............................................................................................................................ 61 3.1. Generation and Characterization of a Cell Line with Stable AR Expression ........................ 61 3.1.1. mRNA and Protein Expression of AR ..................................................................................... 62 3.1.2. Receptor Quantification by Liquid Scintillation Counting .................................................... 63 3.2. Comparing Gene Expression and Genomic Binding of AR and GR Cells ........................... 64 3.2.1. RNA-seq Analysis ........................................................................................................................ 64 3.2.2. ChIP-seq Analysis Reveals Distinct Groups of Binding Sites .............................................. 71 3.2.3. Intersecting Genomic Binding and Gene Regulation ............................................................ 72 3.2.4. Examples of AR-Specific, GR-Specific and Shared Genes ................................................... 74 3.2.5. ChIP-qPCR Validation of Receptor-Specific Binding Peaks ................................................ 75 3.3. Deletion of DNA Binding Sites of Receptor-Specific