The Role of CHD1 during Mesenchymal Stem Cell Differentiation 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 Simon Baumgart born in Gießen Göttingen, 2015 Members of the Thesis Committee: Prof. Dr. Steven A. Johnsen (Reviewer) Department of General, Visceral and Pediatric Surgery University of Göttingen Medical School, Göttingen Prof. Dr. Heidi Hahn (Reviewer) Department of Human Genetics University of Göttingen Medical School, Göttingen Prof. Dr. Jürgen Wienands Department of Cellular and Molecular Immunology University of Göttingen Medical School, Göttingen Date of oral examination: 22nd of February, 2016 Affidavit I hereby declare that the PhD thesis entitled “The Role of CHD1 during Mesenchymal Stem Cell Differentiation” has been written independently and with no other sources and aids than quoted. Simon Baumgart December, 2015 Göttingen Table of Contents Table of Contents Abbreviations ............................................................................................................... I List of Figures ........................................................................................................... VII Summary ................................................................................................................... IX 1 Introduction .............................................................................................................. 1 1.1 DNA organization .............................................................................................. 1 1.2 Histone modifications ........................................................................................ 2 1.3 Deciphering the “histone code” .......................................................................... 3 1.4 Histone variants ................................................................................................. 4 1.4.1 Histone variant H3.3 ....................................................................................... 5 1.4.2 Histone variant H2A.Z .................................................................................... 5 1.5 Nucleosome remodeling .................................................................................... 6 1.5.1 Nucleosome sliding ........................................................................................ 6 1.5.2 Ejection and histone replacement/removal ..................................................... 9 1.5.3.1 Histone dynamics in transcription ................................................................ 9 1.5.3.2 Histone dynamics at the TSS .................................................................... 10 1.6 CHD1 .............................................................................................................. 11 1.6.1 The role of CHD1 in yeast and drosophila .................................................... 11 1.6.2 Role of CHD1 in higher eukaryotes .............................................................. 12 1.7 Stem cells and differentiation .......................................................................... 14 1.8 MSC and their differentiation potential ............................................................ 16 1.8.1 Adipocyte differentiation ............................................................................... 16 1.8.2 Osteoblast differentiation .............................................................................. 17 1.8.3 MSC in clinical studies ................................................................................. 18 1.9 Aim of the study ............................................................................................... 20 2 Material .................................................................................................................. 21 2.1 Technical equipment ....................................................................................... 21 2.2 Consumable materials ..................................................................................... 22 2.3 Chemicals........................................................................................................ 23 2.4 Kits and reagents ............................................................................................ 25 2.5 Nucleic acids ................................................................................................... 26 2.5.1 Vectors for viral particle production ........................................................... 26 2.5.2 Oligonucleotides ........................................................................................ 26 2.6. Proteins, enzymes, standards ........................................................................ 28 2.6.1 Molecular weight standards ...................................................................... 28 Table of Contents 2.6.2 Enzymes ................................................................................................... 28 2.6.3 Antibodies ................................................................................................. 29 2.7 Cells ................................................................................................................ 29 2.8 ChIP-seq datasets ........................................................................................... 30 2.9 Software .......................................................................................................... 30 2.10 Buffers and media ......................................................................................... 31 3 Methods ................................................................................................................. 33 3.1 Cell culture ...................................................................................................... 33 3.1.1 Cell culturing .......................................................................................... 33 3.1.2 Adipocyte and osteoblast differentiation .................................................... 33 3.1.3 Reverse transfection ................................................................................. 33 3.1.4 Forward transfection ................................................................................. 34 3.1.5 Generation of stable cell lines by lentiviral infection .................................. 34 3.2 Chemical staining ............................................................................................ 35 3.2.1 Oil Red O staining ..................................................................................... 35 3.2.2 Alkaline phosphatase staining ................................................................... 36 3.3 Ectopic bone formation experiment ................................................................. 36 3.4 Molecular biology ............................................................................................ 36 3.4.1 RNA isolation ............................................................................................ 36 3.4.2 complementary DNA synthesis ................................................................. 37 3.4.3 qPCR reaction ........................................................................................... 37 3.4.4 Chromatin immunoprecipitation ................................................................. 38 3.5 Protein analysis ............................................................................................... 40 3.5.1 Sample preparation ................................................................................... 40 3.5.2 Western blot and immunostaining ............................................................. 40 3.6 Next generation sequencing ............................................................................ 41 3.6.1 Library preparation .................................................................................... 41 3.6.2 RNA-library preparation ............................................................................ 41 3.6.3 DNA-library preparation ............................................................................ 41 3.6.4 Sequencing ............................................................................................... 42 3.7 Bioinformatic processing of sequencing data .................................................. 42 3.7.1 Mapping of ChIP-seq reads to the genome ............................................... 42 3.7.2 Peak calling via MACS2 ............................................................................ 42 3.7.3 RNA-seq analysis ...................................................................................... 42 3.7.4 Normalization and calculation of ChIP-seq binding affinities ..................... 43 3.7.5 Visualization by IGV .................................................................................. 43 Table of Contents 3.7.6 Analysis in R ............................................................................................. 43 3.7.7 Cis-regulatory element annotation system ................................................ 43 3.7.8 DAVID based analysis of RNA-sequencing .............................................. 44 3.7.9 DiffBind and calculation of RNA-Pol II stalling ratios ................................. 44 4 Results ................................................................................................................... 45 4.1 CHD1 depletion impairs MSC