Mechanisms of aging-mediated loss of stem cell potency through changes in niche architecture and chromatin accessibility I n a u g u r a l - D i s s e r t a t i o n zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Janis Koester aus Leverkusen 2020 Berichterstatter: Dr. Sara A. Wickström Prof. Dr. Mirka Uhlirova Prüfungsvorsitzender: Prof. Dr. Ulrich Baumann Tag der mündlichen Prüfung: 10.12.2020 2 3 Table of Contents ABSTRACT ....................................................................................................................................... 7 ZUSAMMENFASSUNG ...................................................................................................................... 8 1. INTRODUCTION ........................................................................................................................... 9 1.1 AGING ...................................................................................................................................... 9 1.1.1 AGING THEORIES AND FACTORS ......................................................................................................... 9 1.1.1.1 Reactive oxygen and nitrogen species damage and the oxidative stress theory of aging ........ 10 1.1.1.2 Mitochondrial theory of aging .................................................................................................. 11 1.1.1.3 Senescence ................................................................................................................................ 11 1.1.1.4 Telomere length ........................................................................................................................ 12 1.1.1.5 Nutrient sensing and metabolism ............................................................................................. 12 1.1.1.6 Protein homeostasis ................................................................................................................. 14 1.1.1.7 Genomic Instability ................................................................................................................... 15 1.1.2 STEM CELL THEORY OF AGING .......................................................................................................... 16 1.1.3 AGING AND THE STEM CELL NICHE .................................................................................................... 17 1.2 CHROMATIN ARCHITECTURE AND EPIGENETIC REGULATION OF GENE EXPRESSION .................................... 19 1.2.1 CHROMATIN ................................................................................................................................. 19 1.2.2 THE HISTONE CODE ....................................................................................................................... 20 1.2.3 PROMOTERS, ENHANCERS AND REGULATION OF TRANSCRIPTION BY RNA POLYMERASE II .......................... 22 1.2.4 BIVALENCY MAINTAINS GENES “READY TO BE TRANSCRIBED” ................................................................. 23 1.2.5 AGING AND EPIGENETICS ................................................................................................................ 25 1.3 MECHANOTRANSDUCTION AND ITS ROLE IN EPIGENETIC - AND STEM CELL -REGULATION ........................... 26 1.4 MAMMALIAN SKIN .................................................................................................................... 29 1.4.1 HAIR FOLLICLE MORPHOGENESIS AND CYCLING ................................................................................... 30 1.4.2 HAIR FOLLICLE STEM CELLS ............................................................................................................. 32 1.4.3 SIGNALING DURING HF CYCLE .......................................................................................................... 34 1.4.4 THE EXTRACELLULAR MATRIX ........................................................................................................... 36 1.4.5 ECM STRUCTURES IN THE DERMIS .................................................................................................... 38 1.4.6 THE SKIN BASEMENT MEMBRANE ..................................................................................................... 39 1.4.7 AGING IN THE SKIN ........................................................................................................................ 39 2 AIM OF THE THESIS .................................................................................................................. 42 3 MATERIALS AND METHODS ..................................................................................................... 43 3.1 CHEMICALS AND REAGENTS ......................................................................................................... 43 3.2 MICE ..................................................................................................................................... 43 3.3 ISOLATION OF PRIMARY EPIDERMAL CELLS ...................................................................................... 43 3.4 HFSC ORGANOIDS AND HYDROGELS .............................................................................................. 44 3.4.1 3C HFSC ORGANOIDS .................................................................................................................... 44 3.4.2 COLLAGEN HYDROGELS ................................................................................................................... 44 3.5 SIRNA KNOCKDOWN AND COLONY FORMING ASSAY ......................................................................... 45 3.6 CHEMICAL TREATMENTS ............................................................................................................. 46 3.7 DEPILATION AND EDU INJECTIONS ................................................................................................ 46 3.8 FLOW CYTOMETRY .................................................................................................................... 46 3.9 IMMUNOFLUORESCENCE AND CONFOCAL MICROSCOPY ..................................................................... 48 4 3.9.1 PARAFFIN SECTIONS ....................................................................................................................... 48 3.9.2 CRYO-SECTIONS ............................................................................................................................ 49 3.9.3 HYDROGELS .................................................................................................................................. 51 3.9.4 EDU STAINING .............................................................................................................................. 51 3.9.5 H&E STAINING ............................................................................................................................. 52 3.9.6 CONFOCAL MICROSCOPY ................................................................................................................ 52 3.9.7 IMAGE ANALYSES ........................................................................................................................... 53 3.10 ATOMIC FORCE MICROSCOPY ..................................................................................................... 53 3.10.1 ATOMIC FORCE INDENTATION SPECTROSCOPY OF HYDROGELS ............................................................. 53 3.10.2 ATOMIC FORCE INDENTATION SPECTROSCOPY OF HAIR FOLLICLE BASEMENT MEMBRANES ........................ 54 3.11 RNA ISOLATION ..................................................................................................................... 54 3.12 RNA SEQUENCING AND ANALYSIS ............................................................................................... 55 3.12.1 RNA-SEQ LIBRARY CONSTRUCTION ................................................................................................. 55 3.12.2 BIOINFORMATIC ANALYSIS ............................................................................................................ 55 3.13 ATAC SEQUENCING AND ANALYSIS ............................................................................................. 56 3.13.1 ATAC-LIBRARY CONSTRUCTION ..................................................................................................... 56 3.13.2 BIOINFORMATIC ANALYSIS ............................................................................................................ 57 3.14 QPCR ................................................................................................................................... 57 3.15 NASCENT RNA ANALYSIS .......................................................................................................... 59 3.16 CHROMATIN IMMUNOPRECIPITATION .......................................................................................... 60 3.17 MASS SPECTROMETRY .............................................................................................................. 63 3.17.1 PROTEIN AND PEPTIDE SAMPLE PREPARATION ................................................................................... 63 3.17.2 TMT LABELING, MASS SPECTROMETRY AND ANALYSIS ........................................................................ 63 3.18 SKIN DECELLULARIZATION ......................................................................................................... 64 3.19 STATISTICS AND REPRODUCIBILITY .............................................................................................