A non-conventional nuclear import pathway for circadian clock proteins Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin by Dipl. Biochem. SANDRA KORGE born in Schkeuditz 2016 This thesis was conducted from 2012 to 2016, supervised by Prof. Dr. rer. nat. Achim Kramer, at the Laboratory of Chronobiology, Institute for Medical Immunology, Charité, Berlin 1st Reviewer: Prof. Dr. rer. nat. Achim Kramer, Charité Berlin 2nd Reviewer: Prof. Dr. rer. nat. Florian Heyd, Freie Universität Berlin Date of thesis defense: 18th of November 2016 Contents Contents Contents ......................................................................................................................... v List of Figures ............................................................................................................... ix List of Tables ............................................................................................................... xii List of Appendices ...................................................................................................... xiii Summary ....................................................................................................................... xv Zusammenfassung ..................................................................................................... xvii 1 Introduction ............................................................................................................ 1 1.1 Circadian rhythms ................................................................................................................... 1 1.1.1 Short introduction to the history of Chronobiology ............................................... 1 1.1.2 Properties of circadian rhythms .................................................................................. 2 1.1.3 The mammalian timing system ................................................................................... 3 1.1.4 The molecular clock in non-mammalian species ...................................................... 6 1.1.5 Cellular functions of the circadian clock ................................................................... 9 1.2 Nucleo-cytoplasmic translocation ...................................................................................... 10 1.2.1 The nuclear envelope (NE) ....................................................................................... 10 1.2.2 The global gate in and out of the nucleus ............................................................... 12 1.2.3 Passing through the nuclear pore ............................................................................. 13 1.2.4 Active nucleo-cytoplasmic translocation by karyopherins .................................... 15 1.2.5 Nucleo-cytoplasmic translocation of circadian clock proteins ............................. 21 1.3 Redox homeostasis and oxidative stress ........................................................................... 24 1.3.1 Endogenously generated ROS is essential for normal intracellular homeostasis .......... ……………………………………………………………………………...25 1.3.2 Exogenous stimulation of ROS ................................................................................ 26 1.3.3 Antioxidants and cellular oxidative stress responses ............................................. 27 1.3.4 Cross-talk of the circadian clock and the cellular redox system ........................... 29 1.4 Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/CRISPR- associated (Cas9) genome engineering .............................................................................. 30 1.4.1 Summary of pioneering genome editing tools ........................................................ 30 1.4.2 CRISPR/Cas9 genome engineering ......................................................................... 31 v Contents 1.5 Aim of this study .................................................................................................................. 33 2 Material and Methods ........................................................................................... 35 2.1 Materials ................................................................................................................................. 35 2.1.1 Bacterial strains ............................................................................................................ 35 2.1.2 Human cell lines .......................................................................................................... 35 2.1.3 Self-prepared buffers and media ............................................................................... 36 2.1.4 Commercially available buffers ................................................................................. 38 2.1.5 Composition of cell culture media ........................................................................... 39 2.1.6 Antibiotics .................................................................................................................... 39 2.1.7 Antibodies .................................................................................................................... 39 2.1.8 Reagents........................................................................................................................ 40 2.1.9 DNA and protein standards; loading buffers ......................................................... 42 2.1.10 Enzymes ..................................................................................................................... 42 2.1.11 Pharmacological inhibitors ...................................................................................... 43 2.1.12 Primer and oligonucleotides .................................................................................... 43 2.1.13 Vector backbones ..................................................................................................... 48 2.1.14 Protein and peptide coding sequences (CDS) ...................................................... 49 2.1.15 RNA-interference (RNAi) constructs .................................................................... 50 2.1.16 Commercially available kits ..................................................................................... 51 2.1.17 Equipment and electronic devices .......................................................................... 52 2.1.18 Databases and online tools ...................................................................................... 54 2.1.19 Software ...................................................................................................................... 55 2.1.20 Company register ...................................................................................................... 56 2.2 Methods ................................................................................................................................. 58 2.2.1 Cell culture assays........................................................................................................ 58 2.2.2 Imaging methods......................................................................................................... 60 2.2.3 Protein assays ............................................................................................................... 61 2.2.4 RNA and DNA techniques ....................................................................................... 64 2.2.5 GFP-silencing .............................................................................................................. 72 2.2.6 RNAi-screen ................................................................................................................ 72 2.2.7 Subcellular distribution assay..................................................................................... 73 vi Contents 2.2.8 CRISPR/Cas9 genome engineering ......................................................................... 73 2.2.9 Bioinformatics and image analysis ............................................................................ 74 3 Results ................................................................................................................... 76 3.1 Pharmacological perturbation of nucleo-cytoplasmic translocation alters circadian dynamics ................................................................................................................................. 76 3.2 Multiple components of the nucleo-cytoplasmic translocation machinery are required for normal circadian dynamics ............................................................................................ 79 3.2.1 Knockdown of nucleo-cytoplasmic translocation-associated genes leads to impaired circadian rhythm generation ............................................................................... 79 3.2.2 RNAi-knockdown of tnpo1 transcripts leads to period shortening of circadian oscillations.............................................................................................................................. 85 3.2.3 CRISPR/Cas9 generated tnpo1 knockout cells show a shortened circadian rhythm .................................................................................................................................... 86 3.3 Bioinformatical identification of putative TNPO1-binding sites in circadian core clock proteins .......................................................................................................................