Molecular Functions of Multi-SUMO- Binding Protein Complexes A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 Rotem Salmi-Leshem Division of Molecular and Cellular Function Table of contents Table of contents ........................................................................................................ 2 List of figures .............................................................................................................. 6 List of tables ............................................................................................................... 7 List of supplementary data ......................................................................................... 7 Abbreviations ............................................................................................................. 8 Abstract.................................................................................................................... 11 Declaration ............................................................................................................... 12 Copyright statement ................................................................................................. 12 Acknowledgments .................................................................................................... 13 1 Introduction ...................................................................................................... 14 1.1 Chromatin and gene regulation ............................................................................ 14 1.2 Transcription factors and coregulators .................................................................. 19 1.3 SUMO - Small ubiquitin-like modifier .................................................................... 21 1.3.1 Introduction to SUMO ........................................................................................................... 21 1.3.2 Genome wide SUMOylation .................................................................................................. 22 1.3.3 SUMO Structure and functionality: ....................................................................................... 22 1.3.4 Reversible SUMOylation ........................................................................................................ 25 1.3.5 SUMO consensus binding sites .............................................................................................. 27 1.3.6 SUMO interacting motifs (SIMs) ............................................................................................ 27 1.3.7 Poly-SUMO chains ................................................................................................................. 28 1.3.8 Multi-SUMOylation ............................................................................................................... 29 1.3.9 Consequences of SUMOylation ............................................................................................. 31 1.4 The Five Friends of Methylated CHTOP ................................................................. 32 1.4.1 The 5FMC complex is recruited to multi-SUMOylated targets ............................................. 32 1.4.2 The 5FMC complex members ................................................................................................ 33 1.4.3 5FMC complex members involvement in other complexes .................................................. 34 1.4.4 5FMC complex and transcription regulation ......................................................................... 35 1.5 Aims .................................................................................................................... 38 1.5.1 Aim 1: Identify and characterise proteins recruited to different multi-SUMOylated targets. .. ............................................................................................................................................... 38 1.5.2 Aim 2: How does the 5FMC complex affect transcription? ................................................... 39 1.5.3 Aim 3: What is the interplay between the 5FMC complex and SUMO on chromatin? ......... 39 2 Materials and methods ..................................................................................... 40 2.1 Bacterial cloning methods .................................................................................... 40 2.1.1 Bacterial transformation ....................................................................................................... 40 2.1.2 Glycerol stock preparation .................................................................................................... 40 2.1.3 Plasmid DNA purification ...................................................................................................... 42 2.1.4 Restriction digests ................................................................................................................. 42 2 2.1.5 Ligation reactions .................................................................................................................. 42 2.1.6 Plasmid DNA mutagenesis ..................................................................................................... 42 2.1.7 Plasmid sequencing ............................................................................................................... 44 2.2 Protein expression methods ................................................................................. 44 2.2.1 Bacterial protein expression .................................................................................................. 44 2.2.2 Bacterial protein purification ................................................................................................ 45 2.2.3 In-Vitro protein production ................................................................................................... 45 2.3 Mammalian cell extraction methods ..................................................................... 45 2.3.1 Total cell protein lysates........................................................................................................ 45 2.3.2 Nuclear extracts .................................................................................................................... 45 2.3.3 DNA extraction ...................................................................................................................... 46 2.3.4 RNA extraction ...................................................................................................................... 47 2.4 GST pull down assays ........................................................................................... 47 2.5 Mass-spectrometry .............................................................................................. 47 2.6 Immunoblotting ................................................................................................... 48 2.7 Polymerase chain reaction (PCR) .......................................................................... 50 2.7.1 Amplification of DNA fragments for cloning ......................................................................... 50 2.7.2 Whole plasmid PCR for site directed mutagenesis................................................................ 50 2.7.3 Colony PCR ............................................................................................................................ 51 2.7.4 qPCR ...................................................................................................................................... 51 2.7.5 RT-qPCR ................................................................................................................................. 52 2.8 Immunoprecipitation ........................................................................................... 54 2.9 Chromatin immunoprecipitation .......................................................................... 55 2.9.1 Sonication efficiency test ...................................................................................................... 55 2.9.2 SUMO2/3 ChIP with protein A beads .................................................................................... 55 2.10 RNA-seq ............................................................................................................... 56 2.11 Mammalian cell culture methods ......................................................................... 56 2.11.1 Sub-culturing cell lines ...................................................................................................... 56 2.11.2 Freezing and thawing cells ................................................................................................ 57 2.11.3 Cell transfection ................................................................................................................ 57 2.11.4 EGF stimulation of cells .................................................................................................... 59 2.12 Clustered, regularly interspaced, short palindromic repeats (CRISPR) .................... 59 2.12.1 Construct and homology arm design ................................................................................ 59 2.12.2 Cell culture and nucleofection .......................................................................................... 62 2.12.3 Homologous recombination validation ...........................................................................