The effects of disease mutations on the transcription factor Interferon Regulatory Factor 6 A thesis to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Science 2011 Ling-I Su - 0 - Contents Contents ........................................................................................................................... 1 List of Figures .................................................................................................................. 4 List of Tables ................................................................................................................... 7 List of Abbreviations....................................................................................................... 8 Abstract .......................................................................................................................... 12 Declaration ..................................................................................................................... 13 Copyright statement...................................................................................................... 13 Acknowledgements ........................................................................................................ 14 Chapter 1 ................................................................................................ 15 Introduction ................................................................................................................... 15 1. Introduction ............................................................................................................... 16 1.1. Normal development of the lip and palate ........................................................... 17 1.2. Orofacial clefting .................................................................................................... 21 1.2.1. Cleft lip and cleft palate ............................................................................... 21 1.2.2. Syndromic forms of orofacial clefting ......................................................... 21 1.2.3. Non-syndromic forms of orofacial clefting ................................................. 23 1.2.4. Environmental factors .................................................................................. 24 1.2.5. Genetic factors ............................................................................................. 24 1.2.5.1. Polarizing signalling molecules ...................................................... 25 1.2.5.2. Growth factors and their receptors .................................................. 26 1.2.5.3. Cell adhesion molecules .................................................................. 27 1.2.5.4. Extracellular matrix components .................................................... 27 1.2.5.5. Transcription factors ....................................................................... 28 1.3. Van der Woude and Popliteal Pterygium syndromes ......................................... 30 1.4. Interferon Regulatory Factor 6............................................................................. 33 1.4.1. Mutations in IRF6 cause VWS and PPS ..................................................... 33 1.4.2. Expression of Interferon Regulatory Factor 6 in mice ................................ 37 1.4.3. IRF6 function ............................................................................................... 41 1.5. The Interferon Regulatory Factor family ............................................................ 42 1.5.1. Members of the IRF family ......................................................................... 42 1.5.1.1. IRF1 and IRF2 ................................................................................. 44 1.5.1.2. IRF3 and IRF7 ................................................................................. 44 1.5.1.3. IRF4 and IRF8 ................................................................................. 45 1.5.1.4. IRF5 ................................................................................................. 47 1.5.1.5. IRF9 ................................................................................................. 47 1.5.2. Functional domains of IRFs ........................................................................ 48 1.5.3. Post-translational modifications of IRFs ..................................................... 52 1.5.3.1. Phosphorylation ............................................................................... 52 1.5.3.2. Acetylation ...................................................................................... 54 1.5.3.3. Ubiquitination.................................................................................. 54 1.5.4. Subcellular localization of IRFs .................................................................. 55 1.5.4.1 Nuclear localization signals in IRFs ................................................. 55 1.5.4.2. Nuclear export signals in IRFs ........................................................ 56 1.6. Aims and Objectives .............................................................................................. 58 - 1 - Chapter 2 ................................................................................................ 60 Methods and Materials ................................................................................................. 60 2.1. Protein purification of IRF6 proteins ................................................................... 61 2.1.1. Nickel affinity chromatography ................................................................... 61 2.1.2. Heparin sulphate chromatography ............................................................... 62 2.1.3. In vitro translation of proteins ..................................................................... 62 2.1.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis.................... 63 2.2. Electrophoretic mobility shift assay ..................................................................... 63 2.2.1. Generation of double-stranded oligonucleotides ......................................... 63 2.2.2. Radiolabeling oligonucleotides ................................................................... 64 2.2.3. Electrophoretic mobility shift assay ............................................................ 64 2.3. Cloning procedures ................................................................................................ 67 2.3.1. Plasmid construction .................................................................................... 67 2.3.2. Polymerase Chain Reaction ......................................................................... 67 2.3.3. Restriction digestion .................................................................................... 73 2.3.4. Ligation ........................................................................................................ 73 2.3.5. Transformation of Bacteria .......................................................................... 73 2.3.6. Purification of plasmid DNA ....................................................................... 74 2.3.7. Ethanol precipitation of DNA and sequencing ............................................ 75 2.3.8. Site directed mutagenesis ............................................................................ 75 2.4. Transfection Studies............................................................................................... 81 2.4.1. Cell culture .................................................................................................. 81 2.4.2. Transfection ................................................................................................. 81 2.4.3. Luciferase assays ......................................................................................... 81 2.4.4. Statistical analysis ........................................................................................ 82 2.4.5. Nuclei Isolation Assay ................................................................................. 82 2.4.6. Western Blotting .......................................................................................... 82 2.4.7. Immunoprecipitation ................................................................................... 83 2.4.8. Lambda Protein Phosphatase assay ............................................................. 84 2.5. Fluorescent microscopy ......................................................................................... 84 2.5.1. Fluorescent microscopy ............................................................................... 84 2.5.2. Fixation and Mounting ................................................................................ 85 2.6. Mass Spectrometry................................................................................................. 86 2.6.1. Colloidal Coomassie staining ...................................................................... 86 2.6.2. Digestion ...................................................................................................... 86 2.6.3. Mass Spectrometry ...................................................................................... 86 2.6.4. Data Analysis ............................................................................................... 87 Chapter 3 ................................................................................................ 88 3. Introduction ..............................................................................................................