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SOX11 Interactome Analysis: Implication in Transcriptional Control and Neurogenesis

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SOX11 Interactome Analysis: Implication in Transcriptional Control and Neurogenesis SOX11 interactome analysis: Implication in transcriptional control and neurogenesis Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Birgit Heim, geb.Kick aus Augsburg Tübingen 2014 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 12.02.2015 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Olaf Rieß 2. Berichterstatter: Prof. Dr. Marius Ueffing Für meine Familie TABLE OF CONTENTS Table of contents Summary ................................................................................................................ 5 Zusammenfassung ............................................................................................... 7 1. Introduction ...................................................................................... 9 1.1. Adult neurogenesis .................................................................................... 9 1.1.1. Adult neural stem cells and neuronal precursor cells ............................ 9 1.1.2. Neurogenic niches .............................................................................. 11 1.1.3. Regulation of adult neurogenesis ........................................................ 12 1.1.3.1. Extrinsic mechanisms .................................................................. 12 1.1.3.2. Intrinsic mechanisms ................................................................... 13 1.1.3.3. Epigenetic regulation ................................................................... 15 1.1.4. Development and functional integration of new-born neurons ............ 16 1.1.5. Function of adult neurogenesis ........................................................... 19 1.1.5.1. SVZ-derived neurons and olfaction .............................................. 19 1.1.5.2. Hippocampal neurogenesis and learning and memory, stress and depression ................................................................................... 19 1.1.6. Influence on CNS disorders ................................................................ 20 1.2. Immature neuronal markers DCX and STMN1 ....................................... 22 1.3. The SOX transcription factors................................................................. 24 1.3.1. Characteristics of SOX proteins .......................................................... 24 1.3.2. Functional implications of SOX proteins in the nervous system .......... 26 1.3.2.1. Functions in the peripheral nervous system ................................. 26 1.3.2.2. Functions in the central nervous system ...................................... 27 1.3.2.2.1. Embryonic neurogenesis .......................................................... 27 1.3.2.2.2. The role of SOXB1 in adult neurogenesis ................................ 31 1.3.2.2.3. The role of SOX11 in adult neurogenesis ................................ 32 1.3.3. Involvement of SOX proteins in reprogramming assays ..................... 34 2. Aim of the study ............................................................................. 37 3. Material and Methods .................................................................... 40 3.1. Material ...................................................................................................... 40 3.1.1. Equipment ........................................................................................... 40 3.1.2. Consumables and Labware ................................................................ 41 3.1.3. Chemicals ........................................................................................... 43 3.1.4. Special reagents ................................................................................. 44 3.1.5. Buffers, Solutions and Media .............................................................. 45 3.1.5.1. Cell culture ................................................................................... 45 3.1.5.2. E.coli culture ................................................................................ 46 3.1.5.3. Nickel-NTA Purification ................................................................ 47 3.1.5.4. Agarose Gels ............................................................................... 47 3.1.5.5. Nuclear extraction and affinity purification.................................... 48 3.1.5.6. SDS-PAGE, Coomassie staining and Western blot analysis ....... 49 3.1.6. Kits ...................................................................................................... 50 3.1.7. Enzymes ............................................................................................. 50 3.1.8. Oligonucleotides .................................................................................. 50 3.1.9. Plasmids ............................................................................................. 52 3.1.10. E.coli strains ........................................................................................ 53 1 TABLE OF CONTENTS 3.1.11. Antibodies ........................................................................................... 53 3.1.12. Mass spectrometry .............................................................................. 56 3.1.13. Software and databases ..................................................................... 56 3.1.13.1. Software ....................................................................................... 56 3.1.13.2. Databases .................................................................................... 56 3.2. Methods .................................................................................................... 57 3.2.1. Cell culture .......................................................................................... 57 3.2.1.1. Maintenance and growth of cells .................................................. 57 3.2.1.2. Cryopreservation and thawing of cells ......................................... 57 3.2.1.3. Transient transfection .................................................................. 57 3.2.1.3.1. Effectene .................................................................................. 57 3.2.1.3.2. PEI ........................................................................................... 58 3.2.1.4. Stable isotope labelling of amino acids in cell culture (SILAC) ..... 58 3.2.1.5. Generation and maintenance of stable expression cell lines ....... 59 3.2.2. Molecular biology ................................................................................ 59 3.2.2.1. Enzymatic DNA treatments .......................................................... 59 3.2.2.1.1. DNA restriction ......................................................................... 59 3.2.2.1.2. A-tailing of DNA fragments ....................................................... 60 3.2.2.1.3. Annealing of DNA fragments .................................................... 60 3.2.2.1.4. Phosphorylation of DNA fragments .......................................... 61 3.2.2.1.5. Ligation .................................................................................... 61 3.2.2.2. E.coli culture and plating .............................................................. 61 3.2.2.3. Transformation and cryoconservation of E.coli ............................ 61 3.2.2.4. DNA isolation from E.coli ............................................................. 62 3.2.2.5. Polymerase chain reactions ......................................................... 63 3.2.2.6. Cloning of plasmid expression vectors ......................................... 64 3.2.2.6.1. Classical cloning ...................................................................... 64 3.2.2.6.2. TOPO cloning........................................................................... 64 3.2.2.6.3. Gateway cloning ....................................................................... 64 3.2.2.7. Induction of protein expression in BL-21 cells .............................. 66 3.2.2.8. Nickel-NTA purification................................................................. 67 3.2.2.9. Dialysis ......................................................................................... 67 3.2.3. Gene expression analysis ................................................................... 68 3.2.3.1. Isolation of total RNA ................................................................... 68 3.2.3.2. Qualitative and quantitative evaluation of RNA ............................ 68 3.2.3.3. cDNA synthesis ............................................................................ 68 3.2.3.4. Quantitative Real-Time PCR ........................................................ 68 3.2.4. Protein chemistry ................................................................................ 70 3.2.4.1. Extraction of nuclear lysates ........................................................ 70 3.2.4.2. Quantification of protein concentration ......................................... 70 3.2.4.3. SDS-polyacrylamide electrophoresis (SDS-PAGE) ..................... 71 3.2.4.4. Coomassie Staining of SDS-gels ................................................. 72 3.2.4.5. Western blot analysis and detection ............................................ 72 3.2.5. Analysis of protein-protein interactions ............................................... 73 3.2.5.1.
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