The Tip60 chromatin remodeling complex is required for maintenance and polarity of Drosophila neural stem cells Dissertation for the award of the degree “Doctor rerum naturalium” (Dr. rer. nat.) Division of Mathematics and Natural Sciences of the Georg-August-University Göttingen submitted by Katja Rust from Hanover Göttingen, 2016 Members of the Thesis Committee Prof. Dr. Andreas Wodarz Institute I of Anatomy (Referee and Thesis Supervisor) Department of Molecular Cell Biology University of Cologne Prof. Dr. Tomas Pieler University Medical Center (Co-Referee) Department of Developmental Biochemistry Georg-August-University, Göttingen PD Dr. Halyna Shcherbata Max Planck Institute for Biophysical Chemistry Gene Expression and Signaling Göttingen Further Members of the Examination Board Prof. Dr. Matthias Dobbelstein University Medical Center Institute for Molecular Oncology Georg-August-University, Göttingen Dr. Roland Dosch University Medical Center Deparment of Developmental Biochemistry Georg-August-University, Göttingen Prof. Dr. Ernst Wimmer Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology Department of Developmental Biology Georg-August-University, Göttingen Date of the oral examination: 18th November 2016 Affidavit Affidavit I hereby declare that I prepared the thesis “The Tip60 chromatin remodeling complex is required for maintenance and polarity of Drosophila neural stem cells” on my own and with no other sources and aids than quoted. Katja Rust Göttingen, September 30th, 2016 Table of contents Table of contents Abstract ....................................................................................................................... 1 1. Introduction .......................................................................................................... 3 1.1. Drosophila neurogenesis .................................................................................................. 3 1.1.1. The embryonic epithelium: Parent tissue of the Drosophila neuroblast ..................... 3 1.1.2. Neural stem cells in Drosophila development ............................................................. 6 1.1.3. Neuroblast polarity and asymmetric division .............................................................. 8 1.1.4. Drosophila neuroblasts terminate neurogenesis ....................................................... 12 1.2. Drosophila intestinal stem cells ...................................................................................... 13 1.3. The Tip60 chromatin remodeling complex ..................................................................... 15 1.3.1. Domino: A chromatin remodeler ATPase .................................................................. 19 1.3.2. Specific interactors define Tip60 complex functions ................................................. 23 1.4. The roles of the transcription factor Myc in stem cells .................................................. 30 1.5. Scope of the thesis .......................................................................................................... 32 2. Materials and Methods ....................................................................................... 33 2.1. Materials ......................................................................................................................... 33 2.1.1. Chemicals, enzymes and kits ...................................................................................... 33 2.1.2. Antibodies .................................................................................................................. 33 2.1.3. Fly stocks .................................................................................................................... 34 2.1.4. Oligonucleotides ........................................................................................................ 38 2.1.5. Vectors ....................................................................................................................... 38 2.1.6. Bacterial strains .......................................................................................................... 38 2.1.7. Buffer and reagent recipes......................................................................................... 39 2.1.8. Microscope and imaging systems .............................................................................. 46 2.1.9. Other systems ............................................................................................................ 47 2.1.10. Software ..................................................................................................................... 47 2.2. Methods .......................................................................................................................... 47 2.2.1. Molecular biology methods ....................................................................................... 47 2.2.2. Biochemical methods ................................................................................................. 52 2.2.3. Histology and cell culture ........................................................................................... 54 2.2.4. Fly work .................................................................................................................... 60 2.2.5. Statistical analysis ...................................................................................................... 61 2.2.6. Next generation sequencing ...................................................................................... 62 3. Results ................................................................................................................ 64 3.1. Domino is required for Drosophila embryonic development ......................................... 64 3.1.1. Domino is expressed ubiquitously during oogenesis and embryogenesis ................ 64 3.1.2. Neuroblasts in domino null mutants are misoriented ............................................... 69 3.1.3. Embryonic epithelial morphology is disturbed in domino mutants........................... 72 3.1.4. domino mutation leads to nuclear fragmentation..................................................... 77 3.1.5. domino mutation does not induce apoptosis in the Drosophila embryo .................. 78 3.2. Domino controls Drosophila imaginal disc development ............................................... 80 3.3. Domino and the Tip60 complex regulate larval neuroblast division .............................. 84 3.3.1. Domino is expressed in the larval central brain ......................................................... 84 3.3.2. Domino and the Tip60 complex are required for neural cell lineages ...................... 86 3.3.3. Lack of neuroblasts upon domino knockdown is independent of apoptosis ............ 97 3.3.4. domino knockdown leads to polarity defects in larval neuroblasts .......................... 98 Table of contents 3.3.5. domino deficient larval neuroblasts display features of termination of neurogenesis ............................................................................................................ 101 3.3.6. Neuroblast division and its asymmetry is disturbed upon domino knockdown ...... 106 3.4. Myc interacts with the Tip60 complex to maintain larval neuroblasts ........................ 109 3.4.1. Identification of potential Domino interactors in the maintenance of Drosophila larval neuroblasts ..................................................................................................... 109 3.4.2. myc depleted neuroblasts exhibit polarity defects and resemble neuroblasts undergoing terminal differentiation ........................................................................ 113 3.4.3. Myc interacts genetically with the Tip60 complex in larval neuroblasts ................. 115 3.4.4. Tip60 histone substrates in larval neuroblasts ........................................................ 118 3.5. Overexpression of single components of the Myc/Tip60 network does not maintain neuroblasts ................................................................................................................... 119 3.6. Domino regulates the expression of a large set of target genes .................................. 121 3.6.1. Domino target genes regulate neuroblast fate ........................................................ 127 3.7. Domino maintains adult midgut precursors ................................................................. 128 4. Discussion ......................................................................................................... 131 4.1. Domino acts in the Tip60 complex to maintain Drosophila neural stem cells ............. 131 4.2. The Tip60 complex interacts with Myc to regulate neuroblast self-renewal ............... 133 4.2.1. Myc and the Tip60 complex potentially interact to regulate gene expression in larval neuroblasts ..................................................................................................... 134 4.2.2. The Tip60 complex and Myc preserve neuroblast polarity and inhibit premature differentiation .......................................................................................................... 139 4.2.3. Links between the Myc/Tip60 network and neuroblast division ............................. 146 4.2.4. The Myc/Tip60 pathway: A general key player of stem cell maintenance? ............ 147 4.3. Domino is potentially required for various processes in Drosophila development ..... 149 5. Conclusion