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Spatial Protein Interaction Networks of the Intrinsically Disordered Transcription Factor C(%3$

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Spatial Protein Interaction Networks of the Intrinsically Disordered Transcription Factor C(%3$ Spatial protein interaction networks of the intrinsically disordered transcription factor C(%3$ Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie/Molekularbiologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin Von Evelyn Ramberger, M.Sc. Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing.Dr. Sabine Kunst Dekan der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm Gutachter: 1. Prof. Dr. Achim Leutz 2. Prof. Dr. Matthias Selbach 3. Prof. Dr. Gunnar Dittmar Tag der mündlichen Prüfung: 12.8.2020 For T. Table of Contents Selbstständigkeitserklärung ....................................................................................1 List of Figures ............................................................................................................2 List of Tables ..............................................................................................................3 Abbreviations .............................................................................................................4 Zusammenfassung ....................................................................................................6 Summary ....................................................................................................................7 1. Introduction ............................................................................................................8 1.1. Disordered proteins .........................................................................................8 1.1.2. Functions of disordered proteins ................................................................10 1.1.3. Protein interactions mediated by disordered regions .................................10 1.2. C/EBP transcription factors ...........................................................................13 1.2.1. Structure of C/EBPs ...................................................................................13 1.2.2. Biological role of C/EBPα ...........................................................................15 1.3. Studying protein-protein interactions with mass spectrometry ......................17 1.3.1. Mass spectrometry based proteomics ........................................................17 1.3.2. Protein interaction studies ..........................................................................19 1.3.2.1. AP-MS .....................................................................................................20 1.3.2.2. Proximity labelling ...................................................................................20 1.3.2.3. Peptide-protein pull-downs ......................................................................21 1.4. Aim of this study ............................................................................................23 2. Materials and Methods ........................................................................................24 2.1. Cell culture ....................................................................................................24 2.2. SILAC labelling of NB4 cells ..........................................................................24 2.3. NB4 differentiation .........................................................................................24 2.4. Surface marker staining and FACS analysis .................................................24 2.5. Wright Giemsa staining .................................................................................25 2.6. RNA extraction ..............................................................................................25 2.7. Microarrays ....................................................................................................25 2.8. Whole cell protein extract preparation ...........................................................26 2.9. Nuclear extract preparation ...........................................................................26 2.10. Determination of protein concentration with a Bradford assay ....................27 2.11. Western blotting ...........................................................................................27 2.12. Stable NB4 cell lines ...................................................................................28 2.13. Protein Interaction Screen on a peptide Matrix ...........................................28 2.14. BioID experiments .......................................................................................33 2.15. On bead digestion .......................................................................................33 2.16. In solution digestion .....................................................................................34 2.17. Desalting with STAGE tips ...........................................................................34 2.18. LC-MS/MS ...................................................................................................34 2.19. Targeted MS analysis of R142 methylation .................................................35 2.20. MS raw data processing with MaxQuant .....................................................36 2.21. Data analysis of mass spec data .................................................................37 2.21.1 PRISMA data .............................................................................................37 2.21.2 BioID data ..................................................................................................37 2.22. Gene ontology (GO) term and analysis .......................................................38 2.23. Single sample gene set enrichment analysis (ssGSEA) .............................38 3. Results.................................................................................................................. 39 3.1. Establishment of the myeloid NB4 cell line as a model system ....................39 3.1.1. Differentiation of NB4 cells can be induced with ATRA and TPA ................39 3.1.2. Proteomic and transcriptomic changes during NB4 differentiation .............41 3.1.3. Expression of C/EBPα-BioID in NB4 cells induced target genes but did not induce terminal differentiation ...............................................................................45 3.1.4. Nuclear extract preparation from NB4 cells ................................................47 3.2. Identification C/EBPα PTM sites ...................................................................47 3.3. C/EBPα interactome studies .........................................................................51 3.3.1. C/EBPα PRISMA screen ............................................................................51 3.3.1.1. PRISMA binding profiles of known C/EBPα interactors ...........................55 3.3.1.2. Differential PRISMA binding profile of TPA/ATRA/control treated cells ...58 3.3.1.3. PTMs modulate peptide-protein interactions in PRISMA ........................60 3.3.2. C/EBPα BioID .............................................................................................62 3.3.3. Overlap of C/EBPα PRISMA and BioID ......................................................65 3.3.3.1. C/EBPα methylation in CR1L enhances interaction with the SWI/SNF complex ................................................................................................................68 3.3.4. Comparison of interactome data from C/EBPα and C/EBPβ PRISMA screens .................................................................................................................70 3.3.5. The isoform-specific C/EBPα interactome ..................................................72 3.4. Gene expression induced by expression of P30- and P42-C/EBPα in NB4 cells ......................................................................................................................75 4. Discussion ...........................................................................................................77 4.1. NB4 cells as a model system for myeloid differentiation and C/EBPα PPI studies ..................................................................................................................77 4.2. Post-translational modifications of C/EBPα ...................................................78 4.3. C/EBPα protein interactions ..........................................................................80 4.3.1. C/EBPα PRISMA screen ............................................................................80 4.3.2. Validation of PRISMA with BioID ................................................................82 4.3.3. Functional roles of conserved C/EBPα regions ..........................................84 4.3.4. C/EBPα isoform-specific interactions .........................................................85 4.3.4.1. P42-C/EBPα specific interactors .............................................................85 4.3.4.2. P30-C/EBPα specific interactors .............................................................86 4.4. Conclusion and outlook .................................................................................88 5. References ...........................................................................................................89 Supplementary Figures and Tables .....................................................................102 Talks and Posters ..................................................................................................110
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