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The Chromatin Architectural Protein DEK: Assessing Its Chromatin Binding Properties

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The Chromatin Architectural Protein DEK: Assessing Its Chromatin Binding Properties The chromatin architectural protein DEK: assessing its chromatin binding properties Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation Vorgelegt von Master of Science Haihong Guo aus Shanxi, China Berichter: Universitätsprofessor Dr. rer. nat. Bernhard Lüscher Universitätsprofessor Dr. rer. nat. Ralph Panstruga Tag der mündlichen Prüfung: 08.03.2016 Dieses Dissertation is auf den Internetseiten der Hochschulbibliothek online verfügbar Publications: Parts of this thesis will be submitted for publication: 1) Guo, H., Prell, M., Königs, H., Waldmann, T., Meister, M., Becker, C., Hermans-Sachweh, B. & Kappes, F. “Interrogating protein function via bacterial growth”. Manuscript in preparation and attached to this thesis (see 7.3 in Appendix). Additional results, not explicitly mentioned in this work but obtained during my doctoral studies, will be also submitted for publication: 2) Smith, E. A., Krumelbeck, E. F., Gole, B., Willis, N., Adams, A., Matrka, M. C. Jegga, A. G., Guo, H., Meetei, A. R., Andreassen P. R., Kappes, F., Scully R., Wiesmüller, L. and Wells, S. I. “Loss of the DEK oncogene disrupts homologous recombination by destabilizing RAD51 filament formation”. Manuscript in preparation. 3) Prell, M., Preisinger, C., Markovitz, D., Guo, H., Schilling, N., and Kappes, F. "Novel chromatin modulatory functions for the TREX component THOC 4 (ALY/Ref)". Manuscript in preparation. 4) Prell, M., Guo, H., Preisinger, C., Ostareck, D., Ostareck-Lederer, A., Naarmann-de Vries, I., and Kappes, F. "Proteomic Interrogation reveals wide-spread functions of the DEK oncogene". Manuscript in preparation. 5) Prell, M., Preisinger, C., Guo, H., Jonak, C., Waidmann, S., and Kappes, F. "The DEK oncogene regulates alternative splicing through selective interaction with SRPK1 and SRPK2". Manuscript in preparation. Poster presentations: Guo, H., and Kappes, F. (2013). A rapid technique to identify and mutate DNA binding domains in proteins using bacterial toxicity as read-out. 44th Annual Conference of the German Genetics Society (GfG): Genetics 2013 (Braunschweig, Germany). Guo, H., Prell, M., Königs, H., Hermanners-Sachweh, B., and Kappes, F. (2014). Studying DNA-binding features of chromatin architectural factors via bacterial growth. Danube Scientific Conference on Epigenetics (2014) (Budapest, Hungary). Acknowledgements I would like to thank my supervisor, Ferdinand Kappes, for giving me the opportunity to work in his group and to work on this amazing project. You have taught me a lot during the last four years and your guidance helped me to develop skill not only for scientific research but also for living in Germany. This was my best experience to be your student. It would not have been possible to complete this work without you. My great gratitude is also to Prof. Dr. Bernhard Lüscher for being my official supervisor and providing a lot of excellent and critical comments. I would like to thank Prof. Dr. Ralph Panstruga for being my “Zweitgutachter”. Many thanks to my colleagues in Lab 15! Thank you, Malte, for being a good partner in the DEK group and always helping and encouraging me. I would like to thank Dr. Jörg Hartkamp for providing a lot of precious advice on my project. Christiane Becker, thank you for managing the excellent scientific environment in the lab, and especially thanks for taking the immunofluorescence photos in my thesis. Many thanks go to every other member in Lab 15, including Fabian, Mathias, Nahleen, Tim, Irina, Sandra, Elke, Jacky and Tobias. I am so delighted and privileged to have worked with you every day. Special thanks to Dr. Juliana Lüscher-Firzlaff and Prof. Dr. Gerhard Mueller-Newen for providing me with a lot of vectors for my work and Hildegard Schmitz-van-de-Leur for helping with cloning problems. And also to my colleagues in the Lüscher group, including Barbara, Carolina, Jörg, Marc, Mareike, Weili, Laura and Patricia, thank you for providing a scientific environment in the lab and the help in the lab and for helping me in the hot lab. Thanks to Hiltrud Königs for taking the EM photos in this thesis. Thanks to Jinyu for helping to edit the photos of protein secondary structure. I also would like to thank other members in the institute. Thanks to Allison for correcting my thesis and always encouraging me. And also Carolin, Susana, Thomas, Willy, Natalie and Marcel, thank you everybody for giving me a lot of help during the last four years and helping me feel happy and welcome in this big team. I am grateful to the China Scholarship Council (CSC) and to START for supporting me the last four years. 最后，我特别感谢我的父母。你们永远是我坚实的后盾，在背后默默地支持我，在我疲 惫，迷茫的时候给我靠岸的港湾。焉得谖草，言树之背，养育之恩，无以回报，你们永 远健康快乐是我最大的心愿。感谢我的姐姐，妹妹还有弟弟，祝你们天天开心，工作顺 利。另外，我也要感谢男朋友黄旭, 在认识的两年中，你给予我很多工作和生活上的关 心和鼓励，我非常的感激。在以后相伴的日子里，希望我们能够互相鼓励互相扶持，也 祝愿你能早日完成课题，顺利毕业。还有我最亲爱的朋友王晓宇同学，从硕士到博士， 从北京到德国，这六年，我们一起努力奋斗，相互鼓励,相互安慰。你就像是我的亲人， 希望你身体健康，早日博士毕业，也祝我们的友情地久天长。感谢杨建花同学，很开心 认识你并且成为你的室友，非常感谢你对我研究课题提出的很宝贵的建议。在论文完成 之际，也特别感谢所有亚琛的朋友们，非常开心能够在德国认识你们，让我在异国他乡 感受到亲人和朋友的温暖，和你们的相处丰富了我的生活，让我不再感到孤单和害怕， 你们的鼓励和支持让我变的更加勇敢，在这里请接受我最真挚的谢意！ Table of Contents I Table of Contents Table of Contents ................................................................................................................................... I List of Figures ...................................................................................................................................... IV List of Tables........................................................................................................................................ VI Abstract ............................................................................................................................................... VII Zusammenfassung ................................................................................................................................ IX 1. Introduction .................................................................................................................................... 1 1.1 Chromatin .................................................................................................................................... 1 1.2 Modifications of chromatin .......................................................................................................... 3 1.2.1 DNA methylation ................................................................................................................. 4 1.2.2 Histone modifications .......................................................................................................... 4 1.2.3 Histone variants ................................................................................................................... 5 1.2.4 ATP-dependent chromatin remodelers................................................................................. 6 1.2.5 Non-histone chromatin architectural proteins ...................................................................... 7 1.3 Protein motifs for DNA binding ................................................................................................. 12 1.3.1 Helix-turn-helix (HTH) motifs .......................................................................................... 13 1.3.2 Zinc finger motifs .............................................................................................................. 13 1.3.3 Leucine zipper ................................................................................................................... 13 1.3.4 Helix-loop-helix (HLH) ..................................................................................................... 14 1.3.5 HMG-box motifs ............................................................................................................... 14 1.3.6 SAP motif .......................................................................................................................... 15 1.4 The DEK protein ........................................................................................................................ 17 1.4.1 Identification of DEK and its association with human diseases ........................................ 17 1.4.2 Structure and molecular functions of DEK ........................................................................ 18 1.4.3 Cellular functions of DEK ................................................................................................. 20 2. Aim of this Thesis ....................................................................................................................... 25 3. Materials and Methods ............................................................................................................... 26 3.1 Materials .................................................................................................................................... 26 3.1.1 Chemicals .......................................................................................................................... 26 3.1.2 General buffers .................................................................................................................. 28 3.1.3 Oligonucleotides ................................................................................................................ 29 3.1.4 Plasmids ............................................................................................................................. 31 3.1.5 Vectors
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