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Gene Regulation During Stress Response Transcription In DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR CHEMIE UND PHARMAZIE DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN GENE REGULATION DURING STRESS RESPONSE TRANSCRIPTION IN SACCHAROMYCES CEREVISIAE: DYNAMIC TRANSCRIPTOME ANALYSIS OF OSMOTIC STRESS RESPONSE AND MEDIATOR PHOSPHORYLATION Christian Miller aus Füssen, Deutschland 2013 EIDESSTATTILCHE VERSICHERUNG ERKLÄRUNG Diese Dissertation wurde im Sinne von §7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Patrick Cramer betreut. EIDESSTATTLICHE VERSICHERUNG Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, am 29.01.2013 Christian Miller Dissertation eingereicht am: 29.01.2013 1. Gutachter: Prof. Dr. Patrick Cramer 2. Gutachter: PD Dr. Dietmar Martin Mündliche Prüfung am: 27.02.2013 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I was growing up in front of the Bavarian alps, and I learned that it takes a few but essential things to reach the summit. First, you have to be part of a team that is strong and motivated enough to accept the challenge. Second, it’s up to you to put in the efford of going upwards. Third, there is no perfect time for attacking the summit, but there are perfect opportunities. In 2007, I became part of the Cramer-team. First of all I would like to thank my supervisor Prof. Dr. Patrick Cramer, the leader of the rope-team, for giving me the opportunity to join his group of young and extraordinary motivated scientists. Patrick, you gave me the freedom to improve my skills and to bring forward new ideas for my challenging projects. You provided uncomplicated help whenever it was required – no matter if it was about scientific work or during my two-year training program in major incident management. Special thank goes to Jesper Olsen and Heidi Feldmann for being members of my PhD advisory board. You gave valuable feedback and supported my scientific work with helpful advices. I would like to thank Francesc Posas and Eulalia de Nadal for answering many questions about the HOG pathway. My trip to the Parc de Recerca Biomédica de Barcelona was one of the highlights. I am also thankful to Matthias Mann for his collaboration and for his feedback to my work on Mediator phosphorylation. Special thank goes to Ivan Matic, who performed all mass spectrometry analysis of my Mediator project and for all the lively discussions. Kerstin, thank you for being such a great collegue. Thank you for support and your extraordernary help throughout the experimental work on dynamic transcriptome analysis and the Mediator phosphorylation. I am also very thankful to Dietmar, for your earnest interest in my experimental ideas and for giving me valuable feedback. I would like to thank Achim (Tresch) and Björn (Schwalb) for taking their time for many discussions, feedbacks and the collaborative work that result in the development of DTA. Since I was part of a strong and motivated rope-team, I would like to thank my teammates. Elmar, for working side by side throughout my lab time, for talking about attitudes, economics, politics, philosophy, all the stuff beyond science and for the many special moments inside and outside the lab as we had so much fun. Rieke, for sharing the deep passion for sports, for all the very welcome discussions during our coffee breaks and for illustrating the contrasts of urban- and country life, which nowhere else could be larger than between Essen and Kirchthal. Tobias for all the help with the Äkta systems, the discussions of cloning problems and nice moments at the barbecue and wine-tasting. Laurent, for your contagious enthusiasm on science issues and your open minded way of problem-solving. Martin for all the helpful discussions about transcription and the Mediator and for performing the in vitro transcription assay. Stefan (Benkert) and Claudia (Buchen) for managing the daily lab routine and for being a source of continuity. Stefan (Dengl) and Stefan (Jennebach) for talking about all the little but serious issues of lab-life. Jasmin for sharing the long days in the lab and for the work on the Pol II in vitro elongation assay. Andreas for all the discussions about high-throughput data and the nice moments during the Harvard young scientist´s forum (…hoping to meet you at Paulaner`s). Claudia (Blattner) for the very welcome interruptions of the daily routine and the varied topics during our coffee-breaks. - I - ACKNOWLEDGEMENTS Lieber Opa Franz, Danke für Deine fortwährende Unterstützung, für das anhaltende Interesse an meiner Arbeit und für Deine Ratschläge, die meine Sicht auf viele Dinge verändert hat. Leider konntest Du den Abschluss dieser Arbeit nicht mehr miterleben. Liebe Oma Elisabeth, Lieber Werner, Danke für Eure große Hilfsbereitschaft und dass Ihr mir so manches ermöglicht habt. Liebe Birgit, lieber Fredi, liebe Margit, Anna und Sophie, ihr habt mir immer großen Rückhalt gegeben und wart zu jeder Zeit für mich da. Danke dafür. Liebe Eva, vielen Dank für Deine so große Unterstützung und Deinen starken Rückhalt während der nun zurückliegenden schwierigen Zeit. Danke für Dein Verständnis. Danke für die vielen schönen Momente, Erlebnisse, Erinnerungen. Ich freue mich so auf unsere Zukunft – Du und ich!. Liebe Mama, lieber Papa, Danke für Eure außerordentlich große Unterstützung. Eure vorbehaltlose Hilfe, Euer Rückhalt und Euer tiefes Vertrauen haben dies alles erst ermöglicht. - II - PUBLICATIONS PUBLICATIONS Parts of this work have been published: Christian Miller*, Björn Schwalb*, Kerstin Maier*, Daniel Schulz, Sebastian Dümcke, Benedikt 2011 Zacher, Andreas Mayer, Jasmin Sydow, Lisa Marcinowski, Lars Dölken, Dietmar E. Martin, Achim Tresch and Patrick Cramer *contributed equally Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast.1,2 Molecular Systems Biology, 7:458, doi:10.1038/msb.2010.112 Author contributions: CM and KM conducted DTA experiments, BS, SD, BZ, and AT developed the statistical workflow and carried out computational analysis, DS and AM conducted ChIP experiments, JS conducted enzymatic assays, LM, LD, and DEM conducted initial experiments, LD and PC initiated the study, DEM supervised experimental work, and AT and PC supervised research and wrote the manuscript. Christian Miller*, Ivan Matic*, Kerstin C. Maier*, Susanne Roether, Björn Schwalb, Katja 2012 Sträßer, Achim Tresch, Matthias Mann, and Patrick Cramer *contributed equally Mediator phosphorylation prevents stress response transcription during non stress conditions. Journal of biological chemistry, 2012, 287(53), 44017-26 Author contributions: CM designed and conducted the SILAC & DTA experiments and final data analysis. CM and PC wrote the manuscript. IM conducted the phospho-proteomics data processing and analysis. KM performed the DTA experiments and cloned the D7P and D30P strains. SR and KS created the strain for phospho-proteomics. BS and AT carried out the computational analysis of DTA/cDTA data. MM and PC supervised the projects. 1 The Molecular Systems Biology journal has published a top ten chart of the articles that have been downloaded most often over the previous 30 days. This article reached the top rank of the top ten charts within 13 days and reached the 6th rank of the of the top 10 most read research articles published in Molecular Systems Biology in 2011. 2 Overview to this work has been published in Cell: Szewczak, L. & Plosky, B. (2011) Leading Edge Select, Cell, 144, 831-832; - III - SUMMARY SUMMARY TOPIC I: DYNAMIC TRANSCRIPTOME ANALYSIS MEASURES RATES OF MRNA SYNTHESIS AND DECAY IN YEAST To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress- induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems. TOPIC II: MEDIATOR PHOSPHORYLATION PREVENTS STRESS RESPONSE TRANSCRIPTION DURING NON STRESS CONDITIONS The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an endpoint of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast S. cerevisiae is phosphorylated at multiple sites a 17 out of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of
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