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Functional Characterization of the Arabidopsis Retinoblastoma Related Protein Research Collection Doctoral Thesis Functional characterization of the Arabidopsis retinoblastoma related protein Author(s): Gutzat, Ruben Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-006023141 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH Nr. 18446 FUNCTIONAL CHARACTERIZATION OF THE ARABIDOPSIS RETINOBLASTOMA RELATED PROTEIN ABHANDLUNG zur Erlangung des Titels DOKTOR DER WISSENSCHAFTEN der ETH ZÜRICH vorgelegt von RUBEN GUTZAT Dipl.-Biol. Univ. Konstanz geboren am 14.07.1977 aus Deutschland Angenommen auf Antrag von Prof. Dr. Wilhelm Gruissem Prof. Dr. Claudia Köhler Prof. Dr. Ueli Grossniklaus Prof. Dr. Ben Scheres 2009 Ruben Gutzat FUNCTIONAL CHARACTERIZATION OF THE ARABIDOPSIS RETINOBLASTOMA RELATED PROTEIN 2 Abstract The retinoblastoma protein (pRB) is a master regulator of cell cycle and differentiation in animal cells and as such an important tumor suppressor. It regulates the G1/S-phase transition via binding to E2F/DP transcription factors and therefore inhibiting expression of S-phase genes. Some studies provide evidence that pRB is also important for cell fate determination. However, whether this is an effect only on some specialized cell types or if pRB has a general effect on cell differentiation remains unresolved. The presence of retinoblastoma-related proteins (RBRs) in plants offers the opportunity to study the function of this protein in a completely different developmental context. For example, plant organs develop after embryogenesis, plants switch from heterotrophy to autotrophy during germination and plants do not develop tumors without infection of specialized pathogens. RBR knockout alleles are gametophytic lethal, which makes it difficult to study the role of RBR for cell cycle, differentiation and development in the sporophyte. Therefore we generated Arabidopsis mutant lines that displayed reduced expression of RBR (RBRcs- mutants). Mutant embryos showed a strongly increased number of cells in all organs but developed otherwise relatively normal. However, after germination RBRcs mutants were developmentally arrested and were not able to switch from an embryonic heterotrophic growth to an autotrophic seedling growth. RBRcs seedlings were able to efficiently take up sucrose, which resulted in massive ectopic cell over-proliferation. Gene profiling revealed a strong derepression of embryonic and seed maturation genes in the presence of sucrose. Our results suggest that 1) Arabidopsis seedlings require RBR to become autotrophic after germination and 2) RBR is required for repression of sucrose inducible embryonic and seed maturation genes after germination; thus RBR connects cell fate switch in seedlings after germination with cell cycle control. Furthermore we generated antibodies against RBR and established co-immuno- precipitation assays to identify potential interaction partners of RBR. Subsequent MS/MS based analysis revealed that RBR binds to all three Arabidopsis E2F and both DP transcription factors. We also found potential new interactions and could accurately map several post-transcriptional modifications of RBR. This enabled us to propose a RBR interaction network, which could be an important resource for future research. Finally we found an intriguing phenomenon. Seedlings with reduced activity of RBR always showed increased levels of RBR transcript. The same was the case for genes necessary for transcriptional gene silencing and DNA de novo methylation. We provide evidence that RBR is functionally connected with the RNA interference/transgene silencing pathway and seems to have a role in regulating certain genes on a posttranscriptional level. 3 Zusammenfassung Das Retinoblastoma protein (pRB) ist ein wichtiger Zell-Zyklus Regulator und als solches ein wichtiger Tumor-suppressor in Säugetierzellen. Es inhibiert E2F/DP Transcriptionsfaktoren welche notwendig sind um S-Phase gene zu induzieren und reguliert so den G1/S-Phase Uebergang. Einige Studien konnten auch zeigen, dass pRB wichtig ist für Zelldifferenzierungsvorgänge. Ob diese Effekte nur in bestimmten Zell- typen wichtig sind, oder ob pRB eine mehr generelle Rolle in Zelldifferenzierung hat ist noch nicht eindeutig geklärt. Die Tatsache, dass Planzenzellen ebenfalls Retinoblastoma-homologe besitzen (RBRs) bietet die Möglichkeit, die Funktion dieser Proteine in einem völlig neuen Entwicklungsbiologischen Zusammenhang zu studieren. Zum Beispiel entwickeln sich Pflanzenorgane erst nach der Embryogenese, Pflanzen ändern ihren Metabolismus von heterotroph zu autotroph nach der Keimung und Pflanzen entwickeln keine Tumore (mit Ausnahme wenn sie von spezialisierten Pathogenen infiziert werden). Da RBR-Null Arabdiopsis mutanten schon als Gametophyten absterben ist es schwierig den Einfluss von RBR auf Zell-Zyklus-Kontrolle, Zell-Differenzierung und Entwicklung im Sporophyten zu erforschen. Deswegen entwickelten wir Arabidopsis Mutanten die weniger RBR produzierten (RBRcs-Mutanten). Embryonen dieser Mutanten entwickelten sich relative normal, zeigten aber eine grössere Anzahl Zellen im Vergleich zum normalen Phänotyp. Nach der Keimung waren RBRcs Mutanten in ihrer Entwicklung arretiert und konnten keinen autotrophen Sämling etablieren. Jedoch konnten RBRcs Mutanten sehr effizient Saccharose aufnehmen und zeigten unter diesen Bedingungen dramatische Zellteilungs-Aktivität. Das Expressionsprofil von RBRcs Mutanten zeigte, dass Samen-und Embryo-spezifische Gene stark durch Saccharose reaktiviert wurden. Unsere Ergebnisse legen nahe, dass 1) Arabidopsis-Sämlinge RBR benötigen um nach der Keimung Autotroph zu werden und 2) RBR Samen-und Embryo-specifische Gene nach der Keimung reprimiert. Das bedeutet, dass RBR Zell-Differenzierungvorgänge mit Zell- Zyklus-Kontrolle in jungen Keimlingen verbindet. Ausserdem produzierten wir hoch-spezifische Antikörper gegen RBR und konnten damit ein Co-Immuno-Präzipitierungs Protokoll entwickeln. Damit war es möglich mit Hilfe von Massenspektrometrischen Methoden Interaktionspartner von RBR zu identifizieren. In diesen Assays interagierte RBR mit allen drei in Arabidopsis vorkommenden E2F und beiden DP transcriptionsfactoren. Ausserdem fanden wir zahlreiche neue Interaktionspartner und Post-Transkriptionelle Modifizierungen von RBR. Mit diesen Erkenntissen war es möglich ein hypothetisches RBR-Interaktions-Netzwerk zu postulieren welches eine wichtige Quelle für künftige Studien sein könnte. Schliesslich entdeckten wir ein interessantes Phänomen; Sämtliche Arabidopsis Mutanten mit 4 reduzierter RBR Aktivität zeigten stets eine höhere Expression von RBR auf Transkriptionsebene. Das gleiche Phänomen fanden wir für Gene die eine wichtige Rolle in RNA Interferenz und DNA-Methylierung haben. Daraus schliessen wir, dass RBR funktionell mit RNA Interferenz und DNA-Methylierung verknüpft ist und auch auf Post- Transkriptioneller Ebene die Aktivität von bestimmten Genen regulieren kann. 5 Acknowledgments Thousand thanks to Boris, who was too short amongst us My dear RBR Colleges: Especially Lorenzo, Yec`han and Johannes for the brilliant working-atmosphere My dear Lab-Worriers (who share my passion for endless lab-nights) Sylvain, Lorenzo (again ;), Herve and Gilles All the other amazing lab-dwellers for making science so much fun: Christina, Judith, Diana, Simon, Lilli, Ernst, Raya, Kim, Doris, Thomas and everybody else!!! It is great to work with you!! All the former lab-dwellers: Especially Eveline, Carmen, Martin and Jonas – I hope to keep contact with you Special thanks also to Katharina, for being always friendly and helpful, Lars, who was helping with the microarray analysis, John Lunn for the metabolite-measurements, Mike Scott from the FGCZ for help with protein work, Sonja for help with MS/MS data and anybody else from the lab I might have forgotten I also would like to thank Claudia Köhler, Ueli Grossniklaus and Ben Scheres for being members of my thesis-Committee Special thanks to Wilhelm Gruissem for giving me the opportunity to do my PhD thesis in his lab and for providing access to top range technologies A BIG thank you also to my family, my parents Beate and Diethard and my sisters Mirjam and Rebecca and my auntie Lydia who always supported me and my interests Now I would like to thank my friends outside of the lab for keeping me straight ;) and giving me such a nice time in Switzerland: Piet – the Pistol – Köttgen – for surviving many smaller or bigger adventures, Claas for showing me the powder, the B-team, the Greenlandic crew – James, Tom, Ged, Toni, Dan and Es, Arnold McGyver, Igor for the balloon, Anita – for my new style;) and of course anybody else I might have forgotten 6 Table of Contents Abstract ............................................................................................................................... 1 Zusammenfassung .............................................................................................................. 4 Acknowledgments ............................................................................................................... 6 1. Introduction .............................................................................................................. 10 On the origin of retinoblastoma-related proteins ........................................................ 10 Structural features of retinoblastoma-like proteins ....................................................
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