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Master Thesis The role of EAPP in cell cycle regulation and DNA damage control Master Thesis For the attainment of the academic degree Master of Science From the University of Applied Sciences FH Campus Wien Submitted by: Sarah Gobetzky Personal identity code 1410544011 Supervisor: Ao. Prof. Dr. Johann Rotheneder Max F. Perutz Laboratories; Medical University of Vienna; Dept. of medical Biochemistry Vienna Austria Submitted on: 10. 04. 2017 1.1 Abstract in English Cell cycle checkpoints are an important tool of eukaryotic cells to ensure the correct and error-free replication of the DNA and division of the cells. A cell undergoes apoptosis or remains in cell cycle arrest until any damage is corrected before re-entering the cell-cycle. Mutations bypassing this system or other interferences often lead to the generation of carcinogenic structures. The E2F associated phosphoprotein (EAPP), discovered by our group, interacts with E2F 1-3, alters in an E2F-dependent manner the activity of cell cycle regulated promotors and is a co-factor in E2F dependent transcription. The levels of this protein are elevated in various human cancers. It could be observed that overexpression but also a knockout of EAPP seems to lead to a slowdown of the cell cycle and great G1 arrest. An interaction of EAPP with p53, MAO B and Chk2 could already be uncovered. Furthermore it could be shown that EAPP levels are elevated after double strand breaks and stimulates p21 expression causing cell cycle arrest in G1. In this Master´s thesis the impact of EAPP was further investigated. Total knockouts of EAPP in NIH 3T3 cells revealed that EAPP is not necessary for survival for those cells like it is for example for U2OS cells. Cells expressing various variants or truncations of EAPP were generated and used for examination on their impact on stability or protein-protein interactions or to determine their functional domains. It could be shown that the loss of EAPP in HAP1 cells leads to a drastic increase and reduced amounts of a truncated version of EAPP lead to a moderate increase in their rate of expansion compared to the wt. In addition it could be observed that cells with higher levels of EAPP show a higher fraction of cells in G1 phase after DNA damage introduced by Etoposide. It could also be observed that truncated versions of EAPP seem to show increased stability in comparison to the wt. Analysis revealed that the loss of EAPP or reduced amounts of a truncated version lead to a dramatic increase of P-Akt levels. This effect is even enhanced when treated 2 with Etoposide. Also U2OS cells show elevated P-Akt levels in cells with reduced amounts of EAPP and rather low levels in cells with high EAPP levels indicating a correlation between low P-Akt levels and high EAPP and vice versa. The level of EAPP seems further having an impact on the recovery speed as NIH 3T3 cells lacking EAPP seem to re-enter the cell cycle much faster after serum deprivation than NIH 3T3 cells with normal levels of EAPP. In addition to the obtained results we could identify Helic2, PRP8, E6AP, p53, Sm B/B´/N, GSK 3β, YB1 and AAR2 as putative interaction partners of EAPP. Our results show clearly an increase of cell fractions in G1 arrest after DNA damage with elevated EAPP levels, alterations of expression levels of different proteins not to mention interaction of EAPP with various splice factors underlining the important role EAPP seems to play in the cell cycle and its regulation and control. 3 1.2 Abstract in German Zellzykluskontrollpunkte sind ein wichtiges Mittel eukaryotischer Zellen um sicherzustellen dass sich Zellen in einem einwandfreien Zustand replizieren. Die Zelle unterliegt der Apoptose oder verharrt im Zellzyklusarrest bis die aufgetretenen Schäden behoben sind ehe der Zellzyklus weiter voranschreitet. Mutationen die dieses System umgehen oder anderweitige Störungen führen oft zu der Ausbildung kanzerogener Strukturen. Das in unserer Forschungsgruppe entdeckte E2F associated phosphoprotein (EAPP) interagiert mit E2F 1-3, hat mit deren Hilfe Einfluss auf zellzyklusabhängige Promotoren und ist ein Co-faktor in der E2F abhängigen Transkription. Es ist ein Protein dessen Levels in verschiedensten humanen Krebsarten deutlich erhöht sind. Es konnte sowohl in Zellen mit Überexpression als auch mit knockout eine Verlangsamung des Zellzyklus sowie eine erhöhte Zellzahl in G1 beobachtet werden. Eine Interaktion von EAPP mit p53, MAO B oder auch Chk2 wurde bereits nachgewiesen. Zudem wurde gezeigt dass EAPP Levels nach Doppelstrangbrüchen erhöht sind und durch eine Stimulation der p21 Expression zu Zellzyklusarrest in G1 führen. In dieser Masterarbeit wurde die Bedeutung von EAPP weiter untersucht. Totale Knockouts von EAPP in NIH 3T3 Zellen haben bewiesen dass in diesen Zellen EAPP nicht für ein Überleben notwendig ist, wie es beispielsweise in U2OS Zellen der Fall ist. Zellen welche verschiedenste Varianten oder Verkürzungen von EAPP exprimieren wurden hergestellt und dazu verwendet deren Einfluss auf Stabilität, Protein-Protein Interaktionen festzustellen oder mögliche funktionelle Domänen auszumachen. Es konnte gezeigt werden dass der Verlust von EAPP in HAP1 Zellen zu einer deutlich erhöhten Proliferationsrate und verringerte EAPP Mengen einer verkürzten Form zu einer leicht erhöhten Proliferationsrate führen. Zudem konnte beobachtet werden dass sich nach DNS Schädigung durch Etoposid deutlich mehr Zellen mit erhöhten Mengen an EAPP in G1 befinden. Eine weitere Beobachtung war die Tatsache dass verkürzte EAPP Versionen im Vergleich zum endogenen EAPP stabiler zu sein scheinen. Analysen haben 4 gezeigt dass der Verlust von EAPP oder verringerte Mengen einer verkürzten Form in HAP1 Zellen zu einer drastischen Erhöhung der P-Akt Level führen. Dieser Effekt ist durch Etoposid noch einmal verstärkt. Auch U2OS Zellen zeigen bei verringerten Mengen an EAPP erhöhte Mengen an P-Akt und bei erhöhten EAPP Mengen verringerte P-Akt Mengen, was eine Korrelation zwischen geringem P-Akt und hohem EAPP und umgekehrt vermuten lässt. Das Level an EAPP scheint zudem Auswirkungen auf die Erholungsfähigkeit der Zellen zu haben, da NIH 3T3 Zellen ohne EAPP nach Serumentzug deutlich beschleunigten Wiedereintritt in den Zellzyklus gezeigt haben. Zudem konnten Helic2, PRP8, E6AP, p53, Sm B/B´/N, GSK 3β, YB1 und AAR2 als mögliche Interaktionspartner identifiziert werden. Unsere Ergebnisse zeigen deutlich eine erhöhte Anzahl an Zellen in G1 nach DNS Schädigung durch Etoposid in Zellen mit erhöhten EAPP Levels, Veränderungen in den Expressionslevels verschiedener Proteine sowie Interaktionen von EAPP mit verschiedenen Splicefaktoren die unterstreichen dass EAPP eine wichtige Rolle im Zellzyklus und seiner Kontrolle zu spielen scheint. 5 2 Table of Content 1.1 Abstract in English ......................................................................... 2 1.2 Abstract in German ........................................................................ 4 2 Table of Content ............................................................................... 6 3 List of Abbreviations .......................................................................... 8 4 List of Figures ................................................................................. 11 5 Introduction ................................................................................... 13 5.1 Cell cycle.................................................................................. 13 5.2 p53 and its role in cell cycle ........................................................ 17 5.3 E2F and its role in cell cycle progression ...................................... 18 5.4 EAPP ........................................................................................ 20 5.5 CRISPR/Cas9 ............................................................................ 24 5.6 Aims ........................................................................................ 27 6 Materials and Methods ..................................................................... 28 6. 1 Materials ................................................................................. 28 6.2 Methods ................................................................................... 36 7 Results .......................................................................................... 43 7.1 Generation of cell lines with different version of EAPP ................. 43 7.1.1 Generation of NIH 3T3 cells with knockouts of EAPP or introduction of truncated EAPP versions. ......................................... 43 7.1.2 Generation of U2OS cells expressing HA-EAPP 10-240 or 20-240 in addition to endogenous EAPP ..................................................... 48 7.1.3 Generation of U2OS cells with p53 knockout and re-transfection of p53 ............................................................................................ 49 7.2 Cell proliferation assays ............................................................. 50 7.2.1 Cell proliferation of HAP1 cells with different EAPP knockouts .... 50 7.2.2 Cell proliferation of NIH 3T3 cells with different EAPP versions ... 52 7.3 FACS analysis ........................................................................... 53 7.3.1 FACS analysis of U2OS cells with different EAPP versions after treatment with different concentrations of Etoposide ........................ 53 7.3.2 FACS analysis of NIH 3T3 versions after serum starvation and reactivation ................................................................................. 56 6
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