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Although some PRMT6 substrates have been described, the precise function of this enzyme still remains elusive. The aim of this work was the identification and characterization of novel chromatin functions of PRMT6. At first the in vivo existence and relevance of the H3R2 dimethylation catalyzed by PRMT6 was verified. It was shown that the PRMT6-mediated H3R2 dimethylation counteracts the active histone modification H3K4me3 and thereby represses the expression of a subset of HOXA genes and c-Myc target genes. In the following the underlying mechanism of this antagonism between H3R2me2 and H3K4me3 was uncovered at the HOXA2 gene. PRMT6-mediated H3R2 dimethylation inhibits H3K4 trimethylation by preventing the chromatin recruitment of the components MLL1 and WDR5 of the H3K4 methyltransferase complex. This repressive function of PRMT6 was also found to be relevant for HOXA2 gene expression during neuronal differentiation in the NT2/D1 cell model. In the further course of this work a potential positive cross-talk between PRMT6 and polycomb group proteins was studied. PRMT6 was shown to interact with a subset of PcG proteins and similar to PcGs to be involved in the maintenance of the specific expression pattern of the entire HOXA gene locus during neuronal differentiation. Similar to the depletion of PcG proteins, depletion of PRMT6 was accompanied by hyperactivation of the anterior-localized HOXA genes and derepression of the posterior-localized HOXA genes following ATRA-induced differentiation of NT2/D1 cells. This deregulation of the HOXA genes upon loss of PRMT6 coincided with enhanced enrichment of the active histone modification H3K4me3 and decreased amounts of the repressive histone modification H3K27me3. In summary, these findings identify PRMT6 as a novel transcriptional repressor depending on its H3R2 methyltransferase activity and reveal as molecular mechanism of this repression a cross-talk between histone arginine methylation and lysine methylation. Since PcG proteins have been linked to the regulation of proliferation and senescence by repressing the INK4B-ARF-INK4A locus, it was investigated next whether PRMT6 would similarly control the INK4B-ARF-INK4A expression. The loss of PRMT6 in human embryonic fibroblasts resulted in a proliferation block, which was characterized by an accumulation of G1-phase cells. Moreover, depletion of PRMT6 caused cellular senescence indicated by beta-galactosidase positive cells and led to the upregulation of the CDK-inhibitors p16/INK4A as well as CDKN1A (p21/CIP1). In agreement with these results, the clonogenic growth potential of tumor cells was reduced in PRMT6-depleted cells. In conclusion, these data suggest that PRMT6 is necessary for the proliferation of both human diploid fibroblasts and tumor cells and inhibits cellular senescence as indicated here by controlling the expression of important cell cycle regulators. Finally, an unbiased screen for novel interaction partners of PRMT6 was established using overexpression of TAP (tandem affinity purification)-tagged PRMT6. Subsequent to affinity purification of exogenous PRMT6, co-purifying proteins were identified by mass spectrometry and analyzed by the database STRING and literature search to uncover published connections between the putative interactors, arginine methylation and PRMT6. These results will serve as a starting point for future research extending our knowledge on further nuclear functions of PRMT6. Gene regulation ths Prof. Dr. Renkawitz-Pohl Renate Renkawitz-Pohl, Renate (Prof. Dr.) Stein, Claudia Stein Claudia Transcription PRMT6 gehört zur Familie der Protein-Arginin-Methyltransferasen und wurde 2002 durch genomweite Suche nach neuen PRMT Mitgliedern anhand der konservierten katalytischen Domäne gefunden und als nukleär lokalisiertes Enzym identifiziert. Obwohl bereits einige Substrate von PRMT6 beschrieben wurden, ist über die genaue Funktion des Enzyms wenig bekannt. Die vorliegende Arbeit hatte zum Ziel neue Chromatinfunktionen von PRMT6 aufzudecken und zu charakterisieren. In der vorliegenden Arbeit wurden zunächst das Vorkommen und die Relevanz der PRMT6-vermittelten H3R2 Dimethylierung in vivo nachgewiesen. Es wurde gezeigt, dass die H3R2 Dimethylierung durch PRMT6 die aktive Histonmodifikation H3K4me3 antagonisiert und darüber die Repression einiger HOXA Gene und c-Myc-Zielgene erreicht wird. In nachfolgenden Untersuchungen wurde der Antagonismus zwischen H3R2me2 und H3K4me3 am HOXA2 Gen mechanistisch aufgeklärt. Die PRMT6-vermittelte H3R2 Dimethylierung inhibiert die H3K4 Trimethylierung, indem die Chromatinrekrutierung der Untereinheiten des H3K4-Methyltransferase Komplexes MLL1 und WDR5 verhindert wird. Es wurde gezeigt, dass die transkriptionelle Repression von HOXA2 durch PRMT6 auch bei der neuronalen Differenzierung von NT2/D1 Zellen von Bedeutung ist. Weitere Untersuchungen im Rahmen dieser Arbeit beschäftigten sich mit der Aufklärung einer potentiellen positiven Wechselwirkung zwischen PRMT6 und PcG Proteinen. Dabei wurde gefunden, dass PRMT6 mit PcG Proteinen interagiert und ähnlich wie die PcG Proteine zum Erhalt des spezifischen Expressionsmusters der HOXA Gene während der neuronalen Differenzierung beiträgt. Ähnlich wie die Depletion von PcG Proteinen führte die Depletion von PRMT6 zur Hyperaktivierung der anterior lokalisierten HOXA Gene und zur Derepression der posterior lokalisierten HOXA Gene nach Induktion der Differenzierung in NT2/D1 Zellen mit ATRA. Diese Fehlregulation der HOXA Gene als Folge der PRMT6 Depletion ging mit einem veränderten Histonmodifikationsmuster einher. PRMT6-defiziente Zellen wiesen eine Zunahme der aktiven Histonmodifikation H3K4me3 und einen Verlust der repressiven Histonmodifikation H3K27me3 auf. Zusammenfassend identifizieren diese Daten PRMT6 als transkriptionellen Repressor durch die Katalyse der H3R2 Dimethylierung. Diesem Repressionsmechanismus liegt die Wechselwirkung zwischen Histon-Argininmethylierung und Histon-Lysinmethylierung zugrunde. Aufgrund der Tatsache, dass PcG Proteine durch direkte Regulation des INK4B-ARF-INK4A Lokus mit der Regulation von Proliferation und Seneszenz in Verbindung gebracht werden, wurde im Folgenden untersucht, ob PRMT6 den INK4B-ARF-INK4A Lokus ebenfalls reguliert. Der Verlust von PRMT6 in humanen diploiden Fibroblasten (TIG3-T) führte zu einem Proliferationsarrest, der durch eine Akkumulation von G1-Phase Zellen gekennzeichnet war. Darüber hinaus resultierte die Depletion von PRMT6 in zellulärer Seneszenz, die durch die Aktivität von Seneszenz-assoziierter beta-Galaktosidase nachgewiesen wurde, und in einer Hochregulation der CDK-Inhibitoren p16/INK4A und CDKN1A (p21/CIP1). In Übereinstimmung damit führte eine Depletion von PRMT6 in Tumorzellen zur Reduktion des klonogenen Potentials. Zusammenfassend weisen diese Daten darauf hin, dass PRMT6 für die Proliferation von humanen diploiden Fibroblasten und Tumorzellen benötigt wird und durch transkriptionelle Regulation wichtiger Zellzyklusregulatoren Seneszenz inhibiert. Abschließend wurde ein unvoreingenommener Ansatz zur Aufreinigung neuer Interaktionspartner von PRMT6 unter Verwendung von TAP (Tandem Affinity Purification)-markiertem PRMT6 etabliert. Die coaufgereinigten Interaktionspartner wurden mittels massenspektrometrischer Analyse identifiziert und mit Hilfe der Datenbank STRING sowie Literaturrecherche auf publizierte Zusammenhänge zwischen den möglichen Interaktionspartnern, Argininmethylierung und PRMT6 analysiert. Diese Ergebnisse können als Ausgangspunkt für die zukünftige Erforschung von PRMT6 dienen und zur Erweiterung des Verständnisses weiterer nukleärer Funktionen von PRMT6 beitragen. PRMT6 Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg https://doi.org/10.17192/z2011.0068 Biologie monograph Genregulation Argininmethylierung doctoralThesis 2011-08-08 Arginine methylation https://archiv.ub.uni-marburg.de/diss/z2011/0068/cover.png