We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is ∼0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.

SummaryDifferential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co-regulated genes, an enrichment of preferentially up-or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophila ∆ ∆ ∆ ∆ dotA . One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.

Background: Dictyostelium discoideum is frequently subjected to environmental changes in its natural habitat, the forest soil. In order to survive, the organism had to develop effective mechanisms to sense and respond to such changes. When cells are faced with a hypertonic environment a complex response is triggered. It starts with signal sensing and transduction and leads to changes in cell shape, the cytoskeleton, transport processes, metabolism and gene expression. Certain aspects of the Dictyostelium osmotic stress response have been elucidated, however, no comprehensive picture was available up to now.

Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca2+-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3−, phg2−, and pyk3−/phg2− cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca2+ liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3− and phg2− cells and even further decreased in pyk3−/phg2− cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2− cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues.

Hiermit versichere ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe angefertigt und andere als die in der Dissertation angegebenen Hilfsmittel nicht benutzt habe. Alle Stellen, die wörtlich oder sinngemäß aus veröffentlichten oder unveröffentlichten Schriften anderer Personen entnommen sind, habe ich als solche kenntlich gemacht. Kein Teil dieser Arbeit ist in einem anderen Promotions-oder Habilitationsverfahren verwendet worden. Kassel, den 09.2006 iii Zusammenfassung Dnmt2 DNA Methyltransferasen stellen eine hoch konservierte Proteinfamilie mit enigmatischer Funktion dar. Ziel dieser Arbeit war es, DnmA, die Dnmt2 Methyltransferase aus Dictyostelium discoideum zu charakterisieren und weiterhin ihre Beteiligung an DNA Methylierung und transkriptioneller Genstilllegung zu untersuchen. Das Genom der sozialen Amöbe Dictyostelium kodiert DnmA als die einzige DNA Methyltransferase. Das Enzym enthält alle zehn charakteristischen DNA Methyltransferase Motive in seiner katalytischen Domäne. Mittels RT-PCR konnte gezeigt werden, dass die DnmA mRNA im vegetativen Wachstum exprimiert und während der Entwicklung herabreguliert wird. Untersuchungen mittels Fluoreszenz-Mikroskopie zeigten, dass DnmA-myc und DnmA-GFP Fusionen hauptsächlich im Kern lokalisieren. Die Funktion von DnmA blieb zunächst unklar, jedoch zeigten spätere Experimente, dass das Enzym eine aktive DNA Methyltransferase ist, die für die gesamte DNA (Cytosin) Methylierung in Dictyostelium verantwortlich ist. Weder in Gel-Retardations Untersuchungen noch durch das Yeast Two-hybrid System konnten Anhaltspunkte zur Funktionalität von DnmA gewonnen werden. Jedoch gab der immunologische Nachweis der Methylierungsmarkierung mittels eines anti-5mC Antikörpers einen ersten Hinweis darauf, dass die DNA von Dictyostelium methyliert ist. Zugabe von 5-Aza-Cytidine als demethylierendem Agens zum Dictyostelium Medium und anschließende in vitro Inkubation der aus diesen Zellen gewonnenen DNA mit rekombinanter DnmA zeigte weiterhin, dass das Enzym etwas besser an diese DNA bindet. Zur weiteren Untersuchung des Proteins wurde ein Gen Knock-out von dnmA generiert. Das Gen wurde erfolgreich durch homologe Rekombination unterbrochen, der Knock-out Stamm zeigte jedoch keinen offensichtlichen Phänotyp unter normalen Laborbedingungen. Um Zielsequenzen für die DNA Methylierung zu identifizieren, wurde eine Microarray Analyse durchgeführt. Unter Benutzung eines Grenzwerts von mindestens 1.5facher Veränderung in der Stärke der Genexpression, wurden mehrere dem entsprechende Gene im Knock-out Stamm für weitere Untersuchungen ausgewählt. Unter der hochregulierten Genen waren ESTs, die die gag und RT Gene des Retrotransposons skipper repräsentieren, und Northern Blot Analysen bestätigte die Hochregulierung von skipper im DnmA Knock-out Stamm. iv Bisufitbehandlung und Sequenzierung spezifischer DNA Bereiche von skipper zeigte, dass DnmA für die Methylierung hauptsächlich asymmetrischer Cytosine verantwortlich ist. Außer für skipper wurde dies später auch fü...