DNA methylation in Dictyostelium discoideum

DNA methyltransferases of type Dnmt2 are a highly conserved protein family with enigmatic function. The aim of this work was to characterize DnmA, the Dnmt2 methyltransferase in Dictyostelium discoideum, and further to investigate its implication in DNA methylation and transcriptional gene silencing. The genome of the social amoeba Dictyostelium encodes DnmA as the sole DNA methyltransferase. The enzyme bears all ten characteristic DNA methyltransferase motifs in its catalytic domain. The DnmA mRNA was found by RT-PCR to be expressed during vegetative growth and down regulated during development. Investigations using fluorescence microscopy showed that both DnmA-myc and DnmA-GFP fusions predominantly localised to the nucleus. The function of DnmA remained initially unclear, but later experiment revealed that the enzyme is an active DNA methyltransferase responsible for all DNA (cytosine) methylation in Dictyostelium. Neither in gel retardation assays, nor by the yeast two hybrid system, clues on the functionality of DnmA could be obtained. However, immunological detection of the methylation mark with an α - 5mC antibody gave initial evidence that the DNA of Dictyostelium was methylated. Furthermore, addition of 5-aza-cytidine as demethylating agent to the Dictyostelium medium and subsequent in vitro incubation of the DNA isolated from these cells with recombinant DnmA showed that the enzyme binds slightly better to this target DNA. In order to investigate further the function of the protein, a gene knock-out for dnmA was generated. The gene was successfully disrupted by homologous recombination, the knock-out strain, however, did not show any obvious phenotype under normal laboratory conditions. To identify specific target sequences for DNA methylation, a microarray analysis was carried out. Setting a threshold of at least 1.5 fold for differences in the strength of gene expression, several such genes in the knock-out strain were chosen for further investigation. Among the up-regulated genes were the ESTs representing the gag and the RT genes respectively of the retrotransposon skipper. In addition Northern blot analysis confirmed the up-regulation of skipper in the DnmA knock-out strain. Bisufite treatment and sequencing of specific DNA stretches from skipper revealed that DnmA is responsible for methylation of mostly asymmetric cytosines. Together with skipper, DIRS-1 retrotransposon was found later also to be methylated but was not present on the microarray. Furthermore, skipper transcription was also up-regulated in strains that had genes disrupted encoding components of the RNA interference pathway. In contrast, DIRS 1 expression was not affected by a loss of DnmA but was strongly increased in the strain that had the RNA directed RNA polymerase gene rrpC disrupted. Strains generated by propagating the usual wild type Ax2 and the DnmA knock-out cells over 16 rounds in development were analyzed for transposon activity. Northern blot analysis revealed activation for skipper expression, but not for DIRS-1. A large number of siRNAs were found to be correspondent to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA methylation. In contrast, no siRNAs corresponding to the standard skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene regulation in Dictyostelium and that different, partially overlapping mechanisms control transposon silencing for skipper and DIRS-1. To elucidate the mechanism of targeting the protein to particular genes in the Dictyostelium genome, some more genes which were up-regulated in the DnmA knock-out strain were analyzed by bisulfite sequencing. The chosen genes are involved in the multidrug response in other species, but their function in Dictyostelium is uncertain. Bisulfite data showed that two of these genes were methylated at asymmetrical C-residues in the wild type, but not in DnmA knock-out cells. This suggested that DNA methylation in Dictyostelium is involved not only in transposon regulation but also in transcriptional silencing of specific genes.

Facilitating folding of outer membrane proteins, roles of the periplasmic chaperone Skp and the outer membrane lipoprotein BamD

This thesis describes several important advancements in the understanding of the assembly of outer membrane proteins of Gram-negative bacteria like Escherichia coli. A first study was performed to identify binding regions in the trimeric chaperone Skp for outer membrane proteins. Skp is known to facilitate the passage of unfolded outer membrane proteins (OMPs) through the periplasm to the outer membrane (OM). A gene construct named “synthetic chaperone protein (scp)” gene was used to express a fusion protein (Scp) into the cytoplasm of E. coli. The scp gene was used as a template to design mutants of Scp suitable for structural and functional studies using site-directed spectroscopy. Fluorescence resonance energy transfer (FRET) was used to identify distances in Skp-OmpA complexes that separate regions in Scp and in outer membrane protein A (OmpA) from E. coli. For this study, single cysteine (Cys) mutants and single Cys - single tryptophan (Trp) double mutants of Scp were prepared. For FRET experiments, the cysteines were labeled with the tryptophan fluorescence energy acceptor IAEDANS. Single Trp mutants of OmpA were used as fluorescence energy donors. In the second part of this thesis, the function of BamD and the structure of BamD-Scp complexes were examined. BamD is an essential component of the β-barrel assembly machinery (BAM) complex of the OM of Gram-negative bacteria. Fluorescence spectroscopy was used to probe the interactions of BamD with lipid membranes and to investigate the interactions of BamD with possible partner proteins from the periplasm and from the OM. A range of single cysteine (Cys) and single tryptophan (Trp) mutants of BamD were prepared. A very important conclusion from the extensive FRET study is that the essential lipoprotein BamD interacts and binds to the periplasmic chaperone Skp. BamD contains tetratrico peptide repeat (TPR) motifs that are suggested to serve as docking sites for periplasmic chaperones such as Skp.

On the function of the Dictyostelium Argonaute A protein (AgnA) in epigenetic gene regulation

With molecular biology methods and bioinformatics, the Argonaute proteins in Dictyostelium discoideum were characterized, and the function of the AgnA protein in RNAi and DNA methylation was investigated, as well as cellular features. Also interaction partners of the PAZ-Piwi domain of AgnA (PAZ-PiwiAgnA) were discovered. The Dictyostelium genome encodes five Argonaute proteins, termed AgnA/B/C/D/E. The expression level of Argonaute proteins was AgnB/D/E > AgnA > AgnC. All these proteins contain the characteristic conserved of PAZ and Piwi domains. Fluorescence microscopy revealed that the overexpressed C-terminal GFP-fusion of PAZ-PiwiAgnA (PPWa-GFP) localized to the cytoplasm. Overexpression of PPWa-GFP leaded to an increased gene silencing efficiency mediated by RNAi but not by antisense RNA. This indicated that PAZ-PiwiAgnA is involved in the RNAi pathway, but not in the antisense pathway. An analysis of protein-protein interactions by a yeast-two-hybrid screen on a cDNA library from vegetatively grown Dictyostelium revealed that several proteins, such as EF2, EF1-I, IfdA, SahA, SamS, RANBP1, UAE1, CapA, and GpdA could interact with PAZ-PiwiAgnA. There was no interaction between PAZ-PiwiAgnA and HP1, HelF and DnmA detected by direct yeast-two-hybrid analysis. The fluorescence microscopy images showed that the overexpressed GFP-SahA or IfdA fusion proteins localized to both cytoplasm and nuclei, while the overexpressed GFP-SamS localized to the cytoplasm. The expression of SamS in AgnA knock down mutants was strongly down regulated on cDNA and mRNA level in, while the expression of SahA was only slightly down regulated. AgnA knock down mutants displayed defects in growth and phagocytosis, which suggested that AgnA affects also cell biological features. The inhibition of DNA methylation on DIRS-1 and Skipper retroelements, as well as the endogenous mvpB and telA gene, observed for the same strains, revealed that AgnA is involved in the DNA methylation pathway. Northern blot analysis showed that Skipper and DIRS-1 were rarely expressed in Ax2, but the expression of Skipper was upregulated in AgnA knock down mutants, while the expression of DIRS-1 was not changed. A knock out of the agnA gene failed even though the homologous recombination of the disruption construct occurred at the correct site, which indicated that there was a duplication of the agnA gene in the genome. The same phenomenon was also observed in ifdA knock out experiments.

HelF, a suppressor of RNAi mediated gene silencing in Dictyostelium discoideum

RNA interference (RNAi) is a recently discovered process, in which double stranded RNA (dsRNA) triggers the homology-dependant degradation of cognate messenger RNA (mRNA). In a search for new components of the RNAi machinery in Dictyostelium, a new gene was identified, which was called helF. HelF is a putative RNA helicase, which shows a high homology to the helicase domain of Dicer, to the helicase domain of Dictyostelium RdRP and to the C. elegans gene drh-1, that codes for a dicer related DExH-box RNA helicase, which is required for RNAi. The aim of the present Ph.D. work was to investigate the role of HelF in PTGS, either induced by RNAi or asRNA. A genomic disruption of the helF gene was performed, which resulted in a distinct mutant morphology in late development. The cellular localization of the protein was elucidated by creating a HelF-GFP fusion protein, which was found to be localized in speckles in the nucleus. The involvement of HelF in the RNAi mechanism was studied. For this purpose, RNAi was induced by transformation of RNAi hairpin constructs against four endogenous genes in wild type and HelF- cells. The silencing efficiency was strongly enhanced in the HelF K.O. strain in comparison with the wild type. One gene, which could not be silenced in the wild type background, was successfully silenced in HelF-. When the helF gene was disrupted in a secondary transformation in a non-silenced strain, the silencing efficiency was strongly improved, a phenomenon named here “retrosilencing”. Transcriptional run-on experiments revealed that the enhanced gene silencing in HelF- was a posttranscriptional event, and that the silencing efficiency depended on the transcription levels of hairpin RNAs. In HelF-, the threshold level of hairpin transcription required for efficient silencing was dramatically lowered. The RNAi-mediated silencing was accompanied by the production of siRNAs; however, their amount did not depend on the level of hairpin transcription. These results indicated that HelF is a natural suppressor of RNAi in Dictyostelium. In contrast, asRNA mediated gene silencing was not enhanced in the HelF K.O, as shown for three tested genes. These results confirmed previous observations (H. Martens and W. Nellen, unpublished) that although similar, RNAi and asRNA mediated gene silencing mechanisms differ in their requirements for specific proteins. In order to characterize the function of the HelF protein on a molecular level and to study its interactions with other RNAi components, in vitro experiments were performed. Besides the DEAH-helicase domain, HelF contains a double-stranded RNA binding domain (dsRBD) at its N-terminus, which showed high similarity to the dsRBD domain of Dicer A from Dictyostelium. The ability of the recombinant dsRBDs from HelF and Dicer A to bind dsRNA was examined and compared. It was shown by gel-shift assays that both HelF-dsRBD and Dicer-dsRBD could bind directly to long dsRNAs. However, HelF-dsRBD bound more efficiently to dsRNA with imperfect matches than to perfect dsRNA. Both dsRBDs bound specifically to a pre-miRNA substrate (pre-let-7). The results suggested that most probably there were two binding sites for the proteins on the pre-miRNA substrate. Moreover, it was shown that HelF-dsRBD and Dicer-dsRBD have siRNA-binding activity. The affinities of the two dsRBDs to the pre-let-7 substrate were also examined by plasmon surface resonance analyses, which revealed a 9-fold higher binding affinity of the Dicer-dsRBD to pre-let-7 compared to that of the HelF-dsRBD. The binding of HelF-dsRBD to the pre-let-7 was impaired in the presence of Mg2+, while the Dicer-dsRBD interaction with pre-let-7 was not influenced by the presence of Mg2+. The results obtained in this thesis can be used to postulate a model for HelF function. In this, HelF acts as a nuclear suppressor of RNAi in wild type cells by recognition and binding of dsRNA substrates. The protein might act as a surveillance system to avoid RNAi initiation by fortuitous dsRNA formation or low abundance of dsRNA trigger. If the protein acts as an RNA helicase, it could unwind fold-back structures in the nucleus and thus lead to decreased RNAi efficiency. A knock-out of HelF would result in initiation of the RNAi pathway even by low levels of dsRNA. The exact molecular function of the protein in the RNAi mechanism still has to be elucidated. RNA interferenz (RNAi) ist ein in jüngster Zeit entdeckter Mechanismus, bei dem doppelsträngige RNA Moleküle (dsRNA) eine Homologie-abhängige Degradation einer verwandten messenger-RNA (mRNA) auslösen. Auf der Suche nach neuen Komponenten der RNAi-Maschinerie in Dictyostelium konnte ein neues Gen (helF) identifiziert werden. HelF ist eine putative RNA-Helikase mit einer hohen Homologie zur Helikasedomäne der bekannten Dicerproteine, der Helikasedomäne der Dictyostelium RdRP und zu dem C. elegans Gen drh-1, welches für eine Dicer-bezogene DExH-box RNA Helikase codiert, die am RNAi-Mechanismus beteiligt ist. Das Ziel dieser Arbeit war es, die Funktion von HelF im Zusammenhang des RNAi oder asRNA induzierten PTGS zu untersuchen. Es wurde eine Unterbrechung des helF-Gens auf genomischer Ebene (K.O.) vorgenommen, was bei den Mutanten zu einer veränderten Morphologie in der späten Entwicklung führte. Die Lokalisation des Proteins in der Zelle konnte mit Hilfe einer GFP-Fusion analysiert werden und kleinen Bereichen innerhalb des Nukleus zugewiesen werden. Im Weiteren wurde der Einfluss von HelF auf den RNAi-Mechanismus untersucht. Zu diesem Zweck wurde RNAi durch Einbringen von RNAi Hairpin-Konstrukten gegen vier endogene Gene im Wiltypstamm und der HelF--Mutante induziert. Im Vergleich zum Wildtypstamm konnte im HelF--Mutantenstamm eine stark erhöhte „Silencing“-Effizienz nachgewiesen werden. Ein Gen, welches nach RNAi Initiation im Wildtypstamm unverändert blieb, konnte im HelF--Mutantenstamm erfolgreich stillgelegt werden. Durch sekundäres Einführen einer Gendisruption im helF-Locus in einen Stamm, in welchem ein Gen nicht stillgelegt werden konnte, wurde die Effizienz des Stilllegens deutlich erhöht. Dieses Phänomen wurde hier erstmals als „Retrosilencing“ beschrieben. Mit Hilfe von transkriptionellen run-on Experimenten konnte belegt werden, dass es sich bei dieser erhöhten Stilllegungseffizienz um ein posttranskriptionelles Ereignis handelte, wobei die Stillegungseffizienz von der Transkriptionsstärke der Hairpin RNAs abhängt. Für die HelF--Mutanten konnte gezeigt werden, dass der Schwellenwert zum Auslösen eines effizienten Stillegens dramatisch abgesenkt war. Obwohl die RNAi-vermittelte Genstilllegung immer mit der Produktion von siRNAs einhergeht, war die Menge der siRNAs nicht abhängig von dem Expressionsniveau des Hairpin-Konstruktes. Diese Ergebnisse legen nahe, dass es sich bei der HelF um einen natürlichen Suppressor des RNAi-Mechanismus in Dictyostelium handelt. Im Gegensatz hierzu war die as-vermittelte Stilllegung von drei untersuchten Genen im HelF-K.O. im Vergleich zum Wildyp unverändert. Diese Ergebnisse bestätigten frühere Beobachtungen (H. Martens und W. Nellen, unveröffentlicht), wonach die Mechanismen für RNAi und asRNA-vermittelte Genstilllegung unterschiedliche spezifische Proteine benötigen. Um die Funktion des HelF-Proteins auf der molekularen Ebene genauer zu charakterisieren und die Interaktion mit anderen RNAi-Komponenten zu untersuchen, wurden in vitro Versuche durchgeführt. Das HelF-Protein enthält, neben der DEAH-Helikase-Domäne eine N-terminale Doppelstrang RNA bindende Domäne (dsRBD) mit einer hohen Ähnlichkeit zu der dsRBD des Dicer A aus Dictyostelium. Die dsRNA-Bindungsaktivität der beiden dsRBDs aus HelF und Dicer A wurde analysiert und verglichen. Es konnte mithilfe von Gel-Retardationsanalysen gezeigt werden, dass sowohl HelF-dsRBD als auch Dicer-dsRBD direkt an lange dsRNAs binden können. Hierbei zeigte sich, dass die HelF-dsRBD eine höhere Affinität zu einem imperfekten RNA-Doppelstrang besitzt, als zu einer perfekt gepaarten dsRNA. Für beide dsRBDs konnte eine spezifische Bindung an ein pre-miRNA Substrat nachgewiesen werden (pre-let-7). Dieses Ergebnis legt nah, dass es zwei Bindestellen für die Proteine auf dem pre-miRNA Substrat gibt. Überdies hinaus konnte gezeigt werden, dass die dsRBDs beider Proteine eine siRNA bindende Aktivität besitzen. Die Affinität beider dsRBDs an das pre-let-7 Substrat wurde weiterhin mit Hilfe der Plasmon Oberflächen Resonanz untersucht. Hierbei konnte eine 9-fach höhere Bindeaffinität der Dicer-dsRBD im Vergleich zur HelF-dsRBD nachgewiesen werden. Während die Bindung der HelF-dsRBD an das pre-let-7 durch die Anwesenheit von Mg2+ beeinträchtigt war, zeigte sich kein Einfluß von Mg2+ auf das Bindeverhalten der Dicer-dsRBD. Mit Hilfe der in dieser Arbeit gewonnen Ergebnisse lässt sich ein Model für die Funktion von HelF postulieren. In diesem Model wirkt HelF durch Erkennen und Binden von dsRNA Substraten als Suppressor von der RNAi im Kern. Das Protein kann als Überwachungsystem gegen eine irrtümliche Auslösung von RNAi wirken, die durch zufällige dsRNA Faltungen oder eine zu geringe Häufigkeit der siRNAs hervorgerufen sein könnte. Falls das Protein eine Helikase-Aktivität besitzt, könnte es rückgefaltete RNA Strukturen im Kern auflösen, was sich in einer verringerten RNAi-Effizienz wiederspiegelt. Durch Ausschalten des helF-Gens würde nach diesem Modell eine erfolgreiche Auslösung von RNAi schon bei sehr geringer Mengen an dsRNA möglich werden. Das Modell erlaubt, die exakte molekulare Funktion des HelF-Proteins im RNAi-Mechanismus weiter zu untersuchen.

Molecular studies on a Dictyostelium homolog of the tafazzin gene, the cause of Barth syndrome in humans

Der eukaryotische Mikroorganismus Dictyostelium discoideum lebt als einzellige Amöbe solange ausreichende Nahrungsressourcen zur Verfügung stehen. Sobald Nahrungsmangel eintritt, entwickeln sich die Zellen von einem einzelligen zu einem mehrzelligen Zustand, der mit einem multizellulären Fruchtkörper abschließt. Dieser Prozess wird durch eine Reihe aufeinanderfolgender Signale organisiert, die eine differentielle Genexpression regulieren. Die Gene der Discoidin I Familie gehören zu den Ersten, die im Laufe des Wachstums-Differenzierungs-Übergangs (engl. GDT) aktiviert werden. Sie eignen sich daher vorzüglich als Marker für den Beginn der Entwicklung. Mit Hilfe einer REMI-Mutagenese und Discoidin I als molekularem Marker sind verschiedene Komponenten des Wachstums-Differenzierungs-Übergangs in unserer Arbeitsgruppe identifiziert worden (Zeng et al., 2000 A und B; Riemann und Nellen, persönliche Mitteilung). Mit demselben Ansatz wurde in der vorliegenden Arbeit eine REMI-Mutante identifiziert, die eine Fehl-Expression von Discoidin zeigte und einen axenischen Wachstumsdefekt bei 15 °C aufwies. Das Gen wurde als Homolog zum humanen Tafazzin-Gen identifiziert. Dieses Gen wurde zur Rekonstruktion des Phänotyps über homologe Rekombination erneut disruptiert, was wie erwartet zu dem zuerst beschriebenen Phänotyp führte. Folgerichtig ergab eine Überexpression des Gens in den Mutanten eine Komplementation des Phänotyps. Immunfluoreszenz-Experimente zeigten eine mitochondriale Lokalisation des Dictyostelium discoideum Taffazzin Proteins. Dass ein mitochondriales Protein in Zusammenhang mit dem Wachstums-Differenzierungs-Übergang steht, ist ein unerwarteter Befund, der aber als Hinweis darauf gewertet werden kann, dass Mitochondrien einen direkten Einfluss auf die entwicklungsspezifische Signaltransduktion ausüben. Die Taffazzin Disruptions-Mutante in Dictyostelium führte zu einem abnormalen Cardiolipin Metabolismus. Dieses Phospholipid ist ein charakteristischer Bestandteil der inneren Mitochondrienmembran und für die Funktion verschiedener Enzyme erforderlich. Unsere vorläufigen Analysen des Phospholipid-Gehalts zeigten Übereinstimmung mit Daten von Patienten mit Barth-Syndrom, einer humanen Erkrankung, bei der das Taffazzin-Gen Mutationen aufweist, und mit Hefe-Mutanten dieses Gens. Dies zeigt den Wert von Dictyostelium discoideum als einen weiteren Modelorganismus zur Untersuchung des Barth-Syndroms und zur Erprobung möglicher Therapieansätze.

Nuclear organisation and epigenetic regulation of gene expression in Dictyostelium discoideum

A series of vectors for the over-expression of tagged proteins in Dictyostelium were designed, constructed and tested. These vectors allow the addition of an N- or C-terminal tag (GFP, RFP, 3xFLAG, 3xHA, 6xMYC and TAP) with an optimized polylinker sequence and no additional amino acid residues at the N or C terminus. Different selectable markers (Blasticidin and gentamicin) are available as well as an extra chromosomal version; these allow copy number and thus expression level to be controlled, as well as allowing for more options with regard to complementation, co- and super-transformation. Finally, the vectors share standardized cloning sites, allowing a gene of interest to be easily transfered between the different versions of the vectors as experimental requirements evolve. The organisation and dynamics of the Dictyostelium nucleus during the cell cycle was investigated. The centromeric histone H3 (CenH3) variant serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. A number of Dictyostelium histone H3-domain containing proteins as GFP-tagged fusions were expressed and it was found that one of them functions as CenH3 in this species. Like CenH3 from some other species, Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins. The targeting domain, comprising α-helix 2 and loop 1 of the histone fold is required for targeting CenH3 to centromeres. Compared to the targeting domain of other known and putative CenH3 species, Dictyostelium CenH3 has a shorter loop 1 region. The localisation of a variety of histone modifications and histone modifying enzymes was examined. Using fluorescence in situ hybridisation (FISH) and CenH3 chromatin-immunoprecipitation (ChIP) it was shown that the six telocentric centromeres contain all of the DIRS-1 and most of the DDT-A and skipper transposons. During interphase the centromeres remain attached to the centrosome resulting in a single CenH3 cluster which also contains the putative histone H3K9 methyltransferase SuvA, H3K9me3 and HP1 (heterochromatin protein 1). Except for the centromere cluster and a number of small foci at the nuclear periphery opposite the centromeres, the rest of the nucleus is largely devoid of transposons and heterochromatin associated histone modifications. At least some of the small foci correspond to the distal telomeres, suggesting that the chromosomes are organised in a Rabl-like manner. It was found that in contrast to metazoans, loading of CenH3 onto Dictyostelium centromeres occurs in late G2 phase. Transformation of Dictyostelium with vectors carrying the G418 resistance cassette typically results in the vector integrating into the genome in one or a few tandem arrays of approximately a hundred copies. In contrast, plasmids containing a Blasticidin resistance cassette integrate as single or a few copies. The behaviour of transgenes in the nucleus was examined by FISH, and it was found that low copy transgenes show apparently random distribution within the nucleus, while transgenes with more than approximately 10 copies cluster at or immediately adjacent to the centromeres in interphase cells regardless of the actual integration site along the chromosome. During mitosis the transgenes show centromere-like behaviour, and ChIP experiments show that transgenes contain the heterochromatin marker H3K9me2 and the centromeric histone variant H3v1. This clustering, and centromere-like behaviour was not observed on extrachromosomal transgenes, nor on a line where the transgene had integrated into the extrachromosomal rDNA palindrome. This suggests that it is the repetitive nature of the transgenes that causes the centromere-like behaviour. A Dictyostelium homolog of DET1, a protein largely restricted to multicellular eukaryotes where it has a role in developmental regulation was identified. As in other species Dictyostelium DET1 is nuclear localised. In ChIP experiments DET1 was found to bind the promoters of a number of developmentally regulated loci. In contrast to other species where it is an essential protein, loss of DET1 is not lethal in Dictyostelium, although viability is greatly reduced. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed apparent cell type patterning with a bias towards pre-stalk cell types.