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Item Open Access Mechanistic studies on the DNA methyltransferases DNMT3A and DNMT3B(2021) Dukatz, Michael; Jeltsch, Albert (Prof. Dr.)In this work, both regulatory and catalytic mechanisms of de novo methyltransferases were investigated, which include interactions with other proteins and the specific recognition of the substrate sequence. Another part of this work strived to elucidate how enzymatic generation of 3-methylcytosine by DNMT3A can occur.Item Open Access Studien zur biotechnologischen Anwendung und ökologischen Funktion von Pyrrolochinolinchinon(PQQ)-abhängigen Alkoholdehydrogenasen(2021) Wehrmann, Matthias; Hauer, Bernhard (Prof. Dr.)Item Open Access Molecular dynamics simulations of the substrate- and product specificity and mechanism of DNA- and protein lysine methyltransferases(2024) Schnee, Philipp; Jeltsch, Albert (Prof. Dr.)Protein Lysine Methyltransferases (PKMTs) regulate the epigenetic code of cells and their alteration via somatic mutations are often associated with cancer. The aim of this project is to rationalize the product and substrate specificity of this enzyme family by a combination of biochemical experiments and molecular dynamics simulations. Based on this, a detailed view of the underlying mechanism behind the disease associated mutations shall be gained, which may provide new possibilities for personalized cancer therapies.Item Open Access Development of artificial single and double reading domains to analyze chromatin modification patterns(2018) Mauser, Rebekka; Jeltsch, Albert (Prof. Dr.)The unstructured N-terminal tails of histone proteins carry many different post-translational modifications (PTMs), like methylation, acetylation or phosphorylation. These PTMs can alter the chromatin structure, influence the interaction of adjacent nucleosomes and serve as specific binding sites for histone interacting domains. Currently, the investigation of histone tail PTMs is mainly based on antibodies, however concerns about the specificity of these antibodies and reproducibility of data arouse. Therefore, it was one aim of this thesis to develop alternative approaches to histone tail PTM antibodies. Previous studies already showed that histone modification interacting domains (HiMIDs) can replace histone tail antibodies in a highly effective manner. As part of this work, the TAF3 PHD domain was established as new H3K4me3 specific HiMID. In peptide array binding and Far-western blot assays, the domain showed a specific interaction with H3K4me3 modifications. Also in ChIP like experiments (CIDOP: Chromatin Interacting Domain Precipitation) coupled to qPCR and next generation sequencing, the domain showed a similar performance as validated H3K4me3 antibodies. With the proposal of the histone code hypothesis the question was raised if combinations of histone modifications carry specific biological functions. However, so far, the experimental analysis of the co-occurrence of histone modification on the same nucleosome in a genome-wide manner is a challenging task. For this reason, the main aim of this work was to develop double reading domains in which two histone reading domains are fused together with a flexible linker to achieve simultaneously readout of dual histone tail modifications in a single CIDOP experiment. To validate the concept, the Dnmt3a PWWP domain and the MPP8 Chromo domain were fused together and their specific recognitions of H3K36me2/3 and H3K9me3 histone tail modifications were analyzed. Biochemical investigations like peptide arrays, Far-western blot and western blot experiments showed that both domains specifically interact with their targets and preferentially interact with double modified chromatin. Additionally, the preferred interaction with double modified chromatin could be further verified with binding pocket mutants and methyl-lysine analogues. The newly generated double domain was used in chromatin precipitation experiments to identify genome regions where both modifications are present. The genome-wide distribution of the H3K36me2/3-H3K9me3 showed that this combination of histone marks represents a novel bivalent chromatin state, which is associated with weakly transcribed genes and is enriched for binding sites of ZNF274 and SetDB1. Also in this work, mixed peptide arrays were introduced as new screening method for the efficient analysis of double reading domains. The naturally occurring double reading domain of the BPTF protein was used to demonstrate the capability of this new screening tool. BPTF contains a PHD domain, which binds to H3K4me3 and a Bromo domain, which interacts with acetyl groups of the H4 tail. Synergistic binding to both peptides was shown using the newly developed mixed peptide arrays. Additionally, in the course of this work mixed peptide arrays were used to optimize several of the designed double reading domains. Furthermore, some other double reading domains were generated in this work, like PWWP-ATRX, MPP8 Chromo domain-L-double Tudor and CBX7 Chromo domain-L-MPP8 Chromo domain and analyzed for specific dual readout. Also double reading domains with dual specificity for DNA methylation and histone marks were generated. The firstly used methyl-DNA binding domain of the MBD2 protein showed a strong binding, dominating the effect of the HiMIDs. Therefore, the weaker but still specific methyl-DNA binding domain of the MBD1 protein was used. First experiments with this new fusion constructs showed a simultaneously interaction with chromatin which is associated with DNA methylation and histone PTMs. In summary, the studies with double reading domains showed that with this novel method precipitation of double modified chromatin is possible and that the genome-wide investigation of newly studied bivalent chromatin states is feasible. Therefore, this novel approach makes it possible to analyze many different combinations of histone modifications, investigate their influence on chromatin and gain a deeper understanding of the biological role behind histone tail modification patterns.Item Open Access Identifizierung und Charakterisierung von Influenza-A-Virus Fusionsinhibitoren, die aus einem Doppelmyxovirus Hochdurchsatz-Screen hervorgingen(2019) Weißhaar, Marco; Wolf, Dieter H. (Prof. Dr.)Item Open Access Regulation and readout of mammalian DNA methylation(2018) Lungu, Cristiana; Jeltsch, Albert (Prof. Dr.)The mesmerizing phenotypical and functional diversity of mammalian cell types is to a large extent attributed to epigenetic signals that work together with the DNA sequence to determine gene expression programs. DNA methylation is one of the most important types of epigenetic signals and its paramount role was recognized in early genetic studies. Still, even after decades of active research, a comprehensive understanding of the mechanisms that regulate the chromatin targeting and activity of DNA methyltransferases has not been achieved. In this work, three main directions of research were undertaken, with the ultimate goal of shedding mechanistic and methodological insights into the generation and maintenance of DNA methylation patterns. In the first project of this thesis, a combination of biochemical and cellular experiments was used to assess the cellular role of the putative chromatin remodeler HELLS, an essential cofactor of DNA methyltransferases. By employing chromatin fractionation assays and microscopy-based techniques, I could show that the ATPase activity of HELLS is necessary for the high nuclear mobility of the protein and its ability to get released from compacted chromatin sites. In addition, the H3K9me3 pathway was also found to play an important role in the exchange of HELLS at heterochromatin. Taken together, this work provides the first evidence for a role of ATP hydrolysis in the association between HELLS and chromatin and hints at a model where the fast exchange of HELLS at repetitive DNA sequences might enhance the local recruitment of epigenetic enzymes, such as DNA methyltransferases (DNMTs). This could subsequently lead to the local stabilization of silencing complexes at heterochromatin. In the second project of this thesis, the putative interaction between the de novo DNA methyltransferase DNMT3A and the 5mC-reading protein MeCP2 was addressed. By building on previous data from our laboratory, which documented a direct interaction between the TRD domain of MeCP2 and the ADD domain of DNMT3A, causing an inhibition of DNMT3A activity in vitro, I could show that these proteins also interact in the mouse brain and the inhibitory effect of this interaction is also observed in stable cells lines overexpressing MeCP2. Furthermore, by using conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, I could elucidate the mechanism of the inhibition of DNMT3A by MeCP2. Accordingly, I found that MeCP2 stabilizes an allosterically closed conformation of DNMT3A, an effect that could be successfully relieved by addition of unmodified histone H3. These results were supported by whole genome bisulfite brain methylome analysis of a Mecp2 knockout mouse model. Collectively, the findings derived from this project offer unprecedented insights into the regulation of DNMT3A activity and propose a model in which the enzyme is under the combined control of MeCP2 and H3 tail modifications. Accordingly, depending on the modification status of the H3 tail at target sites, MeCP2 can act as either a repressor or activator of DNA methylation. Finally, in the third project of this thesis, the focus was placed on the development and application of a novel method that would enable for the first time the locus-specific visualization of epigenetic modifications in living mammalian cells. This urgent and unmet technological need was solved by developing a set of modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. In these tools, the high DNA sequence specificity of engineered anchor proteins such as ZFs, TALEs, and CRISPR/Cas9 proteins, was combined with the great versatility of chromatin reading domains as natural detector modules of DNA methylation and histone 3 lysine 9 trimethylation. With this approach, I could detect both of these marks for the first time, at defined, endogenous DNA sequences in different mouse and human cell lines. Furthermore, I could follow the changes in the levels of these epigenetic modifications with locus-specific resolution after treatment with epigenetic inhibitors or the induction of epigenetic enzymes. It is anticipated that either in their present form or in combination with the ongoing developments in genomic targeting and microscopy technologies, these tools will greatly improve our understanding of how specific epigenetic signals, like DNA methylation, are set, erased and maintained during embryonic development or onset of disease. Taken together, the results of this doctoral thesis demonstrate how a synergistic use of biochemical and cellular methods allows to derive deep insights into the epigenetic signaling network centered around the regulation of mammalian DNA methylation.Item Open Access Biochemical analysis of DNA- and protein methyltransferases using recombinant designer nucleosomes(2022) Bröhm, Alexander; Jeltsch, Albert (Prof. Dr.)Item Open Access Data integration and data mining for the exploration of enzymatic sequence-structure-function relationships(2018) Buchholz, Patrick C. F.; Pleiss, Jürgen (Prof. Dr.)Item Open Access Biosynthese von N-Heterocyclen mittels Putrescinoxidase und Iminreduktase(2018) Weinmann, Leonie; Hauer, Bernhard (Prof. Dr.)Die vorliegende Arbeit befasst sich mit der Entwicklung einer neuen Enzymkaskade zur Synthese von N-Heterocyclen. Vor allem chirale N-Heterocyclen bilden wichtige Vorstufen für die Synthese von Pharmazeutika oder Agrochemikalien und sind daher von besonderem industriellem Interesse. Über 90% der potenziellen pharmazeutischen Wirkstoffe beinhalten Stickstoff und viele davon in Form von substituierten Piperidinderivaten, weswegen stetig nach neuen Synthesewegen gesucht wird. Für die Entwicklung einer neuen alternativen Enzymkaskade wurden die Putrescinoxidase von Rhodococcus erythropolis (PuO-Re) und die (R)-selektive Iminreduktase von Streptosporangium roseum (IRED-Sr) oder die (S)-selektive Iminreduktase von Paenibacillus elgii (IRED-Pe) gewählt um chirale N-Heterocyclen ausgehend von Diaminderivaten, die z.B. über die Decarboxylierung von Aminosäuren zugänglich sind, herzustellen. In dieser Kaskade oxidiert die PuO-Re eine Aminogruppe der Diamine, wodurch ein Aminoketon entsteht. Dieses Aminoketon cyclisiert spontan unter Bildung einer Schiff-Base, dieses Imin kann dann von der Iminreduktase mit dem Kofaktor NADPH zum gesättigten N-Heterocyclus reduziert werden. Da die Aktivität der PuO-Re für längerkettige oder verzweigte Diamine im Vergleich zum natürlichen Substrat 1,4-Diaminobutan drastisch abnimmt, wurde ein semi-rationales Design durchgeführt um die Aktivität gegenüber Diaminopentanderivaten zu steigern und das Substratspektrum zu erweitern. Dabei konnten zwei Varianten identifiziert werden, die eine gesteigerte Umsatzzahl für ebendiese Substrate aufweisen. Um eine weitere Steigerung der Aktivität zu erreichen wurde ein rationales Design für die aktive Tasche angewendet, dabei konnte eine Variante erstellt werden, die aliphatische Monoamine als Substrat akzeptiert und weitere zwei Varianten mit gesteigerter Aktivität gegenüber 1,5-Diaminohexan. Mit der Variante, die aliphatische Monoamine als Substrate akzeptiert, konnte gezeigt werden, dass die PuO-Re bei verzweigten Aminen, die weniger gehinderte Aminogruppe oxidiert. Alle positiven PuO-Re Varianten wurden erfolgreich mit den beiden IREDs kombiniert und die beste Umsetzung eines substituierten Diamins resultierte in vitro in 90% Produktbildung. Erstaunlicherweise konnte bei der Umsetzung von 1,5-Diaminohexan zu 2-Methylpiperidin in der Kaskade ein Switch im Enantiomerenüberschuss, zwischen den PuO-Re Varianten aus dem semi-rationalen Design und dem rationalen Design der aktiven Tasche, beobachtet werden. Somit konnte in der aktiven Tasche die Position 206 identifiziert werden, die verantwortlich ist für die Unterscheidung der Enantiomere von 1,5-Diaminohexan. Des Weiteren wurde die Kaskade mit einer der PuO-Re Variante in Kombination mit der IRED-Sr in ein in vivo System übertragen. Dabei konnte durch Variation des E. coli Stammes und durch Reaktionsoptimierung die Produktbildung von 3-Methylpiperidine aus 1,5-Diamino-2-methylpentan von 10% auf 83% gesteigert werden. Eine weitere Optimierung der Reaktionsbedingungen in Kooperation mit Sanofi führte zu einer Umsetzung von fast 30 mM 1,5-Diamino-2-methylpentan zu 3-Methylpiperidin in einem 20 L Reaktor.Item Open Access Enzymatische Hydratisierung kurzkettiger Fettsäuren und Alkene(2018) Demming, Rebecca M.; Hauer, Bernhard (Prof. Dr.)