03 Fakultät Chemie
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Item Open Access Biochemical characterisation of tRNA-Asp methyltransferase Dnmt2 and its physiological significance(2014) Shanmugam, Raghuvaran; Jeltsch, Albert (Prof. Dr.)Methylation of tRNA plays important roles in the stabilisation of tRNAs and accurate protein synthesis in cells. In eukaryotes various tRNA methyltransferases exist, among them DNMT2 which methylates tRNAAsp at position C38 in the anticodon loop. It is also called tRNA-aspartate methyltransferase 1 (Trdmt1) and the enzyme is highly conserved among eukaryotes. In this work, I investigated the mechanism of DNMT2 interaction with tRNAAsp, characterised the function of the only prokaryotic Dnmt2 homolog found in G. sulfurreducens and studied the physiological importance of the C38 methylation of tRNAAsp in mammalian cells. The molecular details of the interaction of DNMT2 and tRNAAsp are unknown due to lack of the co-crystal structure. Here, I characterised the important residues in DNMT2 required for the tRNA binding and catalysis. By site-directed mutagenesis of 20 conserved lysine and arginine residues in DNMT2, I show that 8 of them have a strong effect on the catalytic activity of the enzyme. They map to one side of the enzyme where the catalytic pocket of DNMT2 is located. The binding of most of the mutant enzymes to tRNA was unaffected suggesting a role of these residues in transition state stabilisation. Manual docking of tRNAAsp into the surface cleft decorated by the 8 residues suggested that DNMT2 interacts mainly with the anticodon stem/loop of tRNAAsp. In my second project, I characterised the function of Dnmt2 homolog found in G. sulfurreducens (GsDnmt2). Here, I show that GsDnmt2 methylates tRNAGlu more efficiently than tRNAAsp. I also report the molecular basis for the swapped substrate specificity of GsDnmt2 and show that the variable loops of G.sulfurreducens tRNAAsp and tRNAGlu of eukaryotes contain a -GG- dinucleotide which is not preferred by Dnmt2. Exchange of the variable loop of mouse tRNAAsp to that tRNAGlu led to dramatic decrease in the activity of human DNMT2. This identifies the variable loop of tRNA as a specificity determinant in the recognition by Dnmt2. In my final project, I investigated the physiological importance of the tRNAAsp C38 methylation in aminoacylation and cellular protein synthesis. Here, I report that C38 methylation enhances the rate of aspartylation on tRNAAsp by 4-5 folds. Concomitant with this, a decrease in the charging levels of tRNAAsp was observed in Dnmt2 knockout MEF cells, which also showed a reduced efficiency in the synthesis of proteins containing poly-Asp sequences. A gene ontology searches for proteins with poly-Asp sequences showed that a significant number of these proteins are associated with transcriptional regulation and gene expression functions. With this I propose that the mild phenotype observed with the Dnmt2 KO cells under stress condition could be correlated to a disregulation of protein synthesis.Item Open Access Targeted methylation of the epithelial cell adhesion molecule (EpCAM) promoter to silence its expression in ovarian cancer cells(2014) Nunna, Suneetha; Reinhardt, Richard; Ragozin, Sergey; Jeltsch, AlbertThe Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.Item Open Access Somatic cancer mutations in the MLL1 histone methyltransferase modulate its enzymatic activity and dependence on the WDR5/RBBP5/ASH2L complex(2017) Weirich, Sara; Kudithipudi, Srikanth; Jeltsch, AlbertSomatic missense mutations in the mixed lineage leukemia 1 (MLL1) histone H3K4 methyltransferase are often observed in cancers. MLL1 forms a complex with WDR5, RBBP5, and ASH2L (WRA) which stimulates its activity. The MM-102 compound prevents the interaction between MLL1 and WDR5 and functions as an MLL1 inhibitor. We have studied the effects of four cancer mutations in the catalytic SET domain of MLL1 on the enzymatic activity of MLL1 and MLL1–WRA complexes. In addition, we studied the interaction of the MLL1 mutants with the WRA proteins and inhibition of MLL1–WRA complexes by MM-102. All four investigated mutations had strong effects on the activity of MLL1. R3903H was inactive and S3865F showed reduced activity both alone and in complex with WRA, but its activity was stimulated by the WRA complex. By contrast, R3864C and R3841W were both more active than wild-type MLL1, but still less active than the wild-type MLL1–WRA complex. Both mutants were not stimulated by complex formation with WRA, although no differences in the interaction with the complex proteins were observed. These results indicate that both mutants are in an active conformation even in the absence of the WRA complex and their normal control of activity by the WRA complex is altered. In agreement with this observation, the activity of R3864C and R3841W was not reduced by addition of the MM-102 inhibitor. We show that different cancer mutations in MLL1 lead to a loss or increase in activity, illustrating the complex and tumor-specific role of MLL1 in carcinogenesis. Our data exemplify that biochemical investigations of somatic tumor mutations are required to decipher their pathological role. Moreover, our data indicate that MM-102 may not be used as an MLL1 inhibitor if the R3864C and R3841W mutations are present. More generally, the efficacy of any enzyme inhibitor must be experimentally confirmed for mutant enzymes before an application can be considered.Item Open Access ER-associated protein degradation (ERAD): an unexpected function of Yos9 and the discovery of Mnl2, a new component of the pathway(2011) Martínez Benítez, Elena; Wolf, Dieter H. (Prof. Dr.)In eukaryotes, membrane and soluble secretory proteins are synthesized at the rough endoplasmic reticulum (ER). A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD either by loss of function or by premature degradation of proteins cause a range of severe diseases in humans. In 1989 the gene responsible for the human disease cystic fibrosis (CF), cystic fibrosis transmembrane conductance regulator (CFTR), encoding a chloride channel was found. The mutation ΔF508 present in 80% of the patients provokes the most severe symptoms and shortest life expectancy. The disease is a consequence of the accelerated degradation of the protein CFTRΔF508, which never reaches its site of action. Moreover, wild type CFTR has a 25% success in reaching its final destination. Is of great value to understand the ERAD of CFTR and CFTRΔF508 in order to understand the disease. Many components involved in ERAD of these substrates had been discovered. The first part of this work involves a systematic study of human CFTR degradation in yeast (the turnover of CFTR in yeast cells behaves like CFTRΔF508 in human cells). All components examined were found not to be required for CFTR ERAD or had a very mild effect. The ER protein quality control recognizes misfolded proteins in two ways: via exposure of hydrophobic patches on the surface of a protein and modification of its glycan structure. The majority of the proteins that enter the ER are N-glycosylated. During folding of a protein, several enzymes trim these glycan trees generating a degradation signal, which is recognized by the lectin Yos9. There is little known about proteins that enter the ER but are not glycosylated. The second part of the work refers to findings that shed light onto the differences between glycosylated and non-glycosylated substrates in ERAD. To define the ERAD pathway for non-glycosylated proteins, ERAD deficient mutants were checked in their capacity to deliver a non-glycosylated protein for elimination. It is shown that unglycosylated CPY* (CPY*0000) is degraded by the same pathway as is glycosylated CPY* (ERAD-L). However, the Yos9 protein, known to be the recognition component of glycosylated misfolded proteins in ERAD, is shown in this work to have a tuning role in the ERAD of unglycosylated CPY*0000. Yos9 promotes degradation of glycosylated substrates while it hinders degradation of unglycosylated CPY*0000. Additional ERAD components are still to be discovered. In this work a putative mannosidase was found. This protein was named mannosidase like protein 2 (Mnl2). Mnl2 accelerates CPY* degradation. The effect of the deletion of MNL2 is most notable when its homologue MNL1/HTM1 is absent. Substrate degradation in the deletion strain is affected because the glycan structure on the ERAD substrate is no longer a degradation signal. This section of the work introduces a novel ER quality control component involved in glycan trimming.Item Open Access Investigation of proteins responsible for the establishment and recognition of prominent lysine modifications(2014) Tamas, Raluca; Jeltsch, Albert (Prof. Dr.)Histone post-translational modifications influence chromatin architecture, either by direct effects on the interaction between histones and DNA, or indirectly, by serving as docking places for regulatory proteins, which bind through conserved functional domains termed “reading” domains. Different combinations of histone modifications define various chromatin states, each of which being associated with a particular set of regulatory enzymes. Lysine methylation is an important histone post-translational modification, which can occur at various positions in histones, with different roles in epigenetic regulation. This mark is generally established by SET domain Protein Lysine Methyltransferases (PKMTs). Recently, PKMTs have been reported to also methylate numerous non-histone substrates, which subsequently recruit so called “reading” domains. These domains specifically interact with the methylated lysine in a sequence context-dependent manner. In this work, I tried to establish a Yeast-3-Hybrid method for the identification of methylation-dependent interactors of methylated non-histone proteins. For validation, I attempted to test the interaction between reported PKMT substrates fused to the Gal4-DNA-Binding Domain and methyl-“readers” fused to the Gal4-Activation Domain in yeast, either in the presence or absence of the corresponding PKMTs. Later in the project the known “reading” domains would be replaced by a library of human cDNA, in order to search for novel “readers” of protein lysine methylation marks. Additionally, this work presents the study of the substrate specificities of two SET domain methyltransferases responsible for the methylation of histone 3 lysine 4 (H3K4), which are mutually exclusive members of the same coactivator complex, the human COMPASS. In this study, SET1A, an H3K4 trimethylase, was shown to be active only as part of the core COMPASS complex. This PKMT proved to have a higher preference for some sequences other than histone 3, justifying a search for novel non-histone substrates. MLL2, a member of the mixed lineage leukemia (MLL) family, responsible for H3K4 monomethylation, revealed stimulation of activity when part of the core COMPASS complex, and showed some differences in the substrate specificity when acting alone, compared to the complex. The search for non-histone protein substrates is in progress for SET1A/COMPASS, and also MLL2 alone and within the complex. The targeting of most PKMTs is achieved with the help of histone modification “reading” or DNA-binding domains. The binding specificity of the PHD finger “reading” domains of MLL2, and its paralog MLL3, was investigated during this doctoral study. Although most of the PHD fingers did not bind to histone tails, the MLL2 PHD 3-5 group of domains and the MLL3 PHD 4-6 group of domains bound specifically to modified histone tail peptides. Preference towards both histone 3 (H3) and histone 4 (H4) was identified and the strongest binding was seen on H4 peptides containing acetylation at lysine 16 together with multiple acetylations or methylations. This finding suggested recruitment to active chromatin, which is enriched in acetylation marks, but the specificity needs to be further confirmed and characterized in more detail. I also investigated the histone binding specificity of PHF1, a member of the Polycomb Repressive Complex 2. This complex is responsible for developmental gene repression by the trimethylation of histone 3 lysine 27 (H3K27me3). The tudor domain of PHF1 showed preferred binding to its target, H3K27me3 in the sequence context of testis-identified histone variant H3T, in comparison to the canonical histone H3.1. The specificity for the same mark and histone variant was also identified for the chromodomain of the Polycomb Repressive Complex 1 member, CBX7, while the chromodomain of its paralog, CBX2, did not show discrimination between the histone variants, although it presented the same specificity towards the H3K27me3 mark. We propose that the discrimination between histone variants is a unique feature of some “reading” domains, and the role of this particular function needs to be elucidated. Moreover, the H3K27me3-specific CBX7 chromodomain was used as a tool in the validation of new methods developed by Kungulovski et al., 2014, with the purpose of replacing antibodies raised against specific histone modifications in adaptations of several antibody-based assays. Finally, this PhD work also presents the binding specificity of the chromodomain of the SUV39H1 methyltransferase. SUV39H1 is responsible for histone 3 lysine 9 trimethylation (H3K9me3), and the consequent gene repression and silencing of heterochromatin. I showed that the chromodomain of SUV39H1 bound specifically to H3K9me3, and binding of the chromodomain to its target peptide seemed to inhibit the catalytic activity of the enzyme in our in vitro conditions.Item Open Access Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation(2017) Maier, Johannes A. H.; Möhrle, Raphael; Jeltsch, AlbertEpigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. Here, we designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Triggering by heat, nutrients, ultraviolet irradiation or DNA damaging compounds induces CcrM expression and DNA methylation. In the induced on-state, methylation in the operator of the memory operon prevents zinc finger protein binding leading to positive feedback and permanent activation. Using an mf-Lon protease degradable CcrM variant enables reversible switching. Epigenetic memory systems have numerous potential applications in synthetic biology, including life biosensors, death switches or induction systems for industrial protein production. The large variety of bacterial DNA methyltransferases potentially allows for massive multiplexing of signal storage and logical operations depending on more than one input signal.Item Open Access Development of zinc finger methyltransferase fusion proteins for targeted DNA methylation and gene silencing in human cells(2014) Nunna, Suneetha; Jeltsch, Albert (Prof. Dr.)Epigenetic modifications such as DNA methylation and histone modifications play important roles in the regulation of gene expression. DNA methylation occurs at C5 position of cytosine residues mainly in CpG dinucleotides. In the human genome, about 70% of the CpGs are methylated. Most of the gene promoters are accompanied by CpG islands (regions rich in CpG dinucleotides) and methylation in these regions is inversely correlated with gene expression. Aberrant DNA methylation at the promoter region leads to variety of diseases including cancer. In the present study, we used catalytic domains of the Dnmt3a DNA methyltransferase and the GLP H3K9me3 lysine methyltransferase to silence two important oncogenes by targeted methylation. Zinc-finger proteins with predefined specificity were used as the targeting device. The first target gene was the vascular endothelial growth factor A (VEGF-A), which plays an important role in the vasculogenesis and angiogenesis. A zinc finger (VAZF) binding to the VEGF-A promoter was fused to either the catalytic domain of Dnmt3a (VAZF-Dnmt3aC) or to a fusion of Dnmt3a with its stimulator Dnmt3L (VAZF-Dnmt3a3Lsc). After transient transfection in ovarian cancer cells and magnetic activated cell sorting (MACS), we observed 25% methylation of the target region in the cells transfected with VAZF-Dnmt3aC and 49% in VAZF-Dnmt3a3Lsc transfected cells. VEGF-A expression was measured by quantitative -RTPCR and we observed a 36% reduction of VEGF-A expression in the cells that were transfected with VAZF-Dnmt3aC, and 56% in VAZF-Dnmt3a3Lsc transfected cells. However, transfection yields after MACS were only around 60-80% in these studies such that untransfected cells were still present. The second target gene of my study was the epithelial cell adhesion molecule (EpCAM), which is a transmembrane glycoprotein and is required for homophilic cell-cell adhesion. EpCAM is overexpressed in numerous cancers and its expression is inversely correlated with the promoter methylation status. In the present study, I used a zinc finger binding to the EpCAM promoter region, fused to the catalytic domain of the Dnmt3a DNA methyltransferase (EpZF-Dnmt3aC) for targeted methylation of the EpCAM promoter. After magnetic activated cell sorting, transfection yields of 60-80% were reached. With this approach 29% DNA methylation was achieved at the EpCAM promoter region in SKOV3 ovarian cancer cells. In stable cell lines expressing EpZF-Dnmt3aC, the methylation reached up to 48% which in turn led to 80% reduction of EpCAM protein expression. I also observed a reduction of cell proliferation in these stable cell lines which is a promising result suggesting that EpCAM repression might be an approach in cancer treatment. To improve the efficiency of gene delivery into the ovarian cancer cells, recombinant adenoviral vectors encoding the zinc-finger fused DNA methyltransferases and histone H3K9me3 methyltransferase catalytic domain were generated. Using adenovirus mediated gene delivery we achieved 53% methylation and 80% gene suppression at the VEGF-A promoter in SKOV3 ovarian cancer cells. Similarly, the zinc finger fused to GLP resulted in appearance of H3K9me3 at the promoter and 64% gene repression. At the EpCAM promoter, an EpCAM zinc finger fused to DNA methyltransferases led to 32% of methylation and 90% gene repression in the same ovarian cancer cells. Another important objective of the study was to measure the stability of DNA methylation and gene silencing of VEGF-A. After infection of SKOV3 ovarian cancer cells with adenovirus expressing VAZF-Dnmt3aC or VAZF-GLP, the methylation and gene expression was measured at different time points. In the cells that were infected with VAZF-Dnmt3aC constructs, establishment of DNA methylation was initiated 24 h after infection. The methylation reached to a maximum after five days, but surprisingly afterwards DNA methylation gradually declined to its basal level till day 15. VEGF-A supression correlated with the methylation levels, five days after infection 75% suppression of VEGF-A was observed which gradually declined to 8% after 15 days. In SKOV3 cells that were infected with recombinant adenoviral vectors encoding VAZF-GLP, H3K9 histone methylation peaked at day 5, but like the DNA methylation it gradually was lost afterwards. This result indicates that both silencing marks could not be introduced in a stable manner. As it was shown that DNA methylation and histone methylation acts synergistically, we measured DNA methylation after targeted introduction of histone H3K9 methylation and vice versa. However, in both the experiments we could not observe synergistic effects at the VEGF-A promoter. We started experiments to measure whether the decreased VEGF-A and EpCAM expression has an anti-tumor effect in a mouse model, which will explore the therapeutic potential of targeted methylation in cancer treatment.