Browsing by Author "Jeltsch, Albert (Prof. Dr.)"

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Open Access
Allele-specific epigenome editing : development and clinical application
(2024) Kouroukli, Alexandra; Jeltsch, Albert (Prof. Dr.)
Open Access
Application and engineering of targeted DNA methylation editing tools for the modulation and characterization of the epigenome network
(2022) Broche, Julian; Jeltsch, Albert (Prof. Dr.)
Despite of the large variety of different cell types in humans, all cells carry the same genetic information. In order to enable a cell type-specific expression profile, a multitude of epigenetic signals has to be established and maintained by various epigenetic mechanisms. Histone post-translational modifications and DNA methylation are two major epigenetic signals and parts of the epigenome network, which regulate the activation or silencing of gene expression by modulating the chromatin accessibility. In mammals, DNA methylation patterns are established by DNA methyltransferases, and aberrant methylation patterns have been connected to various disorders including cancer. Thus, it is of particular interest for basic research and medical applications to gain a better understanding of this mark, and to artificially manipulate the DNA methylation state at a certain locus. This can be achieved by the employment of diverse epigenome editing tools, termed ‘EpiEditors’, allowing to install or remove DNA methylation with variable specificity. Aim of the main part of this PhD work was to characterize the stability of DNA methylation at CpG islands (CGIs) after artificial introduction. For installation of DNA methylation, a zinc finger (ZnF) was used as targeting device, which has a short DNA binding motif allowing to target numerous genomic loci simultaneously. By conducting ChIP experiments, more than 15,000 ZnF peaks were identified, demonstrating its promiscuous binding in the genome. The fusion of the DNMT3A catalytic domain (3AC) to the ZnF enabled to deposit DNA methylation at thousands of CGIs. Afterwards, the dynamics of this mark were tracked for up to 11 days by MBD2-seq. Strikingly, the installed DNA methylation was only transient at around 90 % of all previously unmethylated CGIs. However, high levels of residual DNA methylation were observed at 10 % of the CGIs. Intriguingly, these stably methylated CGIs were strongly enriched in H3K27me3, a mark associated with Polycomb group chromatin. The deposition of DNA methylation resulted in a marked reduction of the activating marks H3K4me3 and H3K27ac, which were partially recovered upon DNA methylation loss. Surprisingly, no direct effects of DNA methylation were observed on H3K9me3 and H3K36me3 levels. However, the expression of more than 900 genes was downregulated at least two-fold after DNA methylation editing of the corresponding promoter, demonstrating the high silencing capacity of the mark. Thereby, the expression changes were tightly correlated to the temporal changes promoter methylation levels. In the second project of this thesis, the goal was to develop an improved EpiEditor, capable to introduce DNA methylation with high efficiency at the target locus but with low off-target activity. For this, two dCas9-based targeting strategies in combination with two different effector domains, 3AC and a chimeric construct of 3AC fused to the C-terminal domain of DNMT3L (3AC-3L), were compared. The direct fusion of the effector domains to dCas9 resulted in a lower specificity than a connection of dCas9 and effector domain with a dCas9-SunTag system, which is based on the recruitment of multiple antibody-fused effector domains to an array of GCN4 peptides fused to dCas9. Although a high specificity of the dCas9-SunTag system in combination with 3AC was observed, the DNA methylation efficiency was rather low. In contrast, dCas9-SunTag together with 3AC-3L was more efficient, but on genome-wide scale, the off-target activity was very high. By introducing charge-reversal mutations into basic residues required for the interaction of 3AC-3L with DNA, most of the off-target activity of the EpiEditor could be removed without compromising its on-target activity strongly. In the end, the novel EpiEditor outperformed the previously available dCas9-SunTag construct in combination with 3AC. The third project of this work aimed to reprogram the H19/IGF2 imprinting control region (ICR) by targeted DNA demethylation. To this end, the dCas9-SunTag system in combination with the catalytic domain of TET1 was employed. Multiple CpG-rich motifs within the ICR were targeted by using a multi-sgRNA plasmid. Strikingly, a strong reduction in DNA methylation was obtained. By conducting CTCF-ChIP, it could be demonstrated that the DNA demethylation was accompanied by the recruitment of the methylation-sensitive insulator protein CTCF. The reduced DNA methylation and the increased CTCF occupancy were maintained for almost four weeks, indicating the robustness of the reprogrammed state. All in all, the obtained data provide valuable information on the dynamics of targeted DNA methylation and its regulatory effects. In combination with the development of better EpiEditors, this PhD work may pave the way towards safer and more efficient clinical applications of EpiEditing.
Open Access
Biochemical analysis of DNA- and protein methyltransferases using recombinant designer nucleosomes
(2022) Bröhm, Alexander; Jeltsch, Albert (Prof. Dr.)
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.
Open Access
Biochemical characterization and identification of novel substrates of protein lysine methyltransferases
(2019) Schuhmacher, Maren Kirstin; Jeltsch, Albert (Prof. Dr.)
The methylation of lysine side chains is a prevalent post-translational modification (PTM) of proteins, which is introduced by protein lysine methyltransferases (PKMTs). Histone methylation can have different effects on chromatin structure, lysine methylation of non-histone proteins can regulate protein/protein interactions and protein stability. For most PKMTs currently not all methylation sites are known which limits our understanding of the regulatory role of these enzymes in cells. Therefore, it is an important research aim to gain more information about the substrate spectrum of PKMTs. The identification of the substrate specificity of a PKMT is a very important step on the way to identify new PKMT methylation sites. The focus of this study was the analysis of the substrate specificity of different PKMTs by SPOT peptide arrays and based on this on the identification and validation of possible new methylation substrates. The analysis of the substrate specificity of human SUV39H2 revealed significant differences to its human homolog SUV39H1, although both enzymes methylate the same histone substrate (H3K9). SUV39H2 is more stringent than the SUV39H1, which could be demonstrated by the lack of methylation of SUV39H1 non-histone targets by SUV39H2 and by the fact that it was not possible in this study to identify non-histone substrates for SUV39H2. Kinetic studies showed that SUV39H2 prefers the unmethylated H3K9 as substrate. Moreover, it was shown that the N324K mutation of SUV39H2 which leads to a genetic disease in Labrador retrievers causes a change in folding finally leading to the inactivation of the enzyme. It had been reported by another group that the histone variant H2AX is methylated by SUV39H2. However, the sequence of H2AX K134 does not fit to the substrate specificity profile of SUV39H2 determined in the present work. Follow-up in vitro peptide and protein methylation studies indeed showed that H2AX K134 is not methylated by SUV39H2. This indicates that H2AX methylation by SUV39H2 is most probably a wrong assignment of a substrate to a PKMT. Based on already available specificity data for the SUV39H1 PKMT, the SET8 protein was validated as novel substrate in cellular studies. SET8 is a PKMT itself and it could be shown in this thesis that methylation of SET8 at residue K210 by SUV39H1 stimulated the SET8 activity. In humans, there exist different PKMTs, which methylate H3K36. For example, NSD1, NSD2 and SETD2 which were investigated in this thesis. In literature, it was shown that the oncohistone mutation K36M inactivates NSD2 and SETD2. Steady-state methylation kinetics using a peptide substrate and a K36M peptide as inhibitor revealed that NSD1 is inhibited by this histone oncomutation as well. The steady-state inhibition parameters for all enzymes showed a better binding of the PKMTs to the inhibitor peptide than to the substrate, suggesting some mechanistic similarities in target peptide interaction. The SETD2 is a methyltransferase, which is able to introduce trimethylation of H3K36. During this thesis two substrate specificity motifs of SETD2 were determined using peptide array methylation experiments. Additionally, based on the substrate specificity investigations a super-substrate at peptide and protein level was determined. Furthermore, one novel substrate (FBN1) for SETD2 was discovered and validated. The Legionella pneumophila RomA PKMT was shown previously by our collaborators to methylate H3 at K14. Based on the specificity profile of RomA determined in this study it could be shown that this enzyme methylates seven additional human non-histone proteins. Collaborators tested the methylation of one of the non-histone targets (AROS) and could demonstrate its methylation during the infection of human cells with L. pneumophila. The role of these methylation events in the infection process must be studied in future experiments.
Open Access
Biochemical characterization of protein lysine methyltransferases-regulation, specificity and effect of somatic cancer mutants
(2023) Khella, Mina S.; Jeltsch, Albert (Prof. Dr.)
Open Access
Biochemical investigation of the substrate specificity of protein methyltransferases and the identification of novel substrates
(2016) Kusevic, Denis; Jeltsch, Albert (Prof. Dr.)
Posttranslationale Proteinmodiﬁkationen (PTMs) sind wichtig, um verschiedene Proteinfunktionen, wie z. B. Lokalisation, Aktivität, Stabilität und Protein-Protein Interaktionen zu regulieren. In Proteinen können viele Aminosäuren methyliert werden, darunter auch Lysin, Arginin und Glutamin. Methylierungen sind auf vielen verschieden Protein zu ﬁnden, jedoch sind Histonproteine die bedeutendsten. Die Histonmethylierung beeinﬂusst die Chromatinstrukur und spielt eine große Rolle in der Regulation der Transkription. Die Enzyme, die für den Transfer von Methylgruppen auf die Proteine zuständig sind, werden Protein Methyltransferasen (PMTs) genannt. Sie sind sehr speziﬁsch und methylieren immer nur eine Art von Aminosäuren. Dabei zeigt die schnell steigende Anzahl an Berichten über die Methylierung von Proteinen, dass die Methylierung als posttranslationale Modiﬁkation in den letzten Jahren immer mehr an Bedeutung gewinnt. In dieser Doktorarbeit wurde die Substratspeziﬁtät dreier unterschiedlicher Protein Methyltransferasen untersucht, und zwar von HEMK2, einer Glutamin Methyltransferase, sowie von NSD2 und Clr4, zwei Protein Lysin Methyltransferasen (PKMTs). Die Glutamin Methyltransferase HEMK2 methyliert Q185 des Terminationsfaktors eRF1 (eukaryotic translation release factor 1), der für die Termination der Peptidsynthese und für die Hydrolyse der Polypeptidkette von der tRNA am Ribosom verantwortlich ist. Zur Bestimmung der Substratspeziﬁtät von HEMK2 wurde die Aminosäuresequenz von eRF1 als Vorlage verwendet und die erhaltenen Daten zeigen, dass das Substrat für eine Methylierung ein G-Q-X3-K Sequenzmotiv besitzen muss. Eine Suche nach dieser Sequenz in einer Proteindatenbank ergab, dass mehrere humane Proteine dieses Sequenzmotiv besitzen. Von diesen identiﬁzierten Substratkandidaten wurden 125 von HEMK2 auf Peptidebene methyliert. Außerdem konnte gezeigt werden, dass von diesen 125 Kandidaten 16 auf Proteinebene methyliert werden. Zuletzt wurde eine Methylierung der „Chromodomain helicase DNA binding protein 5“ (CHD5) und „Nuclear protein in Testis“ (NUT) Proteine mit Hilfe eines glutaminspeziﬁschen Antikörpers in menschlichen HEK293 Zellen nachgewiesen. NSD2 ist ein Mitglied der „nuclear receptor SET domain-containing“ Enzymfamilie und dimethyliert Lysin K36 von Histon H3 und Lysin K44 von Histon H4. Es wurde gezeigt, dass eine abnormale Expression von NSD2 zu verschiedenen Arten von Krebs und dem Wolf-Hirschhorn Syndrom führen kann. Die Analyse der Substratspeziﬁtät von NSD2 zeigte, dass dieses Enzym die Aminosäuren G33 bis P38 von H3 erkennt. Dabei werden hydrophobe Aminosäuren an den Positionen -1 und +2 (das Ziellysin wird hierbei als Position 0 deﬁniert) bevorzugt. Mit Hilfe des Speziﬁtätsproﬁls von NSD2 wurden mehrere humane Proteine identiﬁziert, die dieses Sequenzmotiv enthalten. Von diesen identiﬁzierten Substratkandidaten wurden 45 durch NSD2 auf Peptidebene methyliert. Des Weiteren wurde gezeigt, dass 3 Kandidaten (ATRX, FANCM und SET8) auf Proteinebene methyliert wurden und zusätzlich konnte die Methylierung von ATRX und FANCM durch NSD2 in HEK293 Zellen nachgewiesen werden. Da die Methylierungen einen erheblichen Einﬂuss auf die Eigenschaften und Funktionen von Proteinen besitzen, müssen weitere Experimente an den neuen Substraten von HEMK2 (CHD5 und NUT) und NSD2 (ATRX und FANCM) durchgeführt werden, um die Auswirkungen auf die biologischen Funktionen der Methylierung herauszuﬁnden. Abgesehen von den menschlichen Enzymen, wurden ähnliche Untersuchungen auch an der Histon Lysin Methyltransferase Clr4, einem SUV39H1-Homolog aus S. pombe, durchgeführt. Clr4 trimethyliert Lysin K9 des Histonproteins H3. Zur Bestimmung des Speziﬁtätsproﬁls von Clr4 wurde die Aminosäuresequenz von H3 (1 - 18) verwendet. Die Ergebnisse zeigten, dass Clr4 speziﬁsch die Aminosäuren der Positionen -1 bis +3 der Zielsequenz erkennt. Zusätzlich wurden 6 neue Peptidsubstrate aus S. pombe identiﬁzieren, die durch Clr4 methyliert wurden. Um die Detektion von Proteinmethylierungen weiter zu verbessern, wurde eine neue radioaktivitätsfreie, Mikrotiter-Untersuchungsmethode entwickelt, die natürlich vorkommende Lese-Domänen anstelle von methylspeziﬁschen Antikörpern zur Erkennung von Methylierungen auf Histonpeptiden verwendet. Es wurde gezeigt, dass diese Methode erfolgreich die Methyltransferaseaktivität bestimmen und für die Suche nach PKMT Inhibitoren verwendet werden kann.
Open Access
Biochemical investigations of multivalent chromatin reading domains
(2024) Choudalakis, Michel; Jeltsch, Albert (Prof. Dr.)
In eukaryotes, the negatively charged nuclear DNA wraps around cationic histone proteins to form nucleosomes and compact the genetic information. Histones carry several post-translational modifications (PTMs) that appear in combinatorial patterns. These marks are interpreted by non-covalent interactions with proteins containing histone modification interacting domains (HiMIDs), also known as “reader” domains. Thirty years ago, it was proposed that the histone marks act as signals in the regulation of transcription and other chromatin functions. With time, this concept has been refined to suggest that combinatorial patterns of marks represent context-specific signals, termed a 'histone code'. It functions as one of the epigenetic regulatory mechanisms, which control reversible and heritable changes in cellular phenotype. Intermolecular models demonstrate thermodynamic benefits from multivalent engagement of nucleosomes, suggesting their widespread occurrence. However, so far only few multivalent readers are known and dissecting their function has been very challenging. This thesis focuses on HiMIDs with complex roles that simultaneously interact with two histone PTMs or two different substrates. Introducing the theoretical foundation, I discuss the thermodynamic and biological basis of how multivalent interactions can guide effector protein complexes, targeting their functions to distinct regions and chromatin states. Then, I present data from the characterisation of the readers DNMT3A-PWWP, DDX19A, and UHRF1-TTD in the context of multivalent engagement of histone PTMs and biomolecules. Starting with DNMT3A-PWWP, I quantified the binding of the wild-type (WT) and a mutant domain to histone H3K36me2/3 peptides, showing negligible differences, while my colleagues showed that the mutant has drastically reduced binding to DNA and nucleosomal substrates. I, then, studied the R-loop helicase DDX19A to demonstrate a very strong binding to H3K27me3 peptides in the nanomolar range, complementing the findings of a complex functional study. The latter showed that interaction with H3K27me3 is necessary for robust DDX19A-mediated R-loop resolution, and LSD1-target gene silencing. With UHRF1-TTD, I discovered and quantified its preferential binding to H3K4me1-K9me2/3 peptides vs H3K9me2/3 alone and engineered mutants with specific and differential binding changes leading to the discovery of a novel Kme1 read-out mechanism, based on the interaction of R207 methylene groups with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. High-throughput sequencing (HTS) data revealed strong TTD binding at chromatin sites with H3K4me1 peaks and broad H3K9me2/3 signal, which are enriched on enhancers and promoters of cell-type specific genes at the flanks of cell-type specific transcription factor binding sites. Data from the full-length protein in mouse and human cells evidenced the physiological role of the H3K4me1-K9me2/3 double marks in TTD-mediated UHRF1 recruitment. To further illustrate this point, I investigated UHRF1-dependent silencing of repeat elements (RE). To this end, I developed RepEnTools, improving the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, with carefully chosen and validated settings, enhancing accessibility, and adding some key functions. RepEnTools analyses showed that chromatin binding of hUHRF1-TTD and full-length mUHRF1 was strongly enriched on different REs promoters with the H3K4me1-K9me3 double mark where UHRF1 represses their expression. The data suggest a novel functional role for the H3K4me1-K9me3 signal of the histone code that is both sequence independent and conserved in two distinct mammals. Taken together, the work presented here is consistent with and supports the histone code theory, best illustrated by UHRF1-TTD which binds a specific double mark that has a biological meaning going beyond the meaning of the individual marks. In this thesis, I presented various mechanisms that influence epigenomic regulation, including chromatin 3D-architecture, accessibility, transcription factor recruitment, and chromatin marks. Especially in the context of UHRF1-TTD functions, I discussed how DNA, RNA, histones, and covalent modifications thereof interweave to produce the signalling network necessary throughout the lifetime of the mammalian cell, during differentiation, development and every other phase of life. Thus, within the three-dimensional scaffold of chromatin structures these biomolecules and their modifications collectively form the context-specific network of effectors and maintainers of the epigenomic modifications. The ways in which they influence transcription and translation are only now becoming unravelled. Hence, the recent data suggest the existence of not just a histone code, but a 3D-chromatin modification code, which dictates how biomolecules and their modifications collectively implement epigenomic regulation by interactions along the chromatin and through 3D space. As shown in these projects, readers commonly use the mechanism of multivalent interactions to interpret such contextual signals and guide epigenomic effectors to their targets. The tools and workflows that were developed and applied in this work can be employed to reveal more instances of refined read-out among HiMIDs. Additionally, I leveraged my experience with fluorescence spectroscopy and made contributions to another two published studies. The first study demonstrated that the DNMT3A-ADD Zn-finger domain, which is a known H3K4me0 reader, also binds to a domain from the MECP2 protein. The association was quantified, and the specificity demonstrated with a binding deficient triple mutant. This interaction offers complex additional regulation options to DNMT3A and MECP2, in interplay with the histone code. The second study focused on SETD2, a H3K36me3 depositing enzyme, and the mechanism of its preference for a designed “super substrate” peptide. By elegantly combining computational simulations and experimental data, the study demonstrated that an H3 peptide substrate predominantly exists in an extended conformation in solution, while the super substrate forms a hairpin conformation. Upon binding to the enzyme, the hairpin is opened and the super substrate adopts a similar conformation as the canonical substrate. These results highlighted the dynamic nature of solubilised peptides' conformations, their impact on protein-protein interactions, and the significance of dynamic conformational changes in interactions.
Open Access
Deep enzymology studies on the mammalian DNA methyltransferases and methylcytosine dioxygenases
(2022) Adam, Sabrina; Jeltsch, Albert (Prof. Dr.)
Open Access
Design of artificial functional and regulatory systems in bacteria
(2017) Maier, Johannes A. H.; Jeltsch, Albert (Prof. Dr.)
Open Access
Development and application of experimental tools for studying the distribution and dynamics of chromatin modifications
(2015) Kungulovski, Goran; Jeltsch, Albert (Prof. Dr.)
All cells in a multicellular organism carry the same genetic information, and yet throughout their lifetimes they follow unique transcriptional programs, which lead to phenotypical and functional differences. These differential gene expression programs are enacted by highly coordinated epigenetic mechanisms, which include modifications of chromatin, such as DNA methylation and histone post-translational modifications. Their involvement in chromatin-associated processes, association with different genomic elements and the means of their establishment and maintenance are crucial scientific issues. The primary focus of this study was to shed light on the genome-wide distribution of chromatin modifications and the effects of their local establishment. First, we focused our efforts into developing and applying novel affinity reagents for local and genome-wide characterization of histone modifications. We made use of native and engineered recombinant proteins that have an intrinsic ability to interact specifically with modified histones. In a rigorous side-by-side comparison with high quality histone modification antibodies, we successfully applied these novel affinity reagents in approaches such as western blot and chromatin precipitation coupled with quantitative PCR or massively parallel sequencing, following established quality control criteria. By this, we validated the feasibility of this strategy. We also discussed in detail the advantages of using recombinant proteins in lieu of antibodies, such as their cheap production with high yield, ease of protein engineering and consistent quality and reproducibility. Secondly, we wanted to clarify some of the principal mechanisms by which epigenetic modifications operate inside the cell. To this aim, we established and applied an approach based on zinc finger targeted promoter methylation of VEGF-A (vascular endothelial growth factor) in order to study the in vivo effects and dynamics of histone modifications and DNA methylation. Adenoviral constructs made of the targeting zinc finger were fused with catalytic domains from epigenetic enzymes such as DNA and histone methyltransferases and were used to infect cells. By these means we were able to successfully follow in detail the establishment, effects and kinetics of the installed chromatin modifications. Our data indicate that local chromatin editing of a target locus can change its intial chromatin state and consequently modulate its transcriptional output, albeit for a short period of time, before it returns to its native configuration. In conclusion, in this body of work we were able to successfully develop and apply novel affinity reagents for studying the distribution of histone modifications. Along the same lines, we also successfully developed and applied a strategy for targeted chromatin editing, which allowed us to study the dynamics of establishment, downstream effects and maintenance of chromatin modifications.
Open Access
Development and application of fluorescent reporter assays for the investigation of chromatin regulation
(2021) Pinter, Sabine; Jeltsch, Albert (Prof. Dr.)
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.
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.
Open Access
Enzymatic characterization of protein lysine methyltransferases
(2017) Weirich, Sara; Jeltsch, Albert (Prof. Dr.)
Histone lysine methylation is an epigenetic mechanism that is involved in the regulation of many biological processes. Over the last decade, the global interest in protein lysine methylation events increased drastically, because several protein lysine methyltransferases (PKMTs) and lysine methylation sites were identified in the genomes and proteomes of many organisms also including non-histone proteins functioning as substrates for PKMTs. The fast development of this field has moved the understanding of the biological outcome of lysine methylation into the center of research. Most urgently, it is necessary to improve our knowledge about lysine methylation by connecting specific target sites with the responsible PKMT and identifying the full substrate spectrum of PKMTs. In this thesis substrate specificity analysis was performed to tackle this challenge. It was shown that methylation of substrate specificity arrays is a good approach to analyze the substrate preference of PKMTs and identify subtle differences between related enzymes with same overall specificity. Furthermore, substrate specificity analysis was shown to be useful for the identification of novel substrates, which was successfully demonstrated for SUV4-20H1, SUV4-20H2, MLL1 and MLL3 in the present study. In vitro methylation experiments indicated that SUV4-20H1 and SUV4-20H2 introduce dimethylation on H4K20 using monomethylated H4K20 as substrate. SUV4-20H1 and SUV4-20H2 have an overlapping sequence motif, but SUV4-20H2 is less specific. This result was supported by the identification of one novel non-histone substrate for SUV4-20H1 and three non-histone targets for SUV4-20H2. MLL1 and MLL3 are H3K4 methyltransferases, but they belong to different MLL subfamilies. MLL1 catalyzes H3K4 trimethylation at promotors of developmental genes, whereas MLL3 introduces H3K4 monomethylation at enhancers. MLL1 and MLL3 are parts of related multi protein complexes also containing WDR5, RBBP5 and ASH2L. Substrate specificity analysis of MLL1 showed that it accepts several other residues at many positions of the target sequence, in addition to the residues in the original sequences of H3. At the protein level two novel substrates (TICRR and ZNF862) were methylated by MLL1. Comparison of the relative activity showed that the H3 protein was the best target in the absence of complex partners, but ZNF862 was preferred in presence of WRA. Finally, my data indicate that the substrate specificity of MLL3-WRA differed slightly from MLL1, suggesting that they may have different non-histone substrates. In several publications, assignments between PKMTs and methylated histone or non-histone target sites have been reported, but in some cases the data are questionable. This could lead to wrong interpretation of biological processes and misleading of follow-up studies. It has been shown for two examples in this study, that substrate specificity analysis can be used to identify problematic assignments between PKMT and methylation events, which need to be studied experimentally to confirm the published findings. Vougiouklakis et al. (2015) reported that SUV4-20H1 methylates ERK1 at K302 and K361, but these target sites do not fit to the specificity profile of SUV4-20H1. Indeed, I could not detect methylation of ERK1 by SUV4-20H1 or SUV4-20H2 at peptide and protein level although positive controls showed the expected methylation. Dhami et al. (2013) reported that Numb protein is methylated by SET8 at K158 and K163, which was not in agreement with the specificity data of SET8. In this thesis, Numb peptide and protein methylation was studied using recombinant SET8 purified from E.coli or HEK293 cells. In both cases, no methylation of Numb could be observed. These data suggest that these assignments of methylation substrates and PKMTs are likely not correct. Whole genome and whole transcriptome sequencing projects have frequently found somatic mutations in epigenetic enzymes in cancers. Somatic cancer mutations can have loss-of-function or gain-of-function effects on the enzymatic properties of PKMTs. Especially gain-of-function effects are a challenge in understanding their role in carcinogenesis. In this study, the effects of somatic cancer mutations found in the SET domain of MLL1 and MLL3 were analyzed. Four somatic cancer mutations of MLL1 and three of MLL3 were selected for analysis on the basis of their location close to binding sites of AdoMet, peptide or the interaction partners. The investigation of somatic cancer mutations in MLL1 and MLL3 indicated that each specific mutation has its unique effect on the enzymatic activity, product or substrate specificity and principle regulatory mechanism indicating that each mutant needs specific in depth experimental investigation in order to understand its carcinogenic effect. Moreover, inhibitor studies demonstrated that each mutant needs to be experimentally studied to allow for the development of mutation specific therapeutic strategies.
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.
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.
Open Access
Mechanistic study on the DNA methyltransferase DNMT3A
(2024) Kunert, Stefan; Jeltsch, Albert (Prof. Dr.)
Open Access
Mechanistic study on the DNA methyltransferase DNMT3A
(2019) Emperle, Max; Jeltsch, Albert (Prof. Dr.)
The phenotypical and functional diversity of mammalian cell types can be attributed to a large extent to epigenetic signals that determine and stabilize gene expression profiles. One of the most important types of epigenetic signals is DNA methylation. This modification is set early in development by the de novo DNA methyltransferases DNMT3A and DNMT3B, and is found predominantly at the C5 position of cytosine bases in a CpG dinucleotide context. The accurate setting of DNA methylation patterns is critical for normal development and is determined by the precise recruitment and control of DNMT activity on chromatin. In this work, four main directions of research were undertaken, with the ultimate goal of shedding novel mechanistic insights into the mechanism of DNMT3A, its regulation by chromatin signals and interaction partners, as well as the dysregulation of this enzyme in cancer. Furthermore, the potential of DNMT3A to generate 3-methylcytosine as a side reaction was explored. The DNA methyltransferase DNMT3A has been shown to multimerize on DNA and to form large multimeric protein/DNA fibers. However, it has also been postulated that this enzyme can methylate DNA in a processive manner, a property incompatible with fiber formation. By using a dedicated set of biochemical experiments, I was able to show that the DNA methylation rate of DNMT3A increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide, which cannot accommodate DNMT3A polymers. Methylation experiments over a range of enzyme concentrations and with substrates containing one or two CpG sites did not provide evidence for a processive mechanism. The addition of a catalytically inactive DNMT3A mutant was found to increase the DNA methylation rate by DNMT3A on the long substrate but not on the short one. Together, these data clearly indicate that DNMT3A binds to DNA in a cooperative reaction and the formation of protein/DNA fibers increases the DNA methylation rate. These results contribute mechanistic insights into the mode by which DNA methylation patterns are established during development. The second project dealt with characterizing the effects of the R882H exchange on DNMT3A. The R882H mutation is found in the DNA binding interface of DNMT3A and is frequently observed in acute myeloid leukemia (AML). By establishing a double-tag affinity purification system, I was able to show that the mutation only leads to a minor reduction in overall DNA methylation activity in mixed R882H/wildtype DNMT3A complexes. However, a pronounced change in flanking sequence preference of the DNMT3A-R882H mutant was found. Accordingly, a substrate designed to contain the target CpG site flanked by sequences preferred by R882H was better methylated by the variant than by the wildtype enzyme. Together, these data strongly argue against a dominant-negative effect of the R882H mutation and rather propose a site-specific gain-of-activity effect. These findings are in agreement with a recently determined structure of DNMT3A in complex with DNA and they might explain the high prevalence of this specific point mutation in AML. The third project was built on previous data from the lab, documenting a strong and direct interaction between the ADD domain of DNMT3A and the TRD domain of the 5mC reading protein MECP2. These experiments revealed that through its binding, MECP2 allosterically stabilizes the autoinhibitory conformation of DNMT3A, resulting in a strong inhibition of enzymatic activity in vitro. The interaction between these two proteins and its associated inhibition could be disrupted by unmodified histone H3. In my work, I further validated the interaction between the ADD and the TRD domains by size exclusion chromatography. Also, by generating cell lines with stable over-expression of MECP2, I could show that MECP2 inhibits DNMT3A activity in cells. Together, the data from this study offer unprecedented insights into the regulation of DNMT3A by the combined action of chromatin modifications and interaction partners. Accordingly, depending on the modification status of the H3 tail at the target site, MECP2 can act as either a repressor or activator of DNA methylation. The last project dealt with the coevolution between DNA methylation and DNA repair systems, a very exciting topic that was addressed in close collaboration with the laboratory of Dr. Peter Sarkies (MRC London). By performing in vitro methylation experiments with the catalytic domain of DNMT3A, I could show that, in addition to 5mC, DNMT3A can also introduce 3mC, a modification which represents an alkylation damage of DNA. This study provides a new evolutionary perspective on the loss of DNA methylation that is observed in many species.
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.