03 Fakultät Chemie
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4
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Item Open Access Analysis of the substrate specificity of the SMYD2 protein lysine methyltransferase and discovery of novel non-histone substrates(2019) Weirich, Sara; Schuhmacher, Maren Kirstin; Kudithipudi, Srikanth; Lungu, Cristiana; Ferguson, Andrew D.; Jeltsch, AlbertThe SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.Item Open Access Mechanistic insights into the allosteric regulation of the Clr4 protein lysine methyltransferase by autoinhibition and automethylation(2020) Khella, Mina S.; Bröhm, Alexander; Weirich, Sara; Jeltsch, AlbertClr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis–Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.Item Open Access Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone(2020) Lutsik, Pavlo; Baude, Annika; Mancarella, Daniela; Öz, Simin; Kühn, Alexander; Toth, Reka; Hey, Joschka; Toprak, Umut H.; Lim, Jinyeong; Nguyen, Viet Ha; Jiang, Chao; Mayakonda, Anand; Hartmann, Mark; Rosemann, Felix; Breuer, Kersten; Vonficht, Dominik; Grünschläger, Florian; Lee, Suman; Schuhmacher, Maren Kirstin; Kusevic, Denis; Jauch, Anna; Weichenhan, Dieter; Zustin, Jozef; Schlesner, Matthias; Haas, Simon; Park, Joo Hyun; Park, Yoon Jung; Oppermann, Udo; Jeltsch, Albert; Haller, Florian; Fellenberg, Jörg; Lindroth, Anders M.; Plass, ChristophThe neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.Item Open Access Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues(2024) Weirich, Sara; Ulu, Gizem T.; Chandrasekaran, Thyagarajan T.; Kehl, Jana; Schmid, Jasmin; Dorscht, Franziska; Kublanovsky, Margarita; Levy, Dan; Jeltsch, AlbertThe HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3-R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.Item Open Access Structure, activity and function of the Suv39h1 and Suv39h2 protein lysine methyltransferases(2021) Weirich, Sara; Khella, Mina S.; Jeltsch, AlbertSUV39H1 and SUV39H2 were the first protein lysine methyltransferases that were identified more than 20 years ago. Both enzymes introduce di- and trimethylation at histone H3 lysine 9 (H3K9) and have important roles in the maintenance of heterochromatin and gene repression. They consist of a catalytically active SET domain and a chromodomain, which binds H3K9me2/3 and has roles in enzyme targeting and regulation. The heterochromatic targeting of SUV39H enzymes is further enhanced by the interaction with HP1 proteins and repeat-associated RNA. SUV39H1 and SUV39H2 recognize an RKST motif with additional residues on both sides, mainly K4 in the case of SUV39H1 and G12 in the case of SUV39H2. Both SUV39H enzymes methylate different non-histone proteins including RAG2, DOT1L, SET8 and HupB in the case of SUV39H1 and LSD1 in the case of SUV39H2. Both enzymes are expressed in embryonic cells and have broad expression profiles in the adult body. SUV39H1 shows little tissue preference except thymus, while SUV39H2 is more highly expressed in the brain, testis and thymus. Both enzymes are connected to cancer, having oncogenic or tumor-suppressive roles depending on the tumor type. In addition, SUV39H2 has roles in the brain during early neurodevelopment.Item Open Access Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate(2020) Schuhmacher, Maren Kirstin; Beldar, Serap; Khella, Mina S.; Bröhm, Alexander; Ludwig, Jan; Tempel, Wolfram; Weirich, Sara; Min, Jinrong; Jeltsch, AlbertSETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.Item Open Access Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state(2017) Mauser, Rebekka; Kungulovski, Goran; Keup, Corinna; Reinhardt, Richard; Jeltsch, AlbertHistone post-translational modifications (PTMs) play central roles in chromatin-templated processes. Combinations of two or more histone PTMs form unique interfaces for readout and recruitment of chromatin-interacting complexes, but the genome-wide mapping of co-existing histone PTMs remains an experimentally difficult task. We introduce here a novel type of affinity reagents consisting of two fused recombinant histone modification interacting domains (HiMID) for direct detection of doubly modified chromatin. To develop the method, we fused the MPP8 Chromodomain and DNMT3A PWWP domain which have a binding specificity for H3K9me3 and H3K36me2/3, respectively. We validate the novel reagent biochemically and in ChIP applications and show its specific interaction with H3K9me3-H3K36me2/3 doubly modified chromatin. Modification specificity was confirmed using mutant double-HiMIDs with inactivated methyllysine binding pockets. Using this novel tool, we mapped co-existing H3K9me3-H3K36me2/3 marks in human cells by chromatin interaction domain precipitation (CIDOP). CIDOP-seq data were validated by qPCR, sequential CIDOP/ChIP and by comparison with CIDOP- and ChIP-seq data obtained with single modification readers and antibodies. The genome-wide distribution of H3K9me3-H3K36me2/3 indicates that it represents a novel bivalent chromatin state, which is enriched in weakly transcribed chromatin segments and at ZNF274 and SetDB1 binding sites.Item Open Access Identification of protein lysine methylation readers with a yeast three-hybrid approach(2018) Rawłuszko-Wieczorek, Agnieszka Anna; Knodel, Franziska; Tamas, Raluca; Dhayalan, Arunkumar; Jeltsch, AlbertBackground: Protein posttranslational modifications (PTMs) occur broadly in the human proteome and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylations sites among histone and non-histone proteins have been characterized, however, the list of readers associated with them is incomplete. Results: This study introduces a modified yeast-three-hybrid system (Y3H) to screen for methyl-lysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like chromodomain of CBX1 or MPP8 as prey. We demonstrate the proof of principle of the method, and, more importantly, we show that an unbiased screen using a library composed of human-specific open reading frames led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei. Conclusions: Our approach introduces a cost-effective method for screening reading domains binding to histone and non-histone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the high capability of the Y3H assay which will allow for proteome wide screens of PTM readers.Item Open Access H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1(2017) Jurkowska, Renata Z.; Qin, Su; Kungulovski, Goran; Tempel, Wolfgang; Liu, Yanli; Bashtrykov, Pavel; Stiefelmaier, Judith; Jurkowski, Tomasz P.; Kudithipudi, Srikanth; Weirich, Sara; Tamas, Raluca; Wu, Hong; Dombrovski, Ludmila; Loppnau, Peter; Reinhardt, Richard; Min, Jinrong; Jeltsch, AlbertSETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.Item Open Access Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2(2018) Rajavelu, Arumugam; Lungu, Cristiana; Emperle, Max; Dukatz, Michael; Bröhm, Alexander; Broche, Julian; Hanelt, Ines; Parsa, Edris; Schiffers, Sarah; Karnik, Rahul; Meissner, Alexander; Carell, Thomas; Rathert, Philipp; Jurkowska, Renata Z.; Jeltsch, AlbertDespite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.