03 Fakultät Chemie
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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 Refined read‐out : the hUHRF1 Tandem‐Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3(2023) Choudalakis, Michel; Kungulovski, Goran; Mauser, Rebekka; Bashtrykov, Pavel; Jeltsch, AlbertUHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.Item Open Access Locus-specific and stable DNA demethylation at the H19/IGF2 ICR1 by epigenome editing using a dCas9-SunTag system and the catalytic domain of TET1(2024) Albrecht, Claudia; Rajaram, Nivethika; Broche, Julian; Bashtrykov, Pavel; Jeltsch, AlbertDNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders.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 Development of an epigenetic tetracycline sensor system based on DNA methylation(2020) Ullrich, Timo; Weirich, Sara; Jeltsch, AlbertBacterial live cell sensors are potentially powerful tools for the detection of environmental toxins. In this work, we have established and validated a flow cytometry readout for an existing bacterial arabinose sensor system with DNA methylation based memory function (Maier et al., 2017, Nat. Comm., 8:15336). Flow cytometry readout is convenient and enables a multiparameter analysis providing information about single-cell variability, which is beneficial for further development of sensor systems of this type in the future. We then designed a tetracycline sensor system, because of the importance of antibiotics pollution in the light of multi-resistant pathogens. To this end, a tetracycline trigger plasmid was constructed by replacing the araC repressor gene and the ara operator of the arabinose trigger plasmid with the tetR gene coding for the tetracycline repressor and the tet operon. After combination with the memory plasmid, the tetracycline sensor system was shown to be functional in E. coli allowing to detect and memorize the presence of tetracycline. Due to a positive feedback between the trigger and memory systems, the combined whole-cell biosensor showed a very high sensitivity for tetracycline with a detection threshold at 0.1 ng/ml tetracycline, which may be a general property of sensors of this type. Moreover, acute presence of tetracycline and past exposure can be detected by this sensor using the dual readout of two reporter fluorophores.Item Open Access Engineering of effector domains for targeted DNA methylation with reduced off-target effects(2020) Hofacker, Daniel; Broche, Julian; Laistner, Laura; Adam, Sabrina; Bashtrykov, Pavel; Jeltsch, AlbertEpigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to future applications of this approach.Item Open Access Charakterisierung der Substratspezifität von Protein-Methyltransferasen(2023) Schnee, Philipp; Weirich, Sara; Jeltsch, AlbertThe regulation of cellular activities is a key hallmark in the development of complex life forms. Among other factors, it is facilitated by protein lysine methyltransferases (PKMTs), which modify proteins in a highly specific manner and regulate their biological activities. Here, we describe methods to decipher the PKMT-substrate specificity by biochemical experiments and molecular dynamics simulations. This led to the discovery of novel PKMT substrates and rational design of even better non-natural substrates, which represent a promising starting point for the design of novel PKMT inhibitors.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.Item Open Access The MECP2‐TRD domain interacts with the DNMT3A‐ADD domain at the H3‐tail binding site(2022) Kunert, Stefan; Linhard, Verena; Weirich, Sara; Choudalakis, Michel; Osswald, Florian; Krämer, Lisa; Köhler, Anja R.; Bröhm, Alexander; Wollenhaupt, Jan; Schwalbe, Harald; Jeltsch, AlbertThe DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A‐ADD and MECP2‐TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull‐down, equilibrium peptide binding assays, and structural analyses. The region D529‐D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N‐terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2‐TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.Item Open Access Mechanistic basis of the increased methylation activity of the SETD2 protein lysine methyltransferase towards a designed super-substrate peptide(2022) Schnee, Philipp; Choudalakis, Michel; Weirich, Sara; Khella, Mina S.; Carvalho, Henrique; Pleiss, Jürgen; Jeltsch, AlbertProtein lysine methyltransferases have important regulatory functions in cells, but mechanisms determining their activity and specificity are incompletely understood. Naturally, SETD2 introduces H3K36me3, but previously an artificial super-substrate (ssK36) was identified, which is methylated >100-fold faster. The ssK36-SETD2 complex structure cannot fully explain this effect. We applied molecular dynamics (MD) simulations and biochemical experiments to unravel the mechanistic basis of the increased methylation of ssK36, considering peptide conformations in solution, association of peptide and enzyme, and formation of transition-state (TS) like conformations of the enzyme-peptide complex. We observed in MD and FRET experiments that ssK36 adopts a hairpin conformation in solution with V35 and K36 placed in the loop. The hairpin conformation has easier access into the active site of SETD2 and it unfolds during the association process. Peptide methylation experiments revealed that introducing a stable hairpin conformation in the H3K36 peptide increased its methylation by SETD2. In MD simulations of enzyme-peptide complexes, the ssK36 peptide approached TS-like structures more frequently than H3K36 and distinct, substrate-specific TS-like structures were observed. Hairpin association, hairpin unfolding during association, and substrate-specific catalytically competent conformations may also be relevant for other PKMTs and hairpins could represent a promising starting point for SETD2 inhibitor development.