Browsing by Author "Broche, Julian"

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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
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, Albert
Despite 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.
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, Albert
Epigenome 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.
Open Access
Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression
(2023) Broche, Julian; Köhler, Anja R.; Kühnel, Fiona; Osteresch, Bernd; Chandrasekaran, Thyagarajan T.; Adam, Sabrina; Brockmeyer, Jens; Jeltsch, Albert
While cytosine-C5 methylation of DNA is an essential regulatory system in higher eukaryotes, the presence and relevance of 6-methyladenine (m6dA) in human cells is controversial. To study the role of m6dA in human DNA, we introduced it in human cells at a genome-wide scale at GANTC and GATC sites by expression of bacterial DNA methyltransferases and observed concomitant reductions in cell viability, in particular after global GANTC methylation. We identified several genes that are directly regulated by m6dA in a GANTC context. Upregulated genes showed m6dA-dependent reduction of H3K27me3 suggesting that the PRC2 complex is inhibited by m6dA. Genes downregulated by m6dA showed enrichment of JUN family transcription factor binding sites. JUN binds m6dA containing DNA with reduced affinity suggesting that m6dA can reduce the recruitment of JUN transcription factors to target genes. Our study documents that global introduction of m6dA in human DNA has physiological effects. Furthermore, we identified a set of target genes which are directly regulated by m6dA in human cells, and we defined two molecular pathways with opposing effects by which artificially introduced m6dA in GANTC motifs can directly control gene expression and phenotypes of human cells.
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, Albert
DNA 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.
Open Access
Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites
(2017) Lungu, Cristiana; Pinter, Sabine; Broche, Julian; Rathert, Philipp; Jeltsch, Albert
Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing target epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.