Browsing by Author "Köhler, Anja R."

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Open Access
Development of a BiAD sensor for locus-specific detection of cellular histone acetylation dynamics by fluorescence microscopy
(2025) Köhler, Anja R.; Gutekunst, Nicole; Harsch, Annika; Bashtrykov, Pavel; Jeltsch, Albert
Background: Dynamic changes in histone acetylation play crucial roles during cellular differentiation and disease development, but their detection in living cells is still a challenging task. Objectives: Here, we developed a Bimolecular Anchor Detector (BiAD) sensor for the detection of locus-specific changes in histone acetylation in living cells by fluorescence microscopy. Methods: We used the BRD9 bromodomain cloned as tandem double domain (2xBRD9-BD) as a reader of histone acetylation. It was integrated into a dual-color BiAD chassis that was previously described by us. Results: We identified the gene body of TTC34 as a potential target for our sensor, because it contains dense histone acetylation and 392 local sequence repeats. Using a binding-deficient mutant of 2xBRD9-BD as a negative control, we established a successful readout of histone acetylation at the TTC34 locus. A single-domain reader did not function, indicating the requirement for the double reader to enhance the affinity and specificity of the chromatin interaction via avidity effects. With this sensor, we could detect dynamic increases in histone acetylation at the TTC34 locus after the treatment of cells with the histone deacetylase inhibitor Trichostatin A for 6 h indicating the applicability of the sensor for single-cell epigenome studies. Conclusions: Our data demonstrate that active chromatin modifications can be detected by BiAD sensors using 2xBRD9-BD as a reader. This complements the toolkit of the available BiAD sensors and documents the modularity of BiAD sensors.
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
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, Albert
The 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.
Open Access
Novel approaches to investigate the cellular effects of epigenome modifications
(2024) Köhler, Anja R.; Jeltsch, Albert (Prof. Dr.)
Open Access
Repeat DNA methylation is modulated by adherens junction signaling
(2024) Brenner, Lisa-Marie; Meyer, Florian; Yang, Haiqian; Köhler, Anja R.; Bashtrykov, Pavel; Guo, Ming; Jeltsch, Albert; Lungu, Cristiana; Olayioye, Monilola A.
Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.
Open Access
Sensoren zur lokusspezifischen Visualisierung epigenetischer Modifikationen
(2025) Köhler, Anja R.; Jeltsch, Albert
Dynamic changes of the epigenome at specific gene loci, including alterations of chemical modifications on the DNA or histone proteins, are critical in development and disease. Bimolecular Anchor Detector (BiAD) sensors can be used to visualize these modifications in living cells with locus resolution. Recently, we improved the BiAD technology by enhancing the sensor sensitivity and expanded the range of detectable modifications, enabling a broad application of the BiAD sensors in various contexts.