Browsing by Author "Köhler, Anja R."

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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.)