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Start date: 2020Subject: search.filters.subject.540Institute: search.filters.UBSInstitut.Institut für Biochemie und Technische BiochemieAuthor: search.filters.author.Bröhm, Alexander

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Now showing 1 - 4 of 4
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    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, Albert
    Clr4 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.
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    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.
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    Methylation of recombinant mononucleosomes by DNMT3A demonstrates efficient linker DNA methylation and a role of H3K36me3
    (2022) Bröhm, Alexander; Schoch, Tabea; Dukatz, Michael; Graf, Nora; Dorscht, Franziska; Mantai, Evelin; Adam, Sabrina; Bashtrykov, Pavel; Jeltsch, Albert
    Recently, the structure of the DNMT3A2/3B3 heterotetramer complex bound to a mononucleosome was reported. Here, we investigate DNA methylation of recombinant unmodified, H3KC4me3 and H3KC36me3 containing mononucleosomes by DNMT3A2, DNMT3A catalytic domain (DNMT3AC) and the DNMT3AC/3B3C complex. We show strong protection of the nucleosomal bound DNA against methylation, but efficient linker-DNA methylation next to the nucleosome core. High and low methylation levels of two specific CpG sites next to the nucleosome core agree well with details of the DNMT3A2/3B3-nucleosome structure. Linker DNA methylation next to the nucleosome is increased in the absence of H3K4me3, likely caused by binding of the H3-tail to the ADD domain leading to relief of autoinhibition. Our data demonstrate a strong stimulatory effect of H3K36me3 on linker DNA methylation, which is independent of the DNMT3A-PWWP domain. This observation reveals a direct functional role of H3K36me3 on the stimulation of DNA methylation, which could be explained by hindering the interaction of the H3-tail and the linker DNA. We propose an evolutionary model in which the direct stimulatory effect of H3K36me3 on DNA methylation preceded its signaling function, which could explain the evolutionary origin of the widely distributed “active gene body-H3K36me3-DNA methylation” connection.