Biochemical characterization and biological role of the DNMT3A and DNMT3B de novo DNA methyltransferases

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DNA methylation is an important epigenetic modification that in concert with histone tail modifications is essential for gene regulation. In mammals, the DNA methylation patterns are set during embryogenesis and development but aberrantly altered during the onset and progression of diseases like cancer. The DNMT3A and 3B DNA methyltransferases play central roles in these processes, but it is still largely unknown, how DNA methylation patterns are generated and maintained. In this grant application, we aim to investigate key biochemical properties of DNMT3 enzymes, including their interaction with PTMs of histone tails, multimerization of the enzymes and regulation of their activity by PTMs, interactors and by allosteric mechanisms. Our central goal is to understand the mechanism, cellular role, regulation and targeting of DNMT3A and DNMT3B with the long term goal to learn how DNA methylation patterns are generated and changed in cells.WP1 of this application deals with the role of the PWWP domains of DNMT3 enzymes in their targeting and regulation. Having discovered that the PWWP of DNMT3 enzymes recognizes H3K36me3-modified histone tails and that this interaction is important for targeting the enzyme to chromatin in 2010. Based on the findings that PWWP domains also bind to DNA, we now plan to study the multivalent interaction of the DNMT3 PWWP domains with K36me3 and DNA in chromatin. Afterwards, we will explore the mechanism of the regulation of the enzymatic activity by peptide binding to the PWWP domain in vitro and in cells. Better molecular understanding of this process is very important since it is the mechanistic basis for the correlation of DNA methylation and H3K36me3 in the bodies of active genes.WP2 deals with the important question of regulation of DNMT3 enzymes by post-translational modifications and interacting proteins allowing cells to control their activity to set up and maintain defined DNA methylation patterns. We will investigate the mechanism of regulation of DNMT3A by CK2 mediated phosphorylation, regulation of DNMT3B by subunit interface phosphorylation and the very essential interaction of both DNMT3 enzymes with HELLS, a chromatin remodeller necessary for genome methylation, the exact mechanistic role which is unclear so far.WP3 aims to understand the specific role of DNMT3B in the methylation of major satellite repeat DNA and its involvement in the ICF syndrome. A second aim is to characterize the functional properties of heterotypic DNMT3A, 3B and 3L complexes, a question that has not been tackled so far. By this it will advance our understanding of the mutual cooperation of these two DNMT3 paralogs in human cells.