Interaction of mammalian de novo DNA methyltransferases with DNA and nucleosomes and regulatory proteins

点击翻译按钮获取中文摘要

DNA methylation is an important epigenetic modification that in concert with histone tail modification is essential for gene regulation. In vivo, the DNA methylation pattern is set during embryogenesis and development by the action of de novo DNA methyltransferases Dnmt3a and Dnmt3b. Based on a new structure of Dnmt3a in complex with Dnmt3L and our previous studies, it is the goal of this application to understand the activity of these enzymes at structural level. We will investigate how the enzymes recognize their CG target site, why certain flanks strongly increase catalytic rates and how the arrangement of CG sites on the DNA influences activity. One unexpected finding of our preliminary studies is that Dnmt3a polymerizes on DNA. Based on the Dnmt3a structure, several modes of interaction of the enzyme with naked DNA and nucleosomes could be proposed to explain this finding. We plan to investigate by site directed mutagenesis which of the crystallographic interfaces participates in this process and how it influences the activity of Dnmt3a. We plan to study the methyltransferase on its physiological substrate (DNA bound to histone proteins) to find out if polymerization of Dnmt3a is involved in the interaction with nucleosomes and if it contributes to heterochromatin localization in vivo. We will study the interaction of Dnmt3a with its stimulator Dnmt3L, with chromatin remodeling enzymes like LSH, and with the histone tails via its PHD domain, that has been implicated in histone tail interaction. Thereby, our results will provide detailed insight into the action of Dnmt3a at sub-molecular level in physiological context. 2. State of the art, preliminary