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

DNA甲基化是一种重要的表观遗传修饰，它与组蛋白尾部修饰一起对基因调控至关重要。在体内，DNA甲基化模式在胚胎发生和发育期间通过从头DNA甲基转移酶Dnmt3a和Dnmt3b的作用设定。基于Dnmt3a与Dnmt3L复合的新结构和我们以前的研究，本申请的目标是在结构水平上了解这些酶的活性。我们将研究酶如何识别它们的CG靶位点，为什么某些侧翼强烈地增加催化速率，以及DNA上CG位点的排列如何影响活性。我们初步研究的一个意外发现是Dnmt3a在DNA上聚合。基于Dnmt 3a的结构，可以提出几种酶与裸DNA和核小体相互作用的模式来解释这一发现。我们计划通过定点突变研究哪些晶体界面参与这一过程以及它如何影响Dnmt3a的活性。我们计划研究其生理底物（DNA结合到组蛋白）上的甲基转移酶，以了解Dnmt3a的聚合是否参与与核小体的相互作用，以及它是否有助于体内异染色质定位。我们将研究Dnmt3a与其刺激剂Dnmt3L，染色质重塑酶如LSH，以及通过其PHD结构域与组蛋白尾部的相互作用，这与组蛋白尾部相互作用有关。因此，我们的研究结果将提供详细的洞察Dnmt3a在亚分子水平上的生理背景下的行动。2.最新技术水平，初步

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