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

DNA甲基化是一种重要的表观遗传修饰，与组蛋白尾部修饰一起对基因调控至关重要。在哺乳动物中，DNA甲基化模式在胚胎发生和发育期间设定，但在癌症等疾病的发作和进展期间发生异常改变。DNMT 3A和3B DNA甲基转移酶在这些过程中发挥着核心作用，但DNA甲基化模式如何产生和维持仍然是未知的。在这项资助申请中，我们的目标是研究DNMT3酶的关键生化特性，包括它们与组蛋白尾部的PTM的相互作用，酶的多聚化以及PTM、相互作用物和变构机制对其活性的调节。我们的中心目标是理解DNMT3A和DNMT3B的机制、细胞作用、调节和靶向，长期目标是了解DNA甲基化模式如何在细胞中产生和改变。本申请的WP 1涉及DNMT3酶的PWWP结构域在其靶向和调节中的作用。2010年发现DNMT3酶的PWWP识别H3K36me3修饰的组蛋白尾部，并且这种相互作用对于将酶靶向染色质很重要。基于PWWP结构域也与DNA结合的发现，我们现在计划研究DNMT3 PWWP结构域与染色质中的K36me3和DNA的多价相互作用。之后，我们将探索在体外和细胞中通过肽结合PWWP结构域来调节酶活性的机制。更好地理解这一过程是非常重要的，因为它是DNA甲基化和H3K36me3在活性基因体中的相关性的机制基础。WP 2涉及通过翻译后修饰和相互作用蛋白质调节DNMT 3酶的重要问题，允许细胞控制其活性以建立和维持定义的DNA甲基化模式。我们将研究CK2介导的磷酸化对DNMT3A的调节机制，亚基界面磷酸化对DNMT3B的调节机制，以及两种DNMT3酶与HELLS（基因组甲基化所必需的染色质重塑剂）的非常重要的相互作用，WP3旨在了解DNMT3B在主要卫星重复DNA甲基化中的具体作用及其参与ICF综合征第二个目的是表征异型DNMT 3A、3B和3L复合物的功能特性，这是迄今为止尚未解决的问题。通过这一点，它将促进我们对这两个DNMT3旁系同源物在人类细胞中相互合作的理解。

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.