FOR 1082: Biochemistry and Biological Function of Dnmt2 Methyltransferases

DNA甲基转移酶（Dnmt）家族蛋白在表观遗传基因调控中起着关键作用。这一领域的研究已经并将对我们对基因调控、有机体发育和人类疾病的理解产生重大影响。在Dnmt家族中，Dnmt1（维持性甲基转移酶）和Dnmt3（从头甲基转移酶）的作用相对较好地了解。相比之下，Dnmt2蛋白仍然相当神秘。这主要是由于对其生物化学的理解严重有限，对其生物学功能的定义也很差。在所有Dnmt蛋白中，Dnmt2是最高度保守的，同源物已在多种生物中被描述，如原生生物（内阿米巴原虫，盘齿ostelium），植物，原口动物和哺乳动物。Dnmt2敲除或敲除的表型各不相同，从严重的（斑马鱼）到几乎检测不到的（果蝇、盘齿骨菌、pombe Schizosaccharomyces、小鼠）。尽管该酶具有DNA甲基转移酶的所有氨基酸基序特征，但在体外只能显示出较弱的DNA甲基化活性。然而，有强有力的实验证据表明，一些基因组位点，特别是逆转录因子，在体内被Dnmt2甲基化。最近，第二种活性，tRNA的位点特异性甲基化已被证实。该研究单位旨在对来自人类、果蝇、内阿米巴、盘基ostelium和S. pombe的Dnmt2蛋白进行比较生化分析，以深入了解其对DNA和RNA的酶促功能。这包括底物的定义以及识别和修饰两种不同底物所需的酶的性质。酶活性很可能需要辅因子，因此，研究单位将鉴定与Dnmt2相互作用的蛋白质，并在酵母中分离合成致死突变体。为了确定进一步的靶点，将进行共免疫沉淀，并鉴定相关的DNA和RNA分子。研究了Dnmt2对体内和体外翻译系统中tRNA活性的影响，以及在逆转录因子沉默和迁移中的意义。为了了解Dnmt2的生物学功能，已经设计了体内读出系统。

Proteins of the DNA methyltransferase (Dnmt) family play a pivotal role in epigenetic gene regulation. Research in this area has and will have a strong impact on our understanding of gene regulation, organismal development and human disease. Within the Dnmt family, the roles of Dnmt1 (maintenance methyltransferase) and Dnmt3 (de novo methyltransferases) are relatively well understood. In contrast, Dnmt2 proteins have remained rather enigmatic. This is mostly due to a severely limited understanding of their biochemistry and the poor definition of their biological function. Among all Dnmt proteins, Dnmt2 is the most highly conserved and homologues have been described in organisms as diverse as protists (Entamoeba, Dictyostelium), plants, protostomia and mammals. Phenotypes of Dnmt2 knock-outs or knock-downs vary from severe (zebrafish) to barely detectable (Drosophila, Dictyostelium, Schizosaccharomyces pombe, mouse). Although the enzyme shares all amino acid motifs characteristic for DNA methyltransferases, only a weak DNA-methylation activity of Dnmt2 could be shown in vitro. However, there is strong experimental evidence that some genomic loci, in particular retroelements, are metylated in vivo by Dnmt2. More recently, a second activity, the site-specific methylation of a tRNA has been shown. The Research Unit aims at a comparative biochemical analysis of Dnmt2 proteins from human, Drosophila, Entamoeba, Dictyostelium and S. pombe to obtain insights into the enzymatic function on DNA as well as on RNA. This includes the definition of substrates as well as properties of the enzyme that are required to recognise and modify the two different substrates. It is likely that cofactors are required for enzyme activity, the Research Unit will, therefore, identitify proteins that interact with Dnmt2 and isolate synthetic lethal mutants in yeast. To specify further targets, co-immune precipitations will be done and associated DNA and RNA molecules will be identified. The effect of Dnmt2 on tRNA activity in translation systems in vivo and in vitro is investigated as well as the significance in silencing and mobility of retroelements. In vivo readout systems have been designed in order to understand the biological function of Dnmt2.