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理解基因调控、生物发育和人类疾病产生重大影响。在Dnmt家族中，Dnmt1(维持甲基转移酶)和Dnmt3(从头甲基转移酶)的作用相对较好。相比之下，DNMT2蛋白一直是个谜。这主要是因为对它们的生物化学了解严重有限，对它们的生物学功能的定义也很差。在所有的DNMT蛋白中，DNMT2是最保守的，其同源物在原生动物(内阿米巴、网柄基底菌)、植物、原口动物和哺乳动物中都被描述过。DNMT2基因敲除或敲除的表型从严重的(斑马鱼)到几乎检测不到的(果蝇、Dictyostelials、Schizosaccharmyces pombe、小鼠)不等。虽然该酶具有DNA甲基转移酶所特有的所有氨基酸基序，但在体外只显示了DNMT2的弱DNA甲基化活性。然而，有强有力的实验证据表明，一些基因组座位，特别是逆转录元件，在体内被DNMT2甲基化。最近，第二个活性，tRNA的位点特异性甲基化已经显示出来。该研究组的目标是对来自人、果蝇、内阿米巴、Dictyostelials和S.pombe的DNMT2蛋白进行比较生化分析，以深入了解DNA和RNA上的酶功能。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。酶的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，并分离酵母中合成的致命突变体。为了进一步明确靶点，将进行共同免疫沉淀，并鉴定相关的DNA和RNA分子。研究了DNMT2在体内和体外翻译系统中对tRNA活性的影响及其在逆转录元件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.