哺乳动物中Tet双加氧酶催化5mC产生的5hmC等氧化产物对其DNA去甲基化及生长发育过程都具有重要作用。Tet酶在许多生物中相对保守。我们以单细胞真核生物莱茵衣藻为模式生物，在其中鉴定到八个可能的Tet同源蛋白。令人意外的是，其中一个显示出与哺乳动物Tet完全不同的酶活。虽然同样以DNA上的5mC为底物，但衣藻Tet蛋白催化5mC与维生素C反应并产生新型DNA修饰。在前期工作中，我们已经解析了该DNA修饰的分子结构，并且证实其在衣藻基因组中稳定存在。后续我们将通过对其反应副产物的检测等方式来解析衣藻Tet蛋白的酶活反应机理。更重要的是，我们已经建立了高通量测序手段来检测该DNA修饰在不同时期衣藻基因组中的分布，并将结合转录组与蛋白质组的方法来分析该修饰碱基的潜在功能。同时，我们还将利用已在衣藻中建立的CRISPR/Cas9基因编辑系统，对衣藻Tet蛋白实施敲除或定点突变来研究其生理功能。

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by TET (Ten-eleven translocation) dioxygenases results in a cascade of additional epigenetic marks and plays important roles in developmental regulation of mammals. Tet proteins are modestly conserved in many eukaryotes. In our study of Tet homologs in the green alga Chlamydomonas reinhardtii, we found that the CrTet1 protein could convert 5mC into modification products which are different from all the reported ones. The catalytic activity of CrTet requires Fe(II) and the integrity of its His-x-Asp (HxD) binding motif, which is conserved in Fe-dependent oxygenases. However, CrTet1 utilizes L-ascorbic acid (Vitamin C, VC) rather 2-oxoglutarate (2-OG) as an essential co-substrate. Recently, we have solved the molecular structure of the new DNA modifications, and have also confirmed their existence in the genome of Chlamydomonas via mass spectrometry. In this project, we will investigate the reaction mechanism of CrTet1 mainly by identifying the byproducts of the reaction and by mutational analyses of CrTet1, which is expected to be different from that of mammalian Tet enzymes. We plan to map the new DNA modifications in the genome of Chlamydomonas and reveal its potential function by comparing with the transcriptome and proteome in various stages of Chlamydomonas. We have recently developed a CRISPR/Cas9-based gene editing approach in Chlamydomonas. With this approach, CrTet1 mutant strains will be established to investigate the biological significance of CrTet1 and the novel DNA modifications it catalyzes.