脱氧胞苷酸脱氨酶DCTD在小鼠发育过程中的表观遗传作用

Although genomic 5-methylcytosine (5mC) can be removed passively through DNA replication, several pathways for active demethylation have also been proposed, including those involving oxidation of 5mC to 5-hydroxymethylcytosine (5hmC). It has been reported that 5hmC can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by TET enzymes, and TDG then cleaves 5fC and 5caC to achieve active DNA demethylation. Although the oxidation-dependent demethylation pathway has been described, pathways involving deamination of either 5mC or 5hmC by cytidine deaminase have been proposed but not proven. In this project, we aim to identify a deaminase potentially involved in DNA demethylation. 2'-Deoxycytidylate deaminase (DCTD) can catalyze the deamination of dCMP to dUMP to provide the main nucleotide substrate for thymidylate synthase, which is crucial in DNA synthesis. We found that besides dCMP, DCTD also shows higher deamination activity on methyl-dCMP and hydroxymethyl-dCMP. Most interestingly, it also displays activity on C of ssDNA. We plan to examine its activity on modified C of ssDNA and study its function in mouse development. DCTD is highly expressed in oocytes, early preimplantation embryos and spleen in mouse. In order to investigate its physiological role, we have generated knockout mouse by gene targeting. Dctd deficiency leads to the delay in early embryo development and reduced fertility. We will use the Dctd mutant mouse model to study its physiological significance, and explore its molecular function in epigenetic regulation of mammalian development.

基因组上的5mC可以通过DNA复制发生被动去甲基化，也可以经酶的催化发生主动去甲基化，但后者的机制一直不是很清楚。我们组已有研究发现，5mC可以被TET酶氧化成5hmC以至5caC，5caC又进一步被糖苷酶TDG识别和切除实现DNA的主动去甲基化。除了这条氧化-碱基切除修复途径，目前普遍认为脱氨-碱基切除修复途径也可能引发DNA主动去甲基化。此项目旨在寻找能够将5mC以至5hmC脱氨变成T或5hmU的脱氨酶，并在小鼠水平上阐明其作用机制。 我们的前期工作显示dCMP脱氨酶DCTD对甲基化与羟甲基化胞苷酸的脱氨活性更高，并且对单链DNA上的胞嘧啶也有脱氨酶活性。该蛋白在小鼠卵细胞、早期胚胎以及肝脏中高表达，并且DCTD缺失的突变小鼠的早期胚胎发育明显滞后以致生育能力显著降低。通过本项目，我们试图探索DCTD更广泛的酶学功能，并以敲除小鼠为模型，探索DCTD在动物发育过程中的表观遗传调控作用。

基因组上的5mC可以通过DNA复制发生被动去甲基化，也可以经酶的催化发生主动去甲基化。5mC可以被TET酶氧化成5hmC以至5caC，5caC又进一步被糖苷酶TDG识别和切除实现DNA的主动去甲基化。然而，除了这条氧化-碱基切除修复途径，目前普遍认为脱氨-碱基切除修复途径也可能引发DNA的主动去甲基化。此项目旨在寻找能够将5mC以至5hmC脱氨变成T或5hmU的脱氨酶，并在小鼠水平上阐明其作用机制。.本项目中我们的工作显示dCMP脱氨酶DCTD对甲基化与羟甲基化dCMP的脱氨活性也很高，但对单链DNA上的胞嘧啶并无脱氨酶活性。DCTD缺失的突变小鼠的早期胚胎发育明显滞后，推测是由于DCTD的缺失导致受精卵中dTTP的合成受到一定的影响，以致DNA复制受影响最终影响了早期胚胎发育以致生育能力显著降低。DCTD的缺失最终并未影响受精卵中DNA的甲基化水平。.鉴于DCTD对早期胚胎发育过程中的DNA去甲基化并无作用，在本项目的支撑下，我们从以下两方面继续进行了探索：一方面分析了DCTD脱氨产生的修饰核苷酸对胚胎干细胞的毒性影响；一方面阐明了UNG2糖苷酶促进TET介导的DNA去甲基化的作用及机制，并探索了UNG2在小鼠早期胚胎发育和受精卵中DNA去甲基化中的作用，完善了DNA去甲基化的分子机制。