Role of NEIL DNA glycosylases in DNA demethylation and differentiation

DNA甲基化是一种表观遗传标记，在发育和疾病中起着重要作用。现在人们普遍认为DNA甲基化是动态的，并受到酶去甲基化的影响。在Tet/TDG去甲基化途径中，甲基化的胞嘧啶被Tet双加氧酶反复氧化，未修饰的胞嘧啶被胸腺嘧啶DNA糖基酶(TDG)还原。Tet/TDG去甲基化的一个易受攻击的中间产物是碱基(Ap)。我们最近发现，在Tet/TDG DNA去甲基化过程中，NEIL1和NEIL2DNA糖基酶协调AP位点的处理。我们的分析表明，在DNA氧化脱甲基化过程中，Neils在底物的协调交接中起着调节作用。为了研究Neil DNA糖基酶在DNA去甲基化和DNA修复中的生理相关性和生物学作用，我们通过CRISPR/Cas9、NEIL-1、-2或3突变的小鼠胚胎干细胞以及Neil双突变培养的小鼠胚胎干细胞。本项目的目的是研究这些突变的ES细胞的分化能力、氧化胞嘧啶水平和DNA修复能力。

DNA methylation is an epigenetic mark which plays important roles in development and disease. It is now widely accepted that DNA methylation is dynamic and is subject to enzymatic demethylation. In the TET/TDG demethylation pathway, methylated cytosine is iteratively oxidized by TET dioxygenases and unmodified cytosine is restored by thymine DNA glycosylase (TDG). A vulnerable intermediate in TET/TDG demethylation is the abasic (AP) site. We have shown recently that NEIL1 and NEIL2 DNA glycosylases coordinate AP site processing during TET/TDG DNA demethylation. Our analysis indicates that NEILs function as regulators in the coordinated substrate hand-over during oxidative DNA demethylation. To investigate the physiological relevance and biological role of Neil DNA glycosylases in DNA demethylation and DNA repair we have generated via CRISPR/Cas9, mouse embryonic stem cells which are mutant for Neil1,-2, or 3, as well as Neil double mutants The aim of this project is to characterize these mutant ES cells for their differentiation capacity, oxidized cytosine levels, and DNA repair abilities.