TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation.
TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation.
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DOI：
10.1093/nar/gku552
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发表时间：
2014-07
影响因子：
14.9
通讯作者：
Leonhardt H
中科院分区：
文献类型：
--
作者：
Müller U;Bauer C;Siegl M;Rottach A;Leonhardt H
通过泰特双加氧酶氧化甲基胞嘧啶产生的羟甲基-、甲酰基-和羧基胞嘧啶的发现提出了这些修饰如何有助于表观遗传调控的问题。由于它们在体内受到复杂的调控，我们用体外修饰的报告基因构建体剖析了基因表达的联系。我们使用了Oct 4启动子驱动的报告基因，并证明了在体外甲基化导致基因沉默，而随后的氧化与纯化的催化结构域TET 1导致基因重新激活。为了鉴定参与该途径的蛋白质，我们筛选了泰特相互作用因子，并鉴定了参与碱基切除修复的TDG、PARP 1、XRCC 1和LIG 3。敲除和拯救实验表明，基因的再激活依赖于糖基化酶TDG，而不是MBD 4，而NEIL 1，2和3可以部分挽救TDG的损失。这些结果清楚地表明，甲基胞嘧啶被泰特双加氧酶氧化，随后被TDG或NEIL糖基化酶和BER途径去除，导致表观遗传学沉默基因的再活化。
The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes.
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