Müller Udo, Bauer Christina, Siegl Michael, Rottach Andrea, Leonhardt Heinrich
Department of Biology II, Ludwig-Maximilians University Munich and Center for Integrated Protein Science Munich (CIPSM), 82152 Planegg-Martinsried, Germany.
Department of Biology II, Ludwig-Maximilians University Munich and Center for Integrated Protein Science Munich (CIPSM), 82152 Planegg-Martinsried, Germany
Nucleic Acids Res. 2014 Jul;42(13):8592-604. doi: 10.1093/nar/gku552. Epub 2014 Jun 19.
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.
由TET双加氧酶氧化甲基胞嘧啶产生的羟甲基胞嘧啶、甲酰基胞嘧啶和羧基胞嘧啶的发现，引发了这些修饰如何促进表观遗传调控的问题。由于它们在体内受到复杂的调控，我们利用体外修饰的报告基因构建体剖析了与基因表达的联系。我们使用了一个由Oct4启动子驱动的报告基因，并证明体外甲基化会导致基因沉默，而随后用TET1的纯化催化结构域进行氧化会导致基因重新激活。为了鉴定参与该途径的蛋白质，我们筛选了与TET相互作用的因子，并鉴定出参与碱基切除修复的TDG、PARP1、XRCC1和LIG3。基因敲除和拯救实验表明，基因重新激活依赖于糖基化酶TDG，而不是MBD4，而NEIL1、2和3可以部分挽救TDG的缺失。这些结果清楚地表明，TET双加氧酶对甲基胞嘧啶的氧化以及随后TDG或NEIL糖基化酶和碱基切除修复途径的去除导致了表观遗传沉默基因的重新激活。
