Role and Regulation of DNA Methylation in Cellular Reprogramming

DNA甲基转移酶(DNMT)在表观遗传信息的维持和修饰中起着核心作用。有证据表明，DNA甲基化的改变与细胞重编程有关，但对其潜在机制知之甚少。我们现在已经证明，DNA甲基化在控制多能干细胞中的基因表达方面起着次要的作用，但它是在分化过程中有效和稳定地沉默多能基因所必需的，并且它的缺失促进了Oct4启动子的重新激活。去年，潜在的脱甲基化中间体5-羟甲基胞嘧啶(HMC)和相应的酶(Tet1-3)的发现为表观遗传重新编程开辟了一个全新的视角。我们已经开发了新的工具来量化和绘制这种新的DNA修饰，现在想通过染色质免疫沉淀和特定的核酸酶结合深度测序来研究它在全基因组的分布。此外，我们希望通过超分辨显微镜来确定HMC在不同染色质结构域和其他表观遗传标记上的亚细胞分布。为了测试这种新的修饰的作用，我们将操纵Tet的活性，并测试其对去甲基化和重编程的影响。这个项目应该有助于阐明DNA(去)甲基化在细胞重编程中的作用和调节，并提高其效率。

DNA methyltransferases (Dnmts) play a central role in the maintenance and modification of epigenetic information. There is evidence that changes of DNA methylation correlate with cellular reprogramming but little is known about the underlying mechanisms. We now have shown that DNA methylation plays a minor role in controlling gene expression in pluripotent stem cells but is required for efficient and stable silencing of pluripotency genes during differentiation and its absence facilitates re-activation of the oct4 promoter. Last year, the discovery of 5-hydroxymethylcytosine (hmC), a potential de-methylation intermediate, and the corresponding enzymes (Tet1-3) opened an entirely new perspective on epigenetic reprogramming. We have developed new tools to quantify and map this new DNA modification and now want to study its genome-wide distribution by chromatin immuno-precipitation and specific nucleases in combination with deep sequencing. In addition, we want to determine the subcellular distribution of hmC with respect to different chromatin domains and other epigenetic marks by super-resolution microscopy. To test the role of this novel modification we will manipulate Tet activity and test its effect on demethylation and reprogramming. This project should help elucidate the role and regulation of DNA (de-)methylation during cellular reprogramming and to improve its efficiency.