An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling
An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling
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DOI：
10.3791/56858
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发表时间：
2017-12-01
影响因子：
1.2
通讯作者：
Huang, Yun
中科院分区：
文献类型：
--
作者：
Lee, Minjung;Zhou, Yubin;Huang, Yun
DNA甲基化是哺乳动物基因组中一种稳定的、可遗传的表观遗传修饰，参与调控基因表达以控制细胞功能。DNA甲基化或DNA去甲基化的逆转由双加氧酶的10 - 11易位（泰特）蛋白家族介导。尽管已经广泛报道异常DNA甲基化和去甲基化与发育缺陷和癌症相关，但这些表观遗传变化如何直接导致随后的基因表达或疾病进展的改变仍不清楚，这主要是由于缺乏可靠的工具来以定义的时间和空间分辨率准确地添加或删除基因组中的DNA修饰。为了克服这一障碍，我们设计了一种分裂TET 2酶，通过简单地添加化学物质，能够暂时控制5-甲基胞嘧啶（5 mC）氧化和随后重塑哺乳动物细胞中的表观遗传状态。在这里，我们描述了引入化学诱导的表观基因组重塑工具（CiDER）的方法，基于工程分裂TET 2酶，到哺乳动物细胞和定量的化学诱导生产的5-羟甲基胞嘧啶（5 hmC）与免疫染色，流式细胞术或斑点印迹试验。这种化学诱导的表观基因组重塑工具将在不改变遗传密码的情况下在询问细胞系统以及在探测各种生物系统中的表观基因型-表型关系中找到广泛的用途。
DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype-phenotype relations in various biological systems.