Lee Minjung, Zhou Yubin, Huang Yun
Centre for Epigenetics and Disease Prevention, Department of Molecular and Cellular Medicine, Institute of Biosciences and Technology, College of Medicine, Texas A&M University.
Centre for Translational Cancer Research, Department of Medical Physiology, Institute of Biosciences and Technology, College of Medicine, Texas A&M University;
J Vis Exp. 2017 Dec 18(130):56858. doi: 10.3791/56858.
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.
DNA甲基化是哺乳动物基因组中一种稳定且可遗传的表观遗传修饰，参与调节基因表达以控制细胞功能。DNA甲基化的逆转，即DNA去甲基化，由双加氧酶的十一-易位（TET）蛋白家族介导。尽管已有广泛报道称异常的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改变或疾病进展仍不清楚，这主要是由于缺乏可靠的工具来在定义的时间和空间分辨率下准确地在基因组中添加或去除DNA修饰。为了克服这一障碍，我们设计了一种分裂型TET2酶，通过简单地添加化学物质来实现对5-甲基胞嘧啶（5mC）氧化以及随后哺乳动物细胞表观遗传状态重塑的时间控制。在此，我们描述了将基于工程化分裂型TET2酶的化学诱导表观基因组重塑工具（CiDER）引入哺乳动物细胞，并通过免疫染色、流式细胞术或斑点印迹分析对5-羟甲基胞嘧啶（5hmC）的化学诱导产生进行定量的方法。这种化学诱导表观基因组重塑工具将在不改变遗传密码的情况下广泛应用于研究细胞系统，以及探索各种生物系统中的表观基因型-表型关系。
