Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity.
Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity.
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DOI：
10.1016/bs.mie.2015.12.006
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发表时间：
2016
影响因子：
--
通讯作者：
Kohli RM
中科院分区：
文献类型：
--
作者：
Liu MY;DeNizio JE;Kohli RM
在真核DNA中，胞嘧啶可以被酶促修饰以产生多达四个表观遗传碱基变体。DNA甲基转移酶将胞嘧啶转化为5-甲基胞嘧啶（mC），这在发育过程中的基因调控中起着关键作用。10 - 11易位（泰特）酶可将mC依次氧化成三种产物：5-羟甲基胞嘧啶（hmC）、5-甲酰基胞嘧啶（fC）和5-羧基胞嘧啶（caC）。这些氧化的碱基已经在许多哺乳动物细胞类型中发现，它们可能在其中执行独立的表观遗传功能并有助于DNA去甲基化。为了深入了解泰特家族酶的机制和功能，需要严格的方法来定量细胞中的基因组胞嘧啶修饰并在体外跟踪泰特酶活性。在这里，我们介绍了我们实验室和其他人开发的工具，以报告具有高特异性和灵敏度的五种形式的胞嘧啶（未修饰的，mC，hmC，fC和caC）。我们提供了详细的协议，在基因组DNA中的胞嘧啶修饰的定性和定量分析斑点印迹和LC-MS/MS。然后，我们描述了用于生成合成的寡核苷酸底物的生化研究的方法，提供优化的反应条件，并介绍了几种化学酶测定，以及HPLC，质谱和闪烁计数方法，以量化胞嘧啶修饰体外。这些方法使得泰特活性的机制研究成为可能，这是理解这些酶在表观遗传调控中的作用的关键。
In eukaryotic DNA, cytosine can be enzymatically modified to yield up to four epigenetic base variants. DNA methyltransferases convert cytosine to 5-methylcytosine (mC), which plays critical roles in gene regulation during development. Ten-eleven translocation (TET) enzymes can sequentially oxidize mC to three products: 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC). These oxidized bases have been found in numerous mammalian cell types, where they potentially carry out independent epigenetic functions and aid in DNA demethylation. To gain insight into the mechanisms and functions of TET family enzymes, rigorous approaches are needed to quantify genomic cytosine modifications in cells and track TET enzyme activity in vitro. Here, we present tools developed by our lab and others to report on each of the five forms of cytosine (unmodified, mC, hmC, fC, and caC) with high specificity and sensitivity. We provide detailed protocols for qualitative and quantitative analysis of cytosine modifications in genomic DNA by dot blotting and LC-MS/MS. We then describe methods for generating synthetic oligonucleotide substrates for biochemical studies, provide optimized reaction conditions, and introduce several chemoenzymatic assays, as well as HPLC, mass spectrometry, and scintillation counting methods to quantify cytosine modifications in vitro. These approaches enable mechanistic studies of TET activity, which are key to understanding the role of these enzymes in epigenetic regulation.
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影响因子：
8.8
作者：
Neri, Francesco;Incarnato, Danny;Oliviero, Salvatore
通讯作者：
Oliviero, Salvatore
DOI：
10.1126/science.1170116
发表时间：
2009-05-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
62.1
作者：
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通讯作者：
Balasubramanian, Shankar