Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.
Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.
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DOI：
10.1007/978-1-0716-0876-0_24
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发表时间：
2021
影响因子：
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通讯作者：
Abdulkadir Abakir;Fahad Alenezi;A. Ruzov
中科院分区：
文献类型：
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作者：
Abdulkadir Abakir;Fahad Alenezi;A. Ruzov
DNA甲基化（5-甲基胞嘧啶，5 mC）参与调节广泛的生物过程。泰特蛋白可将5 mC氧化为5-羟甲基胞嘧啶、5-甲酰基胞嘧啶（5 fC）和5-羧基胞嘧啶（5caC）。虽然5 fC和5caC都是主动去甲基化途径的中间体，但越来越多的实验证据表明，这些DNA修饰也可能与特定的阅读蛋白相互作用，因此可能代表真正的表观遗传标记。尽管最近已经提出了一些单碱基分辨率技术用于5 fC/5caC作图，但基于抗体的方法仍然代表了用于分析这些DNA修饰的基因组分布的相对简单和合理的替代方案。在这里，我们描述了一个协议5caC DNA免疫沉淀（5caC DIP），可用于位点特异性和全基因组评估5caC分布。结合基于质谱的技术和单碱基分辨率映射方法，这种方法可能有助于阐明5caC在发育，分化和肿瘤发生中的作用。
DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry–based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.