Labeling and sequencing of 5hmC and 5mC in DNA-Renewal

项目摘要/摘要 DNA胞嘧啶甲基化(5-甲基胞嘧啶或5mC)是一种关键的表观遗传修饰 人类基因表达的调控。它在抑制转录的过程中起着关键作用。 人类基因组的很大一部分，包括重复元件。5mC可以通过 人类Tet家族酶的氧化，它们利用氧气顺序氧化5mC 到5-羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)，最后是5-羧基胞嘧啶 (5caC)。5fC和5caC均可被人胸腺嘧啶DNA糖基酶识别和切除 (TDG)，然后进行碱基切除修复(BER)，用正常的 胞嘧啶，在一个活跃的去甲基化过程中。细胞类型特异性DNA甲基化已经被 作为疾病诊断和预后的生物标志物研究和应用。基于抗体的 MeDIP免疫沉淀和高通量测序是一种常见的方法 DNA 5mC分布的全基因组图谱，但它不是定量的，需要 投入了大量的材料。亚硫酸氢盐测序是广泛使用的黄金标准方法 应用于定量5mC测序；然而，全基因组亚硫酸氢盐测序 价格昂贵，并导致严重的DNA降解。要克服这些关键障碍， 进展，我们建议开发5mC浓缩和定量相结合的新方法 5mC测序，使用有限的起始材料。这些新方法的潜在应用 循环中的无细胞dna(Cfdna)分析可能会使人类疾病发生革命性变化。 诊断和预后。而5mC修饰抑制了大多数 人类基因组，我们和其他人的研究表明，5hmC标记着活跃的基因座。我们 之前开发了使用1000个细胞在全基因组范围内绘制5hmC图谱的强大程序。 然而，确定5HmC的存在和化学计量比的定量方法 单细胞水平仍然缺乏。我们为克服这一挑战所做的持续努力最近导致了 化学溶液，产生碱基分辨率为5hmC的信息和修改化学计量比， 使用有限的输入材料。在这次更新中，我们建议将5hmC的基本分辨率测序在 单细胞水平，使得能够检测否则被遗漏的细胞间差异， 包括表观基因组重塑，它是早期细胞命运启动的基础 胚胎发生。该项目的成功将为5mC和5hmC提供新的方法 绘制地图，以实现基础研究和临床应用方面的突破性发现。

Project Summary/Abstract DNA cytosine methylation (5-methylcytosine or 5mC) is a key epigenetic modification in the regulation of human gene expression. It plays critical roles in suppressing transcription of a large portion of human genome, including repetitive elements. 5mC can be reversed through oxidation by the human TET family enzymes, which utilize dioxygen to sequentially oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally 5-carboxycytosine (5caC). Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase (TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal cytosine, in an active demethylation process. Cell-type specific DNA methylation has been studied and applied as a biomarker for disease diagnosis and prognosis. Antibody-based MeDIP immunoprecipitation followed by high-throughput sequencing is a common method for genome-wide mapping of DNA 5mC distribution, but it is not quantitative and requires a significant amount of input material. Bisulfite sequencing is the gold standard approach, widely applied in quantitative 5mC sequencing; however, whole-genome bisulfite sequencing is expensive and causes severe DNA degradation. To overcome these critical barriers to progress, we propose to develop new methods that combine 5mC enrichment with quantitative 5mC sequencing, using limited starting material. The potential application of these new methods in circulating cell-free DNA (cfDNA) analyses stands to potentially revolutionize human disease diagnosis and prognosis. Whereas the 5mC modification suppress activation of a majority of human genome, studies from us and others revealed that 5hmC marks active loci. We previously developed robust procedures to map 5hmC genome-wide using 1,000 cells. However, quantitative methods that determine the presence and stoichiometry of 5hmC at the single-cell level are still lacking. Our ongoing efforts to overcome this challenge recently led to a chemical solution that yields base-resolution 5hmC information with modification stoichiometry, using limited input material. In this renewal, we propose base resolution sequencing of 5hmC at the single-cell level, enabling the detection of intercellular differences that are otherwise missed, including the epigenome remodeling that underlies the initiation of cell fates during early embryogenesis. The success of this program will provide new methods for 5mC and 5hmC mapping to enable breakthrough discoveries in both basic research and clinical applications.