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

DNA-Renewal 中 5hmC 和 5mC 的标记和测序

• 批准号: 10413668
• 负责人: CHUAN HE
• 金额: $ 62.11万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-07 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10413668
• 关键词: Adopted; Antibodies; Azides; Base Excision Repairs; Basic Science; Binding; Binding Proteins; Biological Markers; Biomedical Research; Biotin; Cell Differentiation process; Cells; Chemicals; Clinical; Cytosine; DNA; DNA Methylation; DNA Modification Process; DNA analysis; DNA mapping; Detection; Development; Dioxygen; Dioxygenases; Disease; Embryonic Development; Enhancers; Enzymes; Epigenetic Process; Family; Gene Expression; Genes; Genetic Transcription; Genomic Imprinting; Gold; Health; Heterochromatin; High-Throughput Nucleotide Sequencing; Human; Human Genome; Immunoprecipitation; Investigation; Iron; Label; Laboratories; Mammals; Maps; Mediating; Methods; Methylation; Modification; Mus; Oxides; Play; Procedures; Process; Proteins; Recombinants; Regulation; Repetitive Sequence; Research; Resolution; Retrotransposon; Role; Sampling; Sensitivity and Specificity; Site; Technology; Thymine DNA Glycosylase; Tissues; Variant; X Inactivation; base; bisulfite sequencing; cell free DNA; cell type; clinical application; clinical biomarkers; clinical diagnosis; clinical prognosis; demethylation; disease diagnosis; disease prognosis; epigenome; gene repression; genome-wide; human disease; human tissue; member; oxidation; prognosis biomarker; programs; promoter; seal; stoichiometry; success; whole genome

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.

项目摘要/摘要 DNA胞嘧啶甲基化（5-甲基胞嘧啶或5MC）是关键的表观遗传学修饰 调节人基因表达。它在抑制A的转录中起着关键作用 人类基因组的大部分，包括重复元素。 5MC可以通过 人类TET家族酶的氧化，该酶利用二氧化物依次氧化5MC 到5-羟基甲基胞嘧啶（5HMC），5-甲基环胞嘧啶（5FC），最后是5-羧基糖苷 （5CAC）。 5FC和5CAC均可通过人胸腺胺DNA糖基酶识别和切除 （TDG），然后进行基础切除修复（BER），以正常 胞嘧啶在活跃的脱甲基化过程中。细胞类型特异性DNA甲基化已有 研究并用作疾病诊断和预后的生物标志物。基于抗体 MedIP免疫沉淀，然后进行高通量测序是一种常见方法 DNA 5MC分布的全基因组映射，但不是定量的，需要 大量的输入材料。亚硫酸盐测序是金标准方法，广泛 应用于定量5MC测序；但是，全基因组亚硫酸盐测序是 昂贵，并导致严重的DNA降解。克服这些关键障碍 进步，我们建议开发将5MC富集与定量结合的新方法 5MC测序，使用有限的起始材料。这些新方法的潜在应用 在循环无细胞的DNA（CFDNA）中，分析具有潜在革命性的人类疾病 诊断和预后。而5MC修饰抑制了大多数的激活 人类基因组，来自我们和其他人的研究表明，5HMC标志着活跃的基因座。我们 以前，使用1,000个细胞开发了鲁棒性程序，用于绘制全面的5HMC基因组。 但是，确定5HMC的存在和化学计量的定量方法 单细胞水平仍然缺乏。我们持续克服这一挑战的努力最近导致了 通过修饰化学计量法获得基础分辨率5HMC信息的化学解决方案， 使用有限的输入材料。在此续约中，我们提出了5HMC的基础分辨率测序 单细胞级别，可以检测否则会遗漏的细胞间差异， 包括表观基因组的重塑，这些重塑是在早期开始的细胞命运的启动 胚胎发生。该程序的成功将为5MC和5HMC提供新的方法 映射以在基础研究和临床应用中实现突破性发现。