Molecular toolkit for single-cell oxi-mC analysis

用于单细胞 oxi-mC 分析的分子工具包

• 批准号: 10267182
• 负责人: Yun Huang
• 金额: $ 19.05万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-21 至 2023-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10267182
• 关键词: 3-Dimensional; ATAC-seq; Aberrant DNA Methylation; Adopted; Antibodies; Architecture; Biochemical Reaction; Bioinformatics; Biology; Carbon; Cardiovascular Diseases; Cells; Chemicals; Chromatin; Chromatin Loop; Clinical; Coupling; Cytosine; DNA; DNA Damage; DNA Methylation; DNA Modification Methylases; DNA Modification Process; DNA Probes; Data; Data Set; Development; Dioxygenases; Disease; Epigenetic Process; Exposure to; Family; Gene Expression; Genetic Transcription; Genome; Genomic DNA; Genomic Segment; Genomics; Goals; Heterogeneity; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Individual; Intervention; Knowledge; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Modification; Molecular; Molecular Biology; Molecular Conformation; Monitor; Mus; Nucleotides; Oligonucleotides; Oxides; Pathologic; Play; Positioning Attribute; Preparation; Protein translocation; Reaction; Research; Research Personnel; Resolution; Role; Sampling; Side; Switch Genes; Techniques; Technology; Testing; Tissues; Treatment Protocols; Variant; analysis pipeline; base; biological systems; biomaterial compatibility; bisulfite; chemical reaction; comparison group; computerized data processing; cost; demethylation; disease diagnosis; embryonic stem cell; epigenome; epigenome editing; functional genomics; genome integrity; genome-wide; human disease; innovation; innovative technologies; methyl group; oxidation; response; technology development; technology research and development; tool

Project Summary / Abstract DNA methylation homeostasis is primarily maintained by coordinated actions of DNA methyltransferases (DNMTs) and the Ten-eleven Translocation (TET) dioxygenase. While DNMTs catalyze the addition of a methyl group to the carbon-5 position of cytosine to generate 5- methylcytosine (5mC), TET proteins mediate the further oxidation of 5mC to successively yield 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These DNA methylation oxidation products, collectively termed as “oxi-mC”, not only serve as critical intermediates during active DNA demethylation, but also act as important epigenetic marks to regulate gene transcription, chromatin accessibility and 3D chromatin organization. With advanced sequencing technology, the genomic distribution and function of individual oxi-mC species are just beginning to be appreciated. However, our current knowledge on oxi-mC is mostly based on sequencing results from bulk cells and tissues. A major current challenge is to efficiently probe single cell oxi-mC at single-base resolution, and to unambiguously assign their correlations with 3D chromatin features. The existing single cell 5hmC and 5fC mapping technologies adopt totally different strategies, including antibody- based enrichment, chemical/enzymatic reactions, and/or bisulfite treatment. The variation in sample treatment protocols adds tremendous complexity for library preparation and introduces significant barriers for data processing and cross-group comparisons. By leveraging complementary expertise in epigenetics, chemical biology and bioinformatics, the PI has assembled a strong team of investigators to tackle this challenge. The goal of this exploratory technology development proposal is to develop an innovative and widely applicable molecular toolset, which will enable paralleled high-resolution mapping and analysis of individual oxi-mC heterogeneity and chromatin architectures in single cells derived from various biological systems. Given the importance of chromatin accessible regions and long-range chromatin loops in governing gene expression, we will focus on unraveling the functional coupling of oxi-mC with 3D chromatin organization in these functional genomic regions. The successful execution of this project will provide a streamlined pipeline to profile oxi-mC at single-base resolution and permit simple side-by-side comparison of 5hmC, 5fC and 5caC dynamics. This toolkit can be widely used to monitor oxi-mC dynamics in normal development and various pathological conditions associated with aberrant DNA methylation, such as cardiovascular disease, immunoinflammatory disorders and cancer.

项目摘要 /摘要 DNA甲基化稳态主要通过DNA的协调作用维持 甲基转移酶（DNMTS）和十个易位（TET）二氧酶。而dnmts 催化甲基添加到胞嘧啶的碳-5位置以产生5-- TET蛋白（5MC）甲基胞嘧啶（5MC）介导5MC的进一步氧化，成功产生5-- 羟基胞嘧啶（5HMC），5-甲基霉素（5FC）和5-羧基霉素（5CAC）。这些DNA 甲基化氧化产物统称为“ Oxi-MC”，不仅是关键中间体 在活性DNA脱甲基化期间，但也充当了调节基因的重要表观遗传标记 转录，染色质可及性和3D染色质组织。带有高级测序 技术，单个Oxi-MC物种的基因组分布和功能才开始成为 感谢。但是，我们目前对OXI-MC的了解主要基于 散装细胞和组织。当前的主要挑战是在单基碱基处有效探测单细胞OXI-MC 分辨率，并明确用3D染色质特征分配其相关性。现有 单细胞5HMC和5FC映射技术采用完全不同的策略，包括抗体 - 基于富集，化学/酶促反应和/或亚硫酸盐治疗。样品的变化 治疗方案增加了图书馆准备的巨大复杂性，并引入了重要的复杂性 数据处理和跨组比较的障碍。通过利用完成专业知识 PI表观遗传学，化学生物学和生物信息学，已经组建了一个强大的研究人员 应对这一挑战。该探索性技术发展建议的目的是开发 创新且广泛适用的分子刀具集，该工具集将实现并行的高分辨率映射 并分析单个细胞中单个细胞中的单个Oxi-MC异质性和染色质结构 各种生物系统。鉴于染色质访问区域和远距离的重要性 染色质循环在管理基因表达中，我们将专注于揭开功能耦合 在这些功能基因组区域中具有3D染色质组织的Oxi-MC。成功的执行 该项目将提供一个简化的管道，以在单基准分辨率下进行轮廓OXI-MC并允许 5HMC，5FC和5CAC动力学的简单并排比较。该工具包可广泛用于 监测正常发育中的Oxi-MC动力学以及与 异常的DNA甲基化，例如心血管疾病，免疫炎症疾病和癌症。