Molecular toolkit for single-cell oxi-mC analysis

项目摘要/摘要 DNA甲基化动态平衡主要由DNA的协调作用来维持 甲基转移酶(DNMT)和Ten-11易位(Tet)双加氧酶。而DNMTS 催化胞嘧啶的碳-5位加成甲基生成5- 甲基胞嘧啶(5 MC)，Tet蛋白介导5 mC的进一步氧化，从而连续生成5-甲基胞嘧啶(5-mC) 羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。这些DNA 甲基化氧化产物，统称为“OXI-MC”，不仅是关键中间体 在活跃的DNA去甲基化过程中，也可以作为重要的表观遗传标记来调节基因 转录、染色质可及性和3D染色质组织。先进的测序技术 技术，单个oxi-mc物种的基因组分布和功能才刚刚开始 感激不尽。然而，我们目前对oxi-MC的了解主要基于以下方面的测序结果 大量细胞和组织。当前的一个主要挑战是在单碱基上有效地探测单细胞oxi-mc。 分辨率，并明确指定它们与3D染色质特征的相关性。现有的 单细胞5hmc和5fc作图技术采用了完全不同的策略，包括抗体- 基于浓缩、化学/酶反应和/或亚硫酸氢盐处理。样本中的变异 处理方案增加了库准备的巨大复杂性，并引入了显著的 数据处理和跨组比较的障碍。通过利用以下方面的互补专业知识 在表观遗传学、化学生物学和生物信息学方面，PI组建了一支强大的调查团队 来应对这一挑战。此探索性技术开发计划的目标是开发一种 创新和广泛应用的分子工具箱，将实现并行的高分辨率测绘 和单个OXI-MC异质性和单个细胞中染色质结构的分析 各种生物系统。鉴于染色质可及区域和远距离的重要性 在控制基因表达的染色质环中，我们将重点解开 Oxi-MC在这些功能基因组区域具有3D染色质组织。成功的执行 将提供一条简化的管道，以单碱基分辨率和许可的方式分析OXI-MC 5hmC、5fC和5caC动力学的简单并列比较。该工具包可广泛应用于 监测OXI-MC在正常发育和与之相关的各种病理情况下的动力学 DNA甲基化异常，如心血管疾病、免疫性炎症和癌症。

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.