Non-destructive epigenetic sequencing with DNA deaminase enzymes

使用 DNA 脱氨酶进行非破坏性表观遗传测序

基本信息

批准号：
9797035
负责人：
Rahul Manu Kohli
金额：
$ 60万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9797035
关键词：
Address Adoption Alkylation Biochemical Biological Biology Biotechnology Brain Cell Lineage Cells Chemicals Clinic Code Coupled CpG dinucleotide Cytosine Cytosine deaminase DNA DNA sequencing Deaminase Deamination Detection Development Discrimination Enzymes Epigenetic Process Exhibits FOXP3 gene Foundations Gene Expression Genetic Fingerprintings Genetic Transcription Genome Genomic DNA Genomics Goals Gold Heterogeneity Immune Immune system Length Link Location Mediating Methods Modification Mus Neurons Pathologic Processes Pattern Physiological Processes Play Population Positioning Attribute Process Refractory Regulatory T-Lymphocyte Reporting Resolution Role Sampling Shapes Signal Transduction Site Stretching Technology Temperature Third Generation Sequencing Time Tissues Work base bisulfite bisulfite sequencing epigenetic profiling epigenome frontal lobe frontier insight multimodality novel nucleobase oxidation pluripotency single cell analysis single-cell RNA sequencing tool transcriptome tumorigenesis

项目摘要

PROJECT SUMMARY This proposal aims to establish DNA cytosine deaminase enzymes as a non-destructive alternative to bisulfite for base-resolution mapping of cytosine modifications. Epigenetic modifications to the genome play an important role in cellular adaptation and in specialization of various cell lineages derived from the same coding sequence. On DNA, these epigenetic changes include modification of cytosine bases at the 5-postion of the nucleobase. The most common modification is 5-methylcytosine (5mC), followed closely by 5- hydroxymethylcytosine (5hmC), a product of TET enzyme-mediated oxidation of 5mC. Transformations involved in development, pluripotency and oncogenesis entail changes in the genomic patterns of 5mC and 5hmC, making it important to have robust methods to localize these modifications. The methods most commonly used to detect these modifications involve treatment of genomic DNA with bisulfite, as the different cytosine modification states have a different propensity for bisulfite-induced deamination which can be analyzed by sequencing. Chemical deamination, however, can degrade the vast majority of starting DNA. As a result, bisulfite-based approaches constrain our ability to understand the landscapes of cytosine modifications in many small or transient cell populations, or to study how changes are coordinated across long stretches of genomic DNA. In this proposal, we will develop and apply DNA deaminase-based sequencing approaches that address the major shortcomings of bisulfite. Our methods rely upon enzymatic, rather than chemical deamination, using APOBEC3A (A3A), a DNA deaminase from the immune system repurposed for these biotechnological applications. In our biochemical studies, we have established that A3A potently discriminates between different cytosine modification states, and, in foundational work leading up to this proposal, we developed APOBEC- Coupled Epigenetic Sequencing (ACE-Seq) as a non-destructive, base resolution sequencing method for localizing 5hmC. Building on this precedent, we propose to advance two new DNA deaminase-based sequencing approaches that can now localize 5mC and 5hmC together, providing a surrogate for bisulfite, or to directly detect 5mC alone through deamination, which is without precedent. We will apply these methods to address important biological questions that are refractory to bisulfite-based approaches, specifically resolving C, 5mC and 5hmC to decipher epigenetic heterogeneity at the single cell level, revealing how in cis changes across loci are coordinated across long stretches of DNA, and reporting on the ‘ternary code’ of all three modifications in a single read. Our proposal therefore aims to establish DNA deaminases as a non-destructive and more reliable means for sequencing that can displace bisulfite and its associated limitations, and to thereby drive the widespread adoption of DNA deaminases in epigenetic sequencing in the clinic and the lab.

项目概要该提案旨在建立 DNA 胞嘧啶脱氨酶作为亚硫酸氢盐的非破坏性替代品胞嘧啶修饰的碱基分辨率图谱对基因组的表观遗传修饰起着重要作用。在细胞适应和源自相同编码的各种细胞谱系的特化中发挥重要作用在 DNA 序列上，这些表观遗传变化包括 5 位胞嘧啶碱基的修饰。最常见的修饰是 5-甲基胞嘧啶 (5mC)，紧随其后的是 5-。羟甲基胞嘧啶 (5hmC)，TET 酶介导的 5mC 转化的产物。参与发育、多能性和肿瘤发生需要 5mC 和 5hmC，因此拥有可靠的方法来定位这些修改非常重要。通常用于检测这些修饰涉及用亚硫酸氢盐处理基因组 DNA，因为不同的胞嘧啶修饰状态对于亚硫酸氢盐诱导的脱氨有不同的倾向，这可以是然而，通过测序分析，化学脱氨可以降解绝大多数起始 DNA。结果，基于亚硫酸氢盐的方法限制了我们理解胞嘧啶修饰景观的能力在许多小型或短暂的细胞群体中，或者研究如何在长距离内协调变化基因组DNA。在本提案中，我们将开发并应用基于 DNA 脱氨酶的测序方法来解决我们的方法依赖于酶促脱氨，而不是化学脱氨。 APOBEC3A (A3A)，一种来自免疫系统的 DNA 脱氨酶，被重新用于这些生物技术在我们的生化研究中，我们已经确定 A3A 能够有效地区分不同的物质。胞嘧啶修饰状态，并且，在导致该提案的基础工作中，我们开发了 APOBEC- 耦合表观遗传测序 (ACE-Seq) 作为一种非破坏性、碱基分辨率测序方法，用于在此先例的基础上，我们建议推进两种新的基于 DNA 脱氨酶的技术。现在可以将 5mC 和 5hmC 一起定位的测序方法，提供亚硫酸氢盐的替代品，或者通过脱氨直接单独检测5mC，这是没有先例的，我们将应用这些方法。解决基于亚硫酸氢盐的方法难以解决的重要生物学问题，特别是解决 C、5mC 和 5hmC 在单细胞水平上破译表观遗传异质性，揭示顺式变化的方式跨基因座在长段 DNA 中协调，并报告所有三个的“三元代码” 因此，我们的建议旨在将 DNA 脱氨酶建立为一种非破坏性的酶。更可靠的测序方法可以取代亚硫酸氢盐及其相关限制，并广泛推动 DNA 脱氨酶在临床和实验室表观遗传测序中的应用。