Non-destructive epigenetic sequencing with DNA deaminase enzymes

项目摘要 这项建议旨在建立DNA胞嘧啶脱氨酶作为一种非破坏性的替代亚硫酸氢盐 用于胞嘧啶修饰的碱基分辨率映射。基因组的表观遗传修饰 在细胞适应和来自相同编码的各种细胞谱系的特化中起重要作用 顺序在DNA上，这些表观遗传变化包括在DNA的5位胞嘧啶碱基的修饰， 核碱基最常见的修饰是5-甲基胞嘧啶（5mC），其次是5-甲基胞嘧啶（5mC）。 羟甲基胞嘧啶（5hmC），是泰特酶介导的5mC氧化的产物。变换 参与发育、多能性和肿瘤发生的基因组模式的改变需要5mC和 5hmC，因此重要的是要有强大的方法来定位这些修饰。方法最 通常用于检测这些修饰的方法包括用亚硫酸氢盐处理基因组DNA， 胞嘧啶修饰状态具有不同的亚硫酸氢盐诱导的脱氨基倾向， 通过测序分析。然而，化学脱氨可以降解绝大多数起始DNA。作为 结果，基于亚硫酸氢盐的方法限制了我们理解胞嘧啶修饰的能力 在许多小的或短暂的细胞群体中，或者研究变化如何在长时间的 基因组DNA 在本提案中，我们将开发和应用基于DNA脱氨酶的测序方法， 亚硫酸氢盐的主要缺点。我们的方法依赖于酶，而不是化学脱氨基，使用 APOBEC3A（A3A），一种来自免疫系统的DNA脱氨酶，用于这些生物技术 应用.在我们的生物化学研究中，我们已经确定A3A可以有效地区分不同的 胞嘧啶修饰状态，并且，在导致这一提议的基础工作中，我们开发了APOBEC- 偶联表观遗传测序（ACE-Seq）是一种非破坏性的碱基分辨率测序方法， 定位于5hmC。基于这一先例，我们提出了两个新的DNA脱氨酶为基础的 测序方法现在可以将5mC和5hmC定位在一起，为亚硫酸氢盐提供替代品，或者 通过脱氨基作用直接单独检测5mC，这是没有先例的。我们将这些方法应用于 解决了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来解释单细胞水平上的表观遗传异质性，揭示顺式 跨基因座的基因在长段DNA中相互协调，并报告所有三个基因座的"三元密码"。 在一个单一的阅读修改。因此，我们的建议旨在建立DNA脱氨酶作为一种非破坏性 和更可靠的测序方法，可以取代亚硫酸氢盐及其相关的限制， 从而推动DNA脱氨酶在临床和实验室的表观遗传测序中的广泛采用。

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.