Advancing Epigenetic Sequencing Through Solid-Phase Enzymatic Approaches

项目摘要 DNA内胞嘧啶碱基的修饰在塑造细胞身份和命运中起重要的表观遗传作用。 在哺乳动物基因组中，这些修饰中最丰富的是5-甲基胞嘧啶（5 mC）， 与基因沉默有关。该领域的一个里程碑式的时刻来自于泰特酶的发现， 反复氧化5 mC以产生5-羟甲基胞嘧啶（5 hmC）和更高度氧化的5 mC碱基。在 与5 mC相反，5 hmC与基因激活有关，突出了解决 不同的DNA修饰状态考虑到它们的生物学意义，在基因组中定位修饰的碱基 一直是该领域的主要焦点。表观遗传测序方法可以阐明DNA的生物学作用 修饰，并且可以鉴定导致疾病的异常修饰。的传统方法 精确定位胞嘧啶修饰涉及使样品DNA与亚硫酸氢盐反应以选择性地脱氨基 未修饰的胞嘧啶，同时保留5 mC和5 hmC。虽然基础，亚硫酸氢盐可以片段DNA，提高 输入DNA要求的挑战，并且该方法混淆了5 mC和5 hmC。新成立 酶法是非破坏性的，但仍然混淆多个碱基，需要过多的纯化步骤 导致DNA丢失。本研究的目的是建立一种酶法进行表观遗传测序的方法， 固相固定化DNA，能够定量捕获输入DNA并解析修饰 状态，从而允许对有限的样品进行有见地的测序。目标1提出， 开发，基准测试和应用方法来研究TET 1如何重塑表观基因组， 原始生殖细胞是一种分析受到低样本量限制的样本。在最初建立一个 固相酶法进行表观遗传测序，本研究随后将利用固相 酶促原理，并将其与化学转化复合，开发出拆分C， 5 mC和5 hmC在同一DNA分子中。目标2提出了一种前所未有的方法的发展 能够定位和鉴定顺式中的表观遗传修饰。方法开发和 验证，该方法将应用于研究Foxp 3基因座，其中5 mC/5 hmC修饰动力学是 已知在调节性T细胞身份中起决定性作用，但目前的方法不能解析顺式修饰 关系。总的来说，本提案中开发的两种新方法将重点应用于生物 洞察力和定位领域的未来应用，旨在解决表观遗传异常，可以是一个 疾病状态的标志。该培训计划将为PI的独立研究生涯做好准备， 疾病的表观遗传驱动因素，并将在宾夕法尼亚大学高度跨学科的地方。

PROJECT SUMMARY Modifications to cytosine bases within DNA play an important epigenetic role in shaping cellular identity and fate. In mammalian genomes, the most abundant of these modifications is 5-methylcytosine (5mC), which has been linked with gene silencing. A landmark moment in the field came from the discovery of TET enzymes, which iteratively oxidize 5mC to yield 5-hydroxymethylcytosine (5hmC) and more highly oxidized 5mC bases. In contrast with 5mC, 5hmC has been associated with gene activation, highlighting the importance of resolving different DNA modification states. Given their biological significance, localizing modified bases within genomes has been a major focus for the field. Epigenetic sequencing approaches can elucidate biological roles for DNA modifications and can identify aberrant modifications that contribute to disease. The traditional method for pinpointing cytosine modifications involves reacting sample DNA with bisulfite to selectively deaminate unmodified cytosines, while preserving 5mC and 5hmC. While foundational, bisulfite can fragment DNA, raising challenges with input DNA requirements, and the method confounds 5mC and 5hmC. Newly established enzymatic methods are non-destructive, but still confound multiple bases and require excessive purification steps that result in loss of DNA. This study aims to develop an enzymatic method for epigenetic sequencing of solid-phase immobilized DNA, enabling quantitative capture of input DNA and resolution of modification states, thus allowing for insightful sequencing to be conducted on limiting samples. Aim 1 proposes the development, benchmarking, and application of the method to study how TET1 reshapes the epigenome in primordial germ cells, a sample where analysis is limited by low sample quantities. After initially establishing a solid-phase enzymatic method for epigenetic sequencing, this study will subsequently leverage solid-phase enzymatic principles and multiplex them with chemical conversion to develop a method for resolving C, 5mC, and 5hmC in the same DNA molecule. Aim 2 proposes the development of an unprecedented method capable of both locating and identifying epigenetic modifications in cis. Following method development and validation, this method will be applied to study the Foxp3 locus where 5mC/5hmC modification dynamics are known to play deterministic roles in regulatory T cell identity, but current methods cannot to parse cis modification relationships. Overall, the two novel methods developed in this proposal will be applied with a focus on biological insights and position the field for future applications aimed at resolving epigenetic abnormalities that can be a hallmark of disease states. This training plan will prepare the PI for an independent research career investigating epigenetic drivers of disease and will take place at the highly interdisciplinary University of Pennsylvania.