Advancing Epigenetic Sequencing Through Solid-Phase Enzymatic Approaches

项目总结 DNA中胞嘧啶碱基的修饰在塑造细胞身份和命运方面发挥着重要的表观遗传学作用。 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(5mC)，它已经被 与基因沉默有关。该领域的一个里程碑式的时刻来自Tet酶的发现，它 反复氧化5mC得到5-羟甲基胞嘧啶(5hmC)和更多高度氧化的5mC碱基。在……里面 与5mC相比，5hmC与基因激活有关，突出了解决问题的重要性 不同的DNA修饰状态。鉴于它们的生物学意义，在基因组中定位修饰的碱基 一直是该领域的主要关注点。表观遗传测序方法可以阐明DNA的生物学作用 并且可以识别导致疾病的异常修饰。传统的方法是 精确定位胞嘧啶的修饰包括使样品DNA与亚硫酸氢盐反应以选择性脱氨 未修饰的胞嘧啶，同时保存5mC和5hmC。虽然基础，亚硫酸氢盐可以碎片DNA，提高 挑战输入DNA要求，该方法混淆了5mC和5hmC。新成立 酶法是非破坏性的，但仍然混淆了多个碱基，需要过多的纯化步骤 这会导致DNA的丢失。本研究旨在建立一种表观遗传测序的酶促方法。 固相固定化DNA，能够定量捕获输入DNA并解析修饰 因此，可以对有限的样本进行有洞察力的排序。目标1提出了 研究TET1如何重塑表观基因组的方法的发展、基准和应用 原始生殖细胞，一种样本，其分析受到低样本量的限制。在最初建立了 表观遗传测序的固相酶方法，本研究将随后利用固相 并将它们与化学转化相结合，开发出一种拆分C， 5mC和5hmC在同一DNA分子中。目标2建议开发一种前所未有的方法 能够定位和识别顺式基因中的表观遗传修饰。遵循方法开发和 验证，该方法将用于研究Foxp3基因的5mC/5hmC修饰动力学 已知在调节性T细胞识别中起决定性作用，但目前的方法不能解析顺式修饰 两性关系。总体而言，本提案中开发的两种新方法将以生物学为重点加以应用 洞察和定位该领域的未来应用，旨在解决可能成为 疾病状态的标志。该培训计划将使PI为独立研究职业生涯调查做好准备 表观遗传疾病的驱动因素，将在高度跨学科的宾夕法尼亚大学举行。

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.