Advancing Epigenetic Sequencing Through Solid-Phase Enzymatic Approaches

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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.