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Advancing Epigenetic Sequencing Through Solid-Phase Enzymatic Approaches

Award Number: F31HG012892
ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 02/01/2023
PERIOD OF PERFORMANCE END DATE: 01/31/2025
AWARD TERMINATION DATE: 04/30/2025

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Advancing Epigenetic Sequencing Through Solid-Phase Enzymatic Approaches - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $0 )
2026	2024	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Human Genome Research	000	2	1/27/2026	TERMINATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST STE 440A	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Human Genome Research	000	2	4/30/2025	TERMINATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $47,694 )
2024	2024	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST STE 440A	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Human Genome Research	000	2	1/9/2024	NON-COMPETING CONTINUATION	$47,694
														Subtotal = $47,694

Issue Date FY: 2023 ( Subtotal = $47,694 )
2023	2023	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST STE 440A	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Human Genome Research	002	1	9/22/2023	NEW	$942
2023	2023	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST STE 440A	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Human Genome Research	001	1	1/20/2023	NEW	$0
2023	2023	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST STE 440A	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Human Genome Research	000	1	1/20/2023	NEW	$46,752
														Subtotal = $47,694

Grand Total All Awards = $95,388

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Advancing Epigenetic Sequencing Through Solid-Phase Enzymatic Approaches

Award Number: F31HG012892

ORGANIZATION: NATIONAL HUMAN GENOME RESEARCH INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 02/01/2023

PERIOD OF PERFORMANCE END DATE: 01/31/2025

AWARD TERMINATION DATE: 04/30/2025

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