# Dissecting Gene Regulatory Roles of TET Enzymes and 5-hydroxymethylcytosine in Mammalian Active DNA Demethylation > **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2021 · $46,036 ## Abstract Project Summary Epigenetic modifications have important roles in cellular functions and in specialization of cell lineages. On DNA, epigenetic modification occurs on the 5-position of cytosine nucleobases. The most common modification is 5- methylcytosine (5mC), and TET-Eleven-Translocation (TET) proteins enzymatically remove DNA methylation by iteratively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). In mammals, TET-mediated active DNA demethylation can be achieved by replication-dependent dilution of 5hmC, or thymine DNA glycosylase (TDG) mediated base excision repair (BER) of 5fC/5caC to regenerate unmodified C. However, the functional significance of these oxidized modifications (ox-mC) and mechanistically distinct active DNA demethylation pathways remains poorly understood, precluding our comprehension on how dysregulated DNA methylation contributes to disease. Indeed, ox-mC has been implicated in crucial biological processes such as gene transcription. However, it has been challenging to ascribe functional roles to ox-mC as it was technically challenging to decouple generation of 5hmC from 5fC/5caC, and unmodified C. Elucidating functional roles of ox-mC will establish a foundational understanding for developing novel therapeutics for various pathologies. To this end, I developed a CRISPR/dCas9 platform that recruits 5hmC-stalling TET-variants to interrogate gene regulatory roles of 5hmC, 5fC/5caC, and TDG/BER in mammalian systems. Preliminary results generated from comprehensive epigenetic sequencing (bisulfite sequencing (BS- Seq)/Bisulfite-assisted APOBEC-Coupled Epigenetic sequencing (bACE-Seq)/Methylase-Assisted Bisulfite sequencing (MAB-Seq)) revealed 5hmC alone could not reactivate a hypermethylated gene promoter in proliferative human cells, and generation of 5fC/5caC was requisite. These results show for the first time, functional distinction between ox-mC. It remains ambiguous how downstream higher ox-mC pathways could reactivate gene expression. I hypothesize 5fC/5caC deposition depletes nucleosome occupancy to facilitate transcription. In aim 1, I will evaluate the role of 5hmC/5fC/5caC/C and TDG in Tet1-3 triple knock out (TKO) and Tet1-3/Tdg quadruple knockout (QKO) mouse embryonic stem cells (mESCs), on gene expression and local chromatin structure. My results also reveal 5hmC alone could not restore unmodified C by replication-dependent dilution of 5hmC suggesting this mechanism of active DNA demethylation is more tightly regulated than previously anticipated. Quantitative genome-wide analysis of how ox-mC bases is mitotically inherited across division is currently lacking. I hypothesize 5hmC is mitotically inherited to nascent strands, while 5fC/5caC is rapidly removed by TDG/BER. In aim 2, I will develop technologies to quantify and profile ox-mC mitotic inheritance at single-base resolution in genetically engineered mESCs. By completing the proposed aims, I will a... ## Key facts - **NIH application ID:** 10251899 - **Project number:** 5F31HG011429-02 - **Recipient organization:** UNIVERSITY OF PENNSYLVANIA - **Principal Investigator:** Alex Tianjiun Wei - **Activity code:** F31 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2021 - **Award amount:** $46,036 - **Award type:** 5 - **Project period:** 2020-06-01 → 2023-05-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10251899 ## Citation > US National Institutes of Health, RePORTER application 10251899, Dissecting Gene Regulatory Roles of TET Enzymes and 5-hydroxymethylcytosine in Mammalian Active DNA Demethylation (5F31HG011429-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10251899. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*