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 afford unprecedented insight into active DNA demethylation pathways and functional roles of ox-mC that will contribute to our understanding of disease inception.
Epigenetic modifications to genomic DNA change its gene regulatory potential and play an important role in diverse processes, including pluripotency, development and oncogenesis. This proposal advances novel epigenome-editing and sequencing methods for dissecting the gene regulatory roles of oxidized DNA modifications. This work will help reveal the mechanisms by which these oxidized DNA modifications modulate gene regulation and establish a mechanistic foundation for the development of targeted therapeutic epigenetic editing.
