Project Summary / Abstract
DNA methylation homeostasis is primarily maintained by coordinated actions of DNA
methyltransferases (DNMTs) and the Ten-eleven Translocation (TET) dioxygenase. While DNMTs
catalyze the addition of a methyl group to the carbon-5 position of cytosine to generate 5-
methylcytosine (5mC), TET proteins mediate the further oxidation of 5mC to successively yield 5-
hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These DNA
methylation oxidation products, collectively termed as “oxi-mC”, not only serve as critical intermediates
during active DNA demethylation, but also act as important epigenetic marks to regulate gene
transcription, chromatin accessibility and 3D chromatin organization. With advanced sequencing
technology, the genomic distribution and function of individual oxi-mC species are just beginning to be
appreciated. However, our current knowledge on oxi-mC is mostly based on sequencing results from
bulk cells and tissues. A major current challenge is to efficiently probe single cell oxi-mC at single-base
resolution, and to unambiguously assign their correlations with 3D chromatin features. The existing
single cell 5hmC and 5fC mapping technologies adopt totally different strategies, including antibody-
based enrichment, chemical/enzymatic reactions, and/or bisulfite treatment. The variation in sample
treatment protocols adds tremendous complexity for library preparation and introduces significant
barriers for data processing and cross-group comparisons. By leveraging complementary expertise in
epigenetics, chemical biology and bioinformatics, the PI has assembled a strong team of investigators
to tackle this challenge. The goal of this exploratory technology development proposal is to develop an
innovative and widely applicable molecular toolset, which will enable paralleled high-resolution mapping
and analysis of individual oxi-mC heterogeneity and chromatin architectures in single cells derived from
various biological systems. Given the importance of chromatin accessible regions and long-range
chromatin loops in governing gene expression, we will focus on unraveling the functional coupling of
oxi-mC with 3D chromatin organization in these functional genomic regions. The successful execution
of this project will provide a streamlined pipeline to profile oxi-mC at single-base resolution and permit
simple side-by-side comparison of 5hmC, 5fC and 5caC dynamics. This toolkit can be widely used to
monitor oxi-mC dynamics in normal development and various pathological conditions associated with
aberrant DNA methylation, such as cardiovascular disease, immunoinflammatory disorders and cancer.