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.

Epigenetic changes can switch genes on or off to determine the fate of human cells. This project focuses on developing innovative technologies to systematically study single cell epigenetic modifications on DNA at single-base resolution. Decoding the dynamics and cell-to-cell variability of DNA modifications will greatly advance our knowledge on the role of epigenetics in biology and human diseases.