Oxidized 5-Methylcytosines and Nucleosome Architecture Cooperatively Influence Thymine DNA Glycosylase Activity

Active DNA demethylation is a fundamental epigenetic process in which the iterative oxidation of 5-methylcytosine (5mC) by TET dioxygenases generates diverse combinations of oxidized 5-methylcytosines within CpG dinucleotides before their excision by thymine DNA glycosylase (TDG). Although heterogeneous CpG oxidation states are expected to arise frequently during this process, how the modification state of the opposing cytosine influences TDG activity remains poorly understood. Here, we developed a chemically defined panel of CpG substrates containing cytosine, 5mC, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) opposite a target 5fC and systematically examined TDG-mediated excision in both naked DNA and nucleosome core particles (NCPs). In naked DNA, opposing-strand cytosine modifications exerted only modest effects on TDG activity when a single excisable base was present within the CpG dyad, whereas dual-substrate CpGs exhibited unexpected differences in strand utilization. In contrast, nucleosome assembly profoundly altered TDG activity, revealing modification-dependent effects that were largely absent in naked DNA and dramatically redistributing strand utilization within dual-substrate CpGs. Together, these findings demonstrate that the oxidation-state composition of a CpG dyad and nucleosome architecture cooperatively regulate TDG activity, establishing heterogeneous CpG oxidation states as functionally distinct substrates for TDG and providing a biochemical framework for understanding how chromatin structure regulates active DNA demethylation.