Removal of methylated cytosine (5mC) from the genome can occur in either a passive or active manner. During active DNA demethylation, the ten-eleven translocation (TET) family of dioxygenases successively oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). These oxidized methylcytosines are then removed by thymine DNA glycosylase (TDG)-mediated excision of 5fC/5caC followed by base excision repair. Like all genomic processes, active DNA demethylation must take place within the structural constraints of chromatin. Consistently, emerging experimental evidence suggests that nucleosome organization and the degree of chromatin compaction are key modulators of TET and TDG activity. However, due to numerous challenges associated with performing detailed biochemical studies on native cellular chromatin, almost nothing is known about how different chromatin environments influence TET/TDG-mediated DNA demethylation, especially at the molecular level. To address these issues, we have used chemical biology methods to assemble nucleosome arrays containing defined arrangements of both modified cytosine bases and histone post-translational modifications<i>. </i>Uncoupled from the more complex cellular environment, these “designer” chromatin substrates enable unprecedented quantitative interrogation of the active DNA demethylation pathway within distinct chromatin states, both structural and chemical. For example, we used this experimental platform to investigate how the unique chromatin environment at gene enhancers regulates the removal of 5fC by TDG. Details regarding these studies will be presented.

Providing a molecular level view of active DNA demethylation using designer chromatin

2019 Fall National Meeting

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