In most organisms 5-methylcytosine (5mC) is an epigenetic marker for transcription inactivation and, typically, genes affected by 5mC reside within densely packed chromosomal structures known as heterochromatin that effectively shield proteins from accessing the underlying DNA. DNA demethylation is a key chemical step preceding the active transcription of genes regulated by 5mC and, although the many factors involved in this process have been identified, very little is known about how the cell’s demethylation machinery gain access to target DNA sequences within inaccessible chromatin environments. In order to address this gap in our current knowledge, we generated synthetic chromatin fibers harboring 5-formylcytosine (5fC), a demethylation intermediate, and performed quantitative measurements on the impact of unique chromatin states on the activity of thymine DNA glycosylase (TDG). Our results demonstrate that TDG-dependent demethylation is highly dependent on nucleosome position and higher order chromatin structure. Furthermore, we found that pioneer transcription factors (TFs) are capable of enhancing 5fC-removal by TDG on condensed chromatin fibers. Details regarding these studies will be presented.

DNA demethylation in reconstituted systems: Effects of higher order chromatin structure on thymine DNA glycosylase

2019 Fall National Meeting

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