Role of NEIL DNA glycosylases in DNA demethylation and differentiation

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DNA methylation is an epigenetic mark which plays important roles in development and disease. It is now widely accepted that DNA methylation is dynamic and is subject to enzymatic demethylation. In the TET/TDG demethylation pathway, methylated cytosine is iteratively oxidized by TET dioxygenases and unmodified cytosine is restored by thymine DNA glycosylase (TDG). A vulnerable intermediate in TET/TDG demethylation is the abasic (AP) site. We have shown recently that NEIL1 and NEIL2 DNA glycosylases coordinate AP site processing during TET/TDG DNA demethylation. Our analysis indicates that NEILs function as regulators in the coordinated substrate hand-over during oxidative DNA demethylation. To investigate the physiological relevance and biological role of Neil DNA glycosylases in DNA demethylation and DNA repair we have generated via CRISPR/Cas9, mouse embryonic stem cells which are mutant for Neil1,-2, or 3, as well as Neil double mutants The aim of this project is to characterize these mutant ES cells for their differentiation capacity, oxidized cytosine levels, and DNA repair abilities.