Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosineDNA glycosylase to a sequence-specific DNA binding domain

Abstract

DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is amajor goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation byTET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of itsoxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiateits substitution for unmethylated C in a base excision repair process. In this work, we have fused thecatalytic domain ofArabidopsisROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain ofyeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequencein vitro. We also found that transientin vivoexpression of GBD-ROS1_CD inhuman cells specifically reactivates transcription of a methylation-silenced reporter gene, and that suchreactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show thatreactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpGsites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can beused for targeted active DNA demethylation in human cells.