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

• Published in: Epigenetics Vol. 12; no. 4; pp. 296 - 303
• Main Authors: Parrilla-Doblas, Jara Teresa, Ariza, Rafael R., Roldán-Arjona, Teresa
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 03.04.2017; Taylor & Francis Group
• Subjects: 5-methylcytosine; 5-Methylcytosine - metabolism; Base excision repair; DNA demethylation; DNA glycosylase; DNA Glycosylases - metabolism; DNA Methylation; epigenetic editing; Gene Silencing; HEK293 Cells; Humans; Plants - enzymology; Research Paper; Transcriptional Activation; transcriptional silencing; epigenetic editing; DNA methylation; 5-methylcytosine; DNA demethylation; DNA glycosylase; transcriptional silencing; Base excision repair
• ISSN: 1559-2294; 1559-2308
• DOI: 10.1080/15592294.2017.1294306

DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequence in vitro. We also found that transient in vivo expression of GBD-ROS1_CD in human cells specifically reactivates transcription of a methylation-silenced reporter gene, and that such reactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show that reactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpG sites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can be used for targeted active DNA demethylation in human cells.