RT Journal Article
T1 Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain.
A1 Parrilla-Doblas, Jara Teresa
A1 Ariza, Rafael R
A1 Roldan-Arjona, Teresa
K1 5-methylcytosine
K1 Base excision repair
K1 DNA demethylation
K1 DNA glycosylase
K1 DNA methylation
K1 Epigenetic editing
K1 Transcriptional silencing
AB 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.
PB Taylor & Francis
YR 2017
FD 2017-02-06
LK http://hdl.handle.net/10668/10948
UL http://hdl.handle.net/10668/10948
LA en
NO Parrilla-Doblas JT, Ariza RR, Roldán-Arjona T. Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain. Epigenetics. 2017 Apr 3;12(4):296-303
NO This work was supported by the Spanish Ministry of Science and Innova-tion and the European Regional Development Fund under grantBFU2013-43269-P; and by the Junta de Andaluc ıa and the EuropeanRegional Development Fund under grant P11-CVI-7576. J.T.P.D. was therecipient of a PhD FPU Fellowship from the Spanish Ministry of Educa-tion. We thank Dr. A. Jeltsch (Stuttgart University, Germany) for kindlyproviding plasmids 5xUAS-TK-Luc and pcDNA3.1-GBD
DS RISalud
RD Apr 10, 2025