Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
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...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2017-04-01
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| Series: | Epigenetics |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/15592294.2017.1294306 |
| _version_ | 1827811666431049728 |
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| author | Jara Teresa Parrilla-Doblas Rafael R. Ariza Teresa Roldán-Arjona |
| author_facet | Jara Teresa Parrilla-Doblas Rafael R. Ariza Teresa Roldán-Arjona |
| author_sort | Jara Teresa Parrilla-Doblas |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T23:07:49Z |
| format | Article |
| id | doaj.art-cb89ea0cd7e1402093012c5e15b73bae |
| institution | Directory Open Access Journal |
| issn | 1559-2294 1559-2308 |
| language | English |
| last_indexed | 2024-03-11T23:07:49Z |
| publishDate | 2017-04-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Epigenetics |
| spelling | doaj.art-cb89ea0cd7e1402093012c5e15b73bae2023-09-21T12:43:12ZengTaylor & Francis GroupEpigenetics1559-22941559-23082017-04-0112429630310.1080/15592294.2017.12943061294306Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domainJara Teresa Parrilla-Doblas0Rafael R. Ariza1Teresa Roldán-Arjona2Maimónides Biomedical Research Institute of Córdoba (IMIBIC)Maimónides Biomedical Research Institute of Córdoba (IMIBIC)Maimónides Biomedical Research Institute of Córdoba (IMIBIC)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.http://dx.doi.org/10.1080/15592294.2017.1294306base excision repairdna demethylationdna glycosylasedna methylationepigenetic editing5-methylcytosinetranscriptional silencing |
| spellingShingle | Jara Teresa Parrilla-Doblas Rafael R. Ariza Teresa Roldán-Arjona Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain Epigenetics base excision repair dna demethylation dna glycosylase dna methylation epigenetic editing 5-methylcytosine transcriptional silencing |
| title | Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain |
| title_full | Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain |
| title_fullStr | Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain |
| title_full_unstemmed | Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain |
| title_short | Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain |
| title_sort | targeted dna demethylation in human cells by fusion of a plant 5 methylcytosine dna glycosylase to a sequence specific dna binding domain |
| topic | base excision repair dna demethylation dna glycosylase dna methylation epigenetic editing 5-methylcytosine transcriptional silencing |
| url | http://dx.doi.org/10.1080/15592294.2017.1294306 |
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