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Proc Natl Acad Sci U S A. 2018 Feb 27;115(9):E2125-E2134. doi: 10.1073/pnas.1716945115. Epub 2018 Feb 14.

Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.

Author information

1
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095.
2
Molecular Biology Institute, University of California, Los Angeles, CA 90095.
3
Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095.
4
Genome Center, Medical Investigation of Neurodevelopmental Disorders Institute, and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616.
5
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095; jacobsen@ucla.edu.
6
Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095.

Abstract

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.

KEYWORDS:

Arabidopsis; CRISPR/dCas9 SunTag; TET1; artificial zinc finger; targeted demethylation

PMID:
29444862
PMCID:
PMC5834696
DOI:
10.1073/pnas.1716945115
[Indexed for MEDLINE]
Free PMC Article

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