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.
Highlights
DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing
To test if TET1 catalytic domain (TET1cd) can be used in plants for targeted demethylation, we fused human TET1cd to ZF108 and expressed the fusion under the control of the constitutive UBIQUITIN 10 (UBQ10) promoter from Arabidopsis (Fig. 1A)
We found that genome-wide CG, CHG, and CHH methylation levels were very similar to the wild-type Columbia-0 ecotype (Col-0) control, indicating that targeted demethylation using ZF108–TET1cd was very specific
Summary
DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. A previous study in Arabidopsis has shown that a fusion of the RdDM component SU(VAR) HOMOLOG 9 (SUVH9) to an artificial zinc finger designed to target the FWA promoter (ZF108) is able to target methylation to the FWA promoter, silencing FWA expression and rescuing the late-flowering phenotype of the fwa-4 epiallele [25]. Complete loss of 5mC in the promoter of the FLOWERING WAGENINGEN (FWA) gene causes stable fwa epialleles that have been found in floweringtime mutant screens [7] and in strong DNA methylation mutants [2, 4, 8] This loss of 5mC at the FWA promoter activates FWA expression that is responsible for the late-flowering phenotype observed in fwa epialleles [4]. DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a homolog of DNA methyltransferase 3 (DNMT3), is involved in the maintenance of CHH at borders of long TEs in pericentromeric heterochromatin as well as small TEs in euchromatin [10, 13,14,15,16], and represents the last step of the de novo methylation pathway in plants, called RNA-directed DNA methyl-
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