Expression of the C-Terminal Domain of Mammalian <i>TET</i>3 DNA Dioxygenase in <i>Arabidopsis thaliana</i> Induces Heritable Methylation Changes at <i>rDNA</i> Loci

Abstract

In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.

TET-mediated epimutagenesis of the Arabidopsis thaliana methylome

“…Instead of relying on the genome-wide demethylation of one of the two founding parents, epimutagenesis introduces random methylation variation via the introduction of a transgene. Here, we describe a novel epimutagenesis approach in A. thaliana using a human ten–eleven translocation methylcytosine dioxygenase 1 (TET1) 11 – 14 , which catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although TET enzymes and their primary product 5hmC are not found in plant genomes 15 , ectopic expression of a human TET enzyme resulted in widespread DNA demethylation and induced phenotypic variation in A. thaliana .…”

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

“…This is followed by either the passive removal of methylation through the failure of DNA methylation maintenance after DNA replication or the active removal of DNA methylation by glycosylase-mediated base excision repair ( 36 ). While plants do not contain TET enzymes, a previous study has shown that overexpression of the human TET3 catalytic domain in Arabidopsis can cause changes in DNA methylation levels at rDNA loci ( 37 ). However, both hypermethylation and hypomethylation were observed in this study, making the results difficult to interpret, and only effects at rDNA loci were examined.…”

EpiRILs

“…For example, the human Ten-eleven translocation (TET) methylcytosine dioxygenases are an enzyme family catalysing the conversion of 5mC in 5-hydroxymethylcytosine (5hmC), and are involved in active DNA demethylation in embryonic stem cells (Tahiliani et al, 2009). The transgenic expression of TET3 catalytic subunit in Arabidopsis was enough to decrease DNA methylation at ribosomal repeats (Hollwey, Watson, & Meyer, 2016). In addition, the transgenic expression of the same TET3 gene in Tomato induced hypomethylation and ectopic expression of the CEN1.1 gene in leaves, promoting vegetative growth (Hollwey, Out, Watson, Heidmann, & Meyer, 2017).…”