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.
Highlights
In mammals and plants, DNA methylation is targeted to transposons, retrotransposons and other repetitive ele-How to cite this paper: Hollwey, E., Watson, M. and Meyer, P. (2016) Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci
The 3’ region of a cDNA of the human TET3 gene, encoding the c-terminal regions of TET3, was cloned behind the 35S promoter in a plant expression vector, and the resulting TET3c construct was transferred into Arabidopsis thaliana
To assess the stability of these DNA methylation patterns, we examined HpaII and MspI restriction patterns over two generations
Summary
DNA methylation is targeted to transposons, retrotransposons and other repetitive ele-How to cite this paper: Hollwey, E., Watson, M. and Meyer, P. (2016) Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci. DNA methylation is targeted to transposons, retrotransposons and other repetitive ele-. (2016) Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci. Methylated repeat regions in plants accumulate heterochromatic histone H3K9me methylation marks, which are closely linked to CNG methylation [4]. The plant-specific CHROMOMETHYLASE3 (CMT3) interacts with the H3 Lys dimethylation (H3K9me2) pathway to maintain DNA methylation at CNG sites whereas the de novo MTase DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2) maintain DNA methylation at CNN sites requiring the active targeting of small interfering RNAs [5]. Like its mammalian homologue DNA-methyltransferase 1 (Dnmt1), plant DNA METHYLTRANSFERASE 1 (MET1) acts as a maintenance methyltransferase for CG methylation [6]. MET1 is essential for maintenance of dense methylation patterns at certain loci, which contain 5 mC marks in CG and non-CG contexts [7]
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