Abstract
DNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the loss of DNA methylation in methyltransferase mutants results in altered gene expression and severe developmental defects. However, many cases of naturally occurring DNA methylation variations have been reported, whereby altered expression of differentially methylated genes is responsible for agronomically important traits. The ability to manipulate plant methylomes to generate epigenetically distinct individuals could be invaluable for breeding and research purposes. Here, we describe “epimutagenesis,” a method to rapidly generate DNA methylation variation through random demethylation of the Arabidopsis thaliana genome. This method involves the expression of a human ten–eleven translocation (TET) enzyme, and results in widespread hypomethylation that can be inherited to subsequent generations, mimicking mutants in the maintenance of DNA methyltransferase met1. Application of epimutagenesis to agriculturally significant plants may result in differential expression of alleles normally silenced by DNA methylation, uncovering previously hidden phenotypic variations.
Highlights
DNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the loss of DNA methylation in methyltransferase mutants results in altered gene expression and severe developmental defects
Different degrees of CG hypomethylation were observed in different independent transgenic plants. This result has important implications for epimutagenesis in economically and agriculturally significant plant species, as it appears feasible to control the degree of DNA hypomethylation by screening for plants with desired levels of demethylation. These results show that the expression of human TET1 catalytic domain (hTET1cd) results in intermediate CG methylation levels when compared to wild-type and met[1] individuals
The discovery that expression of the catalytic domain of the human translocation methylcytosine dioxygenase 1 (TET1) protein in A. thaliana leads to widespread loss of CG methylation enables the creation epimutants without the need for methyltransferase mutants, which often causes lethality in crops
Summary
DNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the loss of DNA methylation in methyltransferase mutants results in altered gene expression and severe developmental defects. The methylation of DNA in all three contexts is essential for transcriptional silencing of transposons, repeat sequences, and certain genes Genes regulated by this mechanism are stably repressed throughout the soma and represent an untapped source of hidden genetic variation if transcriptionally re-activated, as revealed from pioneering studies in the model plant A. thaliana[2,3,4]. The impact of this variation is not observed in wild-type plants, as genes silenced by DNA methylation are not expressed This novel source of genetic variation was uncovered by creating epigenetic recombinant inbred lines (epiRILs) from crosses between a wildtype individual and a mutant defective in maintenance of DNA methylation[2,3,4]. 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
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