TET3-mediated DNA demethylation contributes to the formation of stable epialleles in tomato [low coverage WGBS]

Traditionally, heritable epimutations in plants have been generated using genetic mutants or chemical inhibitors, but these approaches often lack precision or stability. In this study, we investigated the effects of globally altering DNA methylation in tomato, a species with a large, TE-rich genome, through the ectopic expression of the catalytic domain of the human DNA demethylase TEN-ELEVEN TRANSLOCATION3 (hTET3cd).

We found that TET3-mediated demethylation induced stable hypomethylation at CG and CHG sites and that these changes were inherited across multiple generations, including in non-transgenic siblings. Interestingly, demethylation in heterochromatic pericentromeric regions was often accompanied by gains in CHH methylation, suggesting the compensatory activation of the RNA-directed DNA methylation (RdDM) pathway. Differentially methylated region (DMR) analysis revealed that CG and CHG methylation loss was widespread, while CHH DMRs showed complex patterns of gain and loss, particularly near gene-rich regions and transposable elements enriched for 24-nt small RNAs.