Detection of Epialleles by Measuring Differential DNA Methylation Levels Using Plant-Specific DNA Demethylase

Abstract

DNA methylation is one of the crucial epigenetic factors to control gene expression in plants and mammals. Aberrant changes in DNA methylation often lead to the formation of epialleles in plants, and the epimutants that harbor such epialleles sometimes display stable transgenerational inheritance. Therefore, DNA methylation analysis is required to detect epialleles. However, conventional DNA methylation analyses sometimes are not sufficient to detect epialleles. Here we report a novel DNA methylation analysis method combining both 5-methylcytosine (5mC) excision activity of plant-specific DNA demethylase DEMETER (DME) protein and a quantitative real-time PCR (qRT-PCR) technique. Because DME induces a nick in a sequence non-specific manner at the position where 5mC is present, heavily methylated targets cannot be PCR-amplified due to a number of DNA strand breaks, whereas unmethylated or less methylated regions can be easily amplified. We demonstrate the feasibility of DME-qPCR by successfully distinguishing between wild type and two epialleles, fwa and Cnr, derived from Arabidopsis and tomato, respectively. This novel method has versatility over other enzyme-based tools, and at the same time, may overcome several problems that current DNA methylation analysis principles have – such as pretreatment and intrinsic technical bias.