Abstract
In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.
Highlights
DNA methylation is a prominent epigenetic modification in higher eukaryotes crucial for many biological processes such as differentiation, gene imprinting, and X chromosome inactivation [1,2,3]
These results strongly suggest that DME-quantitative PCR (qPCR) can be utilized to analyze the level of DNA methylation at specific regions without concerning the sequence specificity that is regarded as one of the limitations of currently available methylation-sensitive restriction endonucleases (MSREs)-based methods
Current enzyme-based techniques mostly rely on the unique properties of MSREs that recognize but do not digest methylated CpG sites within recognition sequences
Summary
DNA methylation is a prominent epigenetic modification in higher eukaryotes crucial for many biological processes such as differentiation, gene imprinting, and X chromosome inactivation [1,2,3]. DNA methylation changes are often associated with alteration of genes expression causing various diseases and, notably cancer in humans. Changes in DNA methylation patterns of specific gene can serve as biomarkers in cancer diagnosis. Alteration of DNA methylation may contribute to changes in gene expression and phenotypic variations that can be sometimes transmitted to generations. The inherited alleles that maintain differences in DNA methylation and expression patterns are referred to as epialleles [6], and several epialleles are reported to be associated with important traits in plants such as flower architecture, flowering time, fruit ripening, sex determination, starch metabolism, vitamin E accumulation, and oil productivity [7,8,9,10,11,12]
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