In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants.Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.Cytosine methylation is an epigenetic mark present in many eukaryotes, including plants, vertebrates and fungi [1], and plays an important role in various biological processes including regulation of gene expression, stability of the genome, cellular differentiation and development [2]. Transposons and repeats are frequently methylated in a wide range of species [3]. Loss of cytosine methylation induces reactivation and transposition of transposons [4-7], suggesting that cytosine methylation represents the primary mechanism of transposon suppression in host genomes [8]. Cytosine methylation also functions to maintain a repressed chromatin state and stably silence promoter activity [9]. A genome-wide analysis of Arabidopsis thaliana uncovered an interdependence between cytosine methylation and transcription