Our previous studies found strict gene silencing associated with CaMV-35S promoter-specific de novo methylation in transgenic gentian plants. To dissect the de novo methylation machinery, especially in association with histone modification, 35S-driven sGFP-expressing and -silenced gentian cultured cell lines that originated from a single transformation event were produced and used for epigenetic analyses. A sGFP-expressing primarily induced cell suspension culture (PS) was hypomethylated in the 35S promoter region, although a low level of de novo methylation at the 35S enhancer region (-148 to -85) was detected. In contrast, a sGFP-silenced re-induced cell suspension culture (RS), which originated from leaf tissues of a transgenic plant, was hypermethylated in the 35S promoter region. Chromatin immunoprecipitation analysis showed that in RS, histone H3 of the silenced 35S promoter region was deacetylated and also dimethylated on lysine 9. Interestingly, in the silenced 35S promoter 3' region, dimethylation of histone H3 lysine 4 was also observed. When hypomethylation and histone H3 acetylation of the 35S region occurred in PS, de novo methylation at the 35S enhancer region had already taken place. The de novo methylation status was also resistant to 5-aza-2'-deoxycytidine treatment. These results suggest that de novo methylation of the enhancer region is a primitive process of 35S silencing that triggers histone H3 deacetylation.
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