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The initiation of DNA methylation in  Arabidopsis  is controlled by the RNA-directed DNA methylation (RdDM) pathway that uses 24nt siRNAs to recruit  de novo  methyltransferase DRM2 to the target site. We previously described the  REPETITIVE PETUNIA SEQUENCE  (RPS) fragment that acts as a hot spot for  de novo  methylation, for which it requires the cooperative activity of all three methyltransferases MET1, CMT3 and DRM2, but not the RdDM pathway. RPS contains two identical 11nt elements in inverted orientation, interrupted by a 18nt spacer, which resembles the features of a stemloop structure. The analysis of deletion/substitution derivatives of this region showed that deletion of one 11nt element RPS is sufficient to eliminate  de novo  methylation of RPS. In addition, deletion of a 10nt region directly adjacent to one of the 11nt elements, significantly reduced  de novo  methylation. When both 11nt regions were replaced by two 11nt elements with altered DNA sequence but unchanged inverted repeat homology, DNA methylation was not affected, indicating that  de novo  methylation was not targeted to a specific DNA sequence element. These data suggest that  de novo  DNA methylation is attracted by a secondary structure to which the two 11nt elements contribute, and that the adjacent 10nt region influences the stability of this structure. This resembles the recognition of structural features by DNA methyltransferases in animals and suggests that similar mechanisms exist in plants.

An 11bp Region with Stem Formation Potential Is Essential for de novo DNA Methylation of the RPS Element