Methylation of cytosines in nonconventional methylation acceptor sites can contribute to reduced gene expression

Epigenetic silencing of gene expression is often correlated with extensive DNA methylation at cytosine residues in the promoter and the coding region of silenced genes. Increasing evidence indicates that, in such cases, DNA methylation can also occur in sequence contexts other than CG and CNG, resulting in genomic regions with almost complete modification of cytosines. Whether this nonconventional methylation at CNN sites also contributes to gene repression is not known. We constructed genes with a promoter and a coding region devoid of the conventional methylation acceptor sites CG and CNG in addition to constructs with the corresponding wild-type sequences containing these sites. We generated unmethylated and completely methylated DNA by the polymerase chain reaction and performed expression assays in plant protoplasts. Quantification of transcript levels by RNase protection assay demonstrated that DNA methylation at positions other than CG or CNG sites contributes to the reduction in gene expression.