COMPARISON OF THE EFFECTS OF N-6-METHYLDEOXYADENOSINE AND N-5-METHYLDEOXYCYTOSINE ON TRANSCRIPTION FROM NUCLEAR GENE PROMOTERS IN BARLEY

Methylation of deoxycytosine residues in plant nuclear DNA at CpG dinucleotides is generally assumed to suppress transcription, while deoxyadenosine methylation on recombinant reporter gene constructs transiently introduced into plant cells increases expression by uncharacterized mechanisms. A particle bombardment transient expression system was used in intact barley aleurone layers to quantitate the effects of CpG and A methylation on transcription from well-characterized hormone-regulated alpha-amylase promoters. Methylation of C in all CpG pairs had little effect on transcription. In contrast, the presence of methylated A residues in the sequence GATC scattered in the reporter plasmid outside of the promoter increased transcription two- to fivefold, regardless of the strength of the promoter, and proper hormonal regulation of transcription was maintained. The methyl-dA effect was similar when the amount of reporter construct DNA used was varied over a 20-fold range, beginning with an amount that gave only a small increment of expression above background. Similar enhancing effects for methyl-dA were seen with the CaMV 35S, maize Adh1, and maize ubiquitin promoters, though the magnitude varied for each individual promoter. Methyl-dA did not detectably affect plasmid DNA stability in aleurone cells in transient expression experiments because the enhancing effect of methyl-dA on expression was the same regardless of whether the assay was performed at 12 h or 40 h. Several proteins in wheat germ nuclear extracts bound preferentially to methylated DNA as assessed by gel retardation assays; one showed preferential binding to methyl-dA rather than methyl-CpG residues. The data indicate that the presence of methyl-dA in the vicinity of active promoters enhances transcription of nuclear genes in barley in a manner independent of the strength of the promoter. This effect may be mediated by a methyl-dA-binding protein.