Site-specific methylation in gene coding region underlies transcriptional silencing of the Phytochrome A epiallele in Arabidopsis thaliana

Gulab Rangani, Mariya Khodakovskaya, Mohammad Alimohammadi, Ute Hoecker, Vibha Srivastava

DNA methylation in cytosine residues plays an important role in regulating gene expression. Densely methylated transgenes are often silenced. In contrast, several eukaryotic genomes express moderately methylated genes. These methylations are found in the CG context within the coding region (gene body). The role of gene body methylation in gene expression, however, is not clear. The Arabidopsis Phytochrome A epiallele, phyA', carries hypermethylation in several CG sites resident to the coding region. As a result, phyA' is transcriptionally silenced and confers strong mutant phenotype. Mutations in chromatin modification factors and RNAi genes failed to revert the mutant phenotype, suggesting the involvement of a distinct epigenetic mechanism associated with phyA' silencing. Using the forward genetics approach, a suppressor line, termed as suppressor of p hyA' silencing 1 (sps1), was isolated. Genetic and molecular analysis revealed that sps1 mutation reactivates the phyA' locus without altering its methylation density. However, hypomethylation at a specific CG site in exon 1 was consistently associated with the release of phyA' silencing. While gene underlying sps1 mutation is yet to be identified, microarray analysis suggested that its targets are the expressed genes or euchromatic loci in Arabidopsis genome. By identifying the association of phyA' silencing with the methylation of a specific CG site in exon 1, the present work shows that site-specific methylation confers greater effect on transcription than the methylation density within gene-body. Further, as the identified site (exon 1) is not critical for the promoter activity, transcription elongation rather than transcription initiation is likely to be affected by this site-specific CG methylation.