DNA Methylation Regulates Imprinting And Transcription Start Sites

Imprinting is an epigenetic phenomenon that causes parent of origin-specific gene expression irrespective of the direction of a cross. Mechanisms regulating parental allele-specific gene expression of over200recently identified imprinted genes in rice endosperm are poorly understood. Investigation of the chromatin modifications, DNA methylation and trimethylation of histone H3lysine27(H3K27me3) using bisulfite sequencing and chromatin immunoprecipitation, at three loci (Osl2g08780, Os11g36470, Os01g691lO) demonstrated that differential DNA and histone methylation in the promoter and/or gene body can influence allele-specific expression or site of transcript initiation. Paternal expression of an auxin biosynthesis gene (Osl2g08780), OsYUCCA11, required DNA methylation in the gene body while the gene body of the silenced maternal allele was hypomethylated and marked with H3K27trimethylation. The differential marking of parental alleles mirrors that proposed to regulate paternal expression of two Arabidopsis genes, PHERES1and a YUCCA homolog. Os01g69110encodes a small protein with similarity to the Drosophila transcription factor ENHANCER OF YELLOW2. DNA hypomethylation in the upstream flanking region of the maternal yellow2-like allele resulted in a transcript which was longer than the paternal transcript. The region corresponding to maternal transcript initiation was marked by DNA methylation in the paternal allele where transcription initiated from a site-700bp downstream. The paternal allele of an ubiquitin hydrolase gene (Os11g36470) exhibited gene body DNA methylation and produced full-length transcripts. The maternal allele was hypomethylated in the5’ gene body and transcripts initiated from a downstream promoter. Inhibition of DNA methylation by5-azacytidine or zebularine resulted in the appearance of the long transcripts from yellow2-like and enhanced expression of the short transcripts from the ubiquitin hydrolase in seedlings, indicating that DNA methylation prevents transcript initiation from cryptic promoters in plant genes. This observation suggests a new paradigm whereby maternal genome hypomethylation is associated with the production of novel transcripts, potentially diversifying the gene products from the two alleles, rather than uniparental expression of an annotated cDNA.