Gehring et al
] set out to find additional Arabidopsis
genes that show parent-of-origin-specific (imprinted) expression in the endosperm. Their strategy was to identify regions of the genome that have reduced DNA methylation in the endosperm, compared with the embryo, as candidates for imprinted loci. They dissected endosperm and embryo tissues from developing seeds and immunoprecipitated methylated DNA from each sample with an anti-5-methylcytosine antibody. They then used high-throughput sequencing or hybridization to microarrays to determine which sequences were recovered. Analysis of the data revealed widespread reduced DNA methylation of transposons and other repeats that generate small RNAs in the endosperm.
This trend was partially reversed by mutation of DEMETER, indicating a role for active DNA demethylation. The authors then focused on genes that lay close to differentially methylated regions. They validated five new imprinted genes by showing allele-specific expression in endosperms generated by crosses between polymorphic parents. In each case, imprinted expression correlated with reduced DNA methylation on a transposon or repeated sequence within 1 kb of the coding sequence of the im printed gene. On the basis of the characteristics of the validated genes, the authors estimate that approximately 50 genes in total will prove to have imprinted expression in the endosperm.
Hsieh et al.
] used a different approach - shotgun bisulfite sequencing - to examine patterns of DNA methylation in embryo, endosperm and adult plant tissues. Genomic DNA was treated with the mutagen sodium bisulfite to convert cytosines, but not 5-methylcytosines, to uracils. DNA was then subjected to high-throughput deep sequencing to visualize the global patterns of cytosine mutagenesis. The advantage of this approach over immunoprecipitating methylated DNA is that precise patterns of DNA methylation can be determined. This is an especially important consideration in plant genomes, where cytosines in different sequence contexts are methylated by different pathways. In particular, methylation of cytosines in asymmetric contexts is directed by small RNAs that correspond to the methylated sequence [1
Genome-wide bisulfite sequencing of DNA from embryo, endosperm, and adult tissues showed dramatically different trends for methylation of cytosines in the symmetric context CG, which is the most prevalent context for Arabidopsis
DNA methylation, compared with asymmetric contexts [6
]. CG methylation was reduced in the endosperm in both gene and repeat sequences relative to the embryo, adult, and demeter
mutant endosperm. This overall pattern is similar to that described by Gehring et al.
]. However, Hsieh et al
] also found that asymmetric cytosine methylation in repeat sequences was elevated in both endosperm and embryo relative to adult tissues, and that the demeter
mutation strongly depleted asymmetric cytosine methylation in the endosperm.
A likely explanation for these context-specific methylation changes comes from a previous study showing that loss of CG methylation causes transcriptional reactivation of repeat sequences throughout the genome and a burst of new small RNAs, stimulating new methylation of asymmetric cytosines in the complementary regions [12
]. Projecting this cascade onto the female gametophyte, DEMETER would first act genome-wide in the central cell to erase DNA methylation and activate synthesis of small RNAs. These small RNAs would trigger new asymmetric cytosine methylation on the two maternal and one paternal endosperm chromosomes, whereas demethylation in CG contexts on the maternal chromosomes would persist. In demeter
mutants, the small RNAs would never be made, preventing methylation of asymmetric cytosines.
Consistent with this model, a recent small RNA high-throughput sequencing study by Mosher et al.
] found that there is indeed a genome-wide burst of small RNAs from maternal genome transposons and repeats in the seed. A further test of this model will be to determine whether mutations that disrupt production of small RNAs block the increase in asymmetric cytosine methylation seen in endosperm compared to adult tissues.