IC4R002-Epigenomic-2010-20937895

Project Title

• Local DNA hypomethylation activates genes in rice endosperm

The Background of This Project

• Roughly 150 million y ago, flowering plants diverged to form the two dominant extant lineages, monocots and dicots (1). Arabidopsis thaliana, the preeminent plant genetic system, is a dicot, whereas cereal crops, such as rice, wheat, and maize, that feed much of the world are monocots. In both plant groups, pollen grains contain two sperm nuclei, one of which fertilizes a diploid central cell to give rise to triploid endosperm (2). A. thaliana endosperm is consumed by the developing embryo, whereas cereal endosperm persists and makes up the bulk of the mature seed— a developmental difference of particular practical importance (3). Developing seeds are genetic battlegrounds on multiple fronts: parents are proposed to be in conflict over resource allocation (2), whereas the embryo must repress parasitic transposable elements (TEs) to prevent damage to the genome.
• Most of our knowledge about DNA methylation in plant seeds is derived from A. thaliana. Processes involving genetic conflict tend to evolve rapidly (9), and therefore, methylation dynamics in cereal seeds may be quite different. In this project, the researchers use deep bisulfite sequencing to examine DNA methylation in rice seeds. Wild-type rice endosperm methylation patterns—globally reduced non-CG methylation and local CG hypomethylation—resemble those of DME-deficient A. thaliana endosperm, a finding consistent with lack of DME in monocots. Reduced endosperm methylation is common in genes with preferential endosperm expression, in- dicating that demethylation is a major mechanism for gene activation in rice endosperm. Short TEs are hypermethylated at CHH sites in embryo, suggesting that endosperm demethylation func- tions to immunize the embryo against TEs through small RNAs.

Plant Materials & Treatment

• Our custom NimbleGen microarray consists of 2,154,325 45-bp to 85-bp probes that are tiled across the entire sequenced rice genome (Oryza sativa ssp. japonica cultivar Nipponbare, Michigan State University release 5, http://rice.plantbiology.msu.edu) without repeat masking. Each probe is se- lected to have a predicted melting temperature close to 76 °C. The array design is deposited in Gene Expression Omnibus (GEO) with accession number GSE22591. cDNA samples were prepared and labeled as described (24), with hybridization and data extraction preformed at the Fred Hutchinson Cancer Research Center (www.fhcrc.org) DNA array facility (25). Two independent cDNA samples for each tissue were labeled with Cy5 and cohybridized with sonicated genomic DNA labeled with Cy3. The two replicates were averaged, and outlier probes were removed by median smoothing (three-probe window). An expression score for each gene was calculated by averaging the signal of all probes within the gene’s exons.

Labs working on this Project

• Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720

Corresponding Author

• Daniel Zilberman (E-mail: danielz@berkeley.edu.)