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.
  
  

Figure 1.Patterns of DNA methylation in rice tissues. Rice genes (A, C, and E) or TEs (B, D, and F) were aligned at the 5′ end (Left) or the 3′ end (Right), and average methylation levels for each 100-bp interval are plotted. The dashed line represents the point of alignment.

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.
  
  

Figure 2. MITEs are the predominant target of CHH methylation. (A) Box plots showing methylation levels of different TE classes in rice embryo (Em), shoot (St), root (Rt), and endosperm (En). Each box encloses the middle 50% of the distribution, with the horizontal line marking the median. The lines extending from each box mark the minimum and maximum values that fall within 1.5 times the height of the box. MITE mean length = 189 bp; maximum length = 500 bp. SINE mean length = 141 bp; maximum length = 487 bp. LTR mean length = 855 bp; maximum length = 11 kb. (B–D) Rice genes were aligned as in Fig. 1, and TE frequency (B and C) or average methylation levels (D) for each 100-bp interval are plotted. In C and D, genes were grouped into quintiles by transcription.

Research Findings

• To learn how cytosine methylation regulates cereal seed genomes, we quantified methylation in rice embryos, endosperm, and seedling shoots and roots by sequencing bisulfite-converted genomic DNA (bisulfite treatment converts unmethylated cytosine to uracil) to 11- to 15-fold coverage of the nuclear genome. The aggregate methylation patterns in all tissues are very similar to those of mature rice leaves (8) as well as those of A. thaliana (6)—CG methylation is common in gene bodies, except near the transcription start and termination sites, whereas TEs are methylated in all sequence contexts (Fig. 1). Overall CG methyl- ation patterns and levels are virtually indistinguishable between embryos, shoots, roots, and leaves (Fig. 1 A and B). CHG methylation increases modestly with age of the tissue: lowest in em- bryos, higher in young shoots and roots, and highest in mature leaves (Fig. 1 C and D), consistent with reports of increased methylation in older tissues of maize and petunia (10, 11). CHH methylation is also higher in leaves than in seedling tissues (Fig. 1 E and F).
• Short TEs Are Hypermethylated at CHH Sites in Rice Embryo. Average CHH methylation of embryo TEs is higher than in seedlings near the points of alignment but indistinguishable past 1 kb into the element (Fig. 1F), a pattern caused by differential methylation of short and long TEs (Fig. 2A and Fig. S1). TEs longer than 1 kb show the same methylation levels in embryos and seedlings, whereas shorter elements [i.e., miniature inverted-repeat trans- posable elements (MITEs) and short interspersed nuclear elements (SINEs)] are hypermethylated in embryos (Fig. 2A). The abundance of CHH methylation in short TEs led us to examine whether their genomic distribution accounts for the spike in CHH methylation upstream of genes (Fig. 1E). MITEs, the most abundant short elements in rice (some of which are active) (12), preferentially occur near genes (13). MITE distribution in- deed closely parallels that of CHH methylation (Fig. 2B). MITE frequency 5′ and 3′ of genes is directly correlated with gene tran- scription, whereas MITE frequency within genes is inversely cor- related with transcription (Fig. 2C). CHH methylation shows a similar distribution (Fig. 2D), a pattern quite different from CG or CHG methylation (Fig. S2). Thus the distribution of CHH methylation closely follows that of MITEs.

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.)