IC4R004-Epigenomic-2012-22778444

Project Title

• Transcriptome and methylome interactions in rice hybrids

The Background of This Project

• DNA methylation is an epigenetic mark that can often lead to the repression of gene expression (1, 2). It is enriched in heterochromatin and, when present at regulatory sites, usually acts as a repressor of expression, most notably in transposons (3). However, it is also found over coding regions, where it likely does not directly affect transcription and is associated with moderately expressed genes (2, 4–6). In plants, DNA methylation occurs in three different contexts: CG, CHG, and CHH (where H is any nucleotide but G). In Arabidopsis, each context is maintained by different enzymes: MET1 for CG sites, CMT3 for CHG sites and DRM2 for CHH sites. CG and CHG sites are symmetric across the two DNA strands, which is thought to be important for the maintenance of methylation at these sites following DNA repli- cation. In contrast, CHH sites are not symmetric, and their methylation is mediated by RNA-directed DNA methylation pathways (RdDM), which use siRNAs to initiate de novo methylation (3). Cellular methylation states tend to persist during cell division, and recent studies in Arabidopsis have also shown that DNA methylation is faithfully inherited across generations (7, 8). Nonetheless, we are only beginning to understand how different methylation patterns from inbred parents may “interact” during the generation of their hybrid progeny (9, 10).
• In this project, the researchers generated integrative maps of whole-genome cytosine methylation profiles [bisulfite sequencing (BS-seq)] and transcriptional profiles (RNA-seq), to characterize two rice subspecies, Oryza sativa spp japonica [Nipponbare (NPB)] and Oryza sativa spp indica (93–11) and their two reciprocal hybrid off- spring. Using a combination of BS-seq, RNA-seq, and siRNA-seq, we were able to generate allele-specific patterns of methylation and transcription in the hybrids, and thus directly measure the degree to which these are altered between the corresponding parental and F1 chromosomes.

Plant Materials & Treatment

• 　Oryza sativa ssp. japonica (NPB), O.sativa ssp. indica (93–11), and their reciprocal cross F1s (NPB × 93–11 and 93–11 × NPB) were used in this study. Rice seeds were surface sterilized with 40% (vol∕vol) sodium hypochlorite solution and transferred on 1∕2 Murashige and Skoog medium. After germination, rice seedlings were trans- planted into soil and grown at 26 °C∕20 °C under a 10-h light/14-h dark cycle in growth chamber. Fully expanded leaves from 6-wk-old plants were collected for library construction.
• BS-seq libraries were made from genomic DNA isolated from leaf tissues of NPB, 93–11, and their two reciprocal hybrid offspring, 93–11 × NPB and NPB × 93–11, by a previously published method using pre-methylated Illumina adapters

Labs working on this Project

• Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095; b Howard Hughes Medical Institute,
• University of California, Los Angeles, CA 90095; f Molecular Biology Institute, University of California, Los Angeles, CA 90095; c Department of Plant
• Pathology, Ohio State University, Columbus, OH 43210; d Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware,
• Newark, DE 19711; e US Department of Agriculture—Agricultural Research Service Dale Bumpers National Rice Research Center, Stuttgart, AR 72160;
• Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095

Corresponding Author

• Steven E. Jacobsen (E-mail:jacobsen@ucla.edu) & Matteo Pellegrini (E-mail: matteop@mcdb.ucla.edu)