IC4R001-Epigenomic-2008-18263775

Project Title

• High-Resolution Mapping of Epigenetic Modifications of the Rice Genome Uncovers Interplay between DNA Methylation, Histone Methylation, and Gene Expression

The Background of This Project

• In eukaryotic nuclei, DNA associates with proteins to form chro- matin. It first wraps around core histones to form nucleosomes that, in turn, are often organized into higher-ordered structures. Chromatin structure plays an essential role in genome organization, transcriptional activity, and memory of developmental state (Bernstein et al., 2002). While all cells in an individual have the same nuclear genome, each cell type may harbor a distinct epigenome, which relies on heritable, often reversible, DNA methylation at cytosines and histone modifications (Richards, 1997).
• Rice (Oryza sativa) is an important model species for cereals and other monocotyledonous plants. Two prominent features of most rice chromosomes are their clear organization into hetero- chromatic and euchromatic regions and the large amount of pericentromeric heterochromatin. For example, cytological studies using 49,6-diamidino-2-phenylindole staining indicate that approximately half of chromosomes 4 and 10 is the more densely stained heterochromatin, including their entire short arms and the proximal portions of their long arms (Cheng et al., 2001; Yan and Jiang, 2007). Global repression of transcription in rice heterochromatin has been observed, but the molecular basis is unknown (Jiao et al., 2005; Li et al., 2006). Completion of the rice genome sequence (International Rice Genome Sequencing Project, 2005) provides an unprecedented opportunity to examine epigenetic modifications comprehensively and correlate them with gene expression.
• In this project, the researchers describe high-resolution mapping of DNA methyla- tion and H3K4me2 and H3K4me3 patterns of rice (spp japonica cv Nipponbare) chromosomes 4 and 10 using tiling-path micro- arrays. We compare two developmental states: undifferentiated suspension-cultured cells and young light-grown shoots. The large heterochromatic regions on these chromosomes allow a genome-scale investigation of DNA methylation and histone modifications in heterochromatin. The completely sequenced rice centromeres of chromosomes 4 and 8 were also included in this analysis (Nagaki et al., 2004; Zhang et al., 2004). This indepth, genome-scale analysis provides unprecedented insights into the epigenetic signatures of the rice genome.

Plant Materials & Treatment

• Plant Materials and Growth Conditions.All plants used in this study were rice strain Oryza sativa ssp japonica cv Nipponbare. Dehusked seeds were surface-sterilized and sown on solidified Murashige and Skoog medium with 3.0% sucrose. Plants were grown in chambers at 288C with continuous white light for 7 d, and the entire shoots were harvested.
• Isolation of Epigenetically Modified Genomic DNA Fragments.Methylated DNA was isolated from total genomic DNA prepared using the DNeasy plant mini kit (Qiagen) by the McrBC digestion method (Lippman et al., 2004). DNA bearing modified histones was isolated by ChIP with antibodies that specifically recognize H3K4me2 (Upstate), H3K4me3 (Abcam), and CenH3 (Nagaki et al., 2004). See Supplemental Methods online for detailed experiment protocols.
• Tiling Microarray Design, Hybridization, Scanning, and Data Analysis. Tiling probes were selected by the NASA Oligonucleotide Selection Algorithm (NOPSA) (Stolc et al., 2005). The algorithm for filtering the repetitive probes is described in the Supplemental Methods online. Microarrays were hybridized with Cy3- or Cy5-labeled DNA for 16 to 20 h at 508C and then washed as described in the Supplemental Methods online. Hybridization images were generated by a GenePix 4200A scanner (Axon). Raw data were sequentially processed by LOESS normaliza- tion and Quantile normalization, and then regions bearing epigenetic modifications were identified using the Wilcoxon signed rank test (see Supplemental Methods online for specific procedure and parameters). For DNA and histone methylation, two or three biological replicates, respectively, of each tissue were performed.

Labs working on this Project

• National Institute of Biological Sciences, Beijing 102206, China
• Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520
• Peking-Yale Joint Research Center of Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences,Peking University, Beijing 100871, China
• Genome Research Facility, NASA Ames Research Center, Moffett Field, California 94035
• Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706

Corresponding Author

• Xing Wang Deng (E-mail: xingwang.deng@yale.edu)