IC4R001-Epigenomic-2008-18263775

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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.

Figure 1. Overview of Epigenetic Modifications of Rice Chromosomes 4 and 10.

Research Findings

  • To identify genomic regions significantly enriched in methylated DNA, H3K4me2, or H3K4me3, a Wilcoxon signed rank test (Hollander and Wolfe, 1999) was applied in a sliding window of 6500 bp across the chromosomal tile paths (see Methods). In brief, a methylated DNA or methylated H3K4 region was defined by combining adjacent probes with a significance threshold of P < 0.05, allowing a maximal gap of 150 bp, and requiring a minimal run of two consecutive probes. Figure 1A shows the analysis of a representative region on chromosome 4. Transposable elements (TEs) in general had highly methylated DNA but little H3K4me2 or H3K4me3, whereas non-TE genes had much less methylated DNA and were enriched for H3K4me2 and H3K4me3. A fully dynamic browser for viewing the DNA and H3K4 methylation patterns observed in this study is publicly available at http://plantgenomics.biology.yale.edu.

Figure 1. Overview of Epigenetic Modifications of Rice Chromosomes 4 and 10.
  • Rice chromosomes 4 and 10 have similar overall patterns of DNA and H3K4 methylation, with some variation between the two tissues studied (Figures 1B to 1D). In shoots, 5494 methylated DNA, 5137 H3K4me2, and 5050 H3K4me3 regions were identified on chromosome 4, covering 16.1, 21.1, and 24.3% of the chromosome, respectively (Figure 1D; see Supplemental Table 1 online). Similarly, 3482 methylated DNA, 3352 H3K4me2, and 3452 H3K4me3 regions were found on chromosome 10 in shoots, covering 15.8, 20.7, and 24.0% of the chromosome, respectively. The median sizes of methylated DNA, H3K4me2, and H3K4me3 regions were 532, 1064, and 913 bp, respectively, while there were 152, 239, and 152 continuous regions longer than 5.0 kb for DNA methylation, H3K4me2, and H3K4me3, respectively, spanning multiple gene loci.

Figure 1. Overview of Epigenetic Modifications of Rice Chromosomes 4 and 10.
  • Further inspection revealed an inverse relationship between the amounts of methylated DNA and methylated H3K4 in heterochromatin and euchromatin on both chromosomes (Figures 1B to 1D; see Supplemental Figure 4 online; Jiao et al., 2005; Li et al., 2006). DNA methylation was more abundant in the heterochro- matic half than in the euchromatic half (18.1% versus 14.2% on chromosome 4 in shoots). However, due to the low coverage of highly repetitive sequences in the heterochromain on our tiling array, the real difference in DNA methylation was expected to be bigger than the numbers obtained here. By contrast, the eu- chromatic half had significantly higher levels of H3K4me2 and H3K4me3 (30.9% versus 11.2% and 35.3% versus 13.2% on chromosome 4 in shoots). Based on the previous genome-wide transcription analysis using tiling microarrays (Li et al., 2007b), transcription of a greater proportion of the DNA was detected in the euchromatic halves of both chromosomes (Figure 1). Ele- vated DNA methylation and reduced transcription in hetero- chromatin correlates with the higher density of TEs and related tandem repeats in heterochromatin, which can influence the expression of nearby genes and their DNA methylation (Martienssen and Colot, 2001; Schotta et al., 2003; Lippman et al., 2004).Conversely, the enrichment of H3K4me2 and H3K4me3 and higher transcriptional activity in euchromatin correlates with the greater abundance of non-TE genes.

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)