Global mapping of epigenetic modifications of histone H3 Lysine 4 di- and trimethylation in Rice

We present high-resolution maps of DNA methylation and H3K4 di- and tri-methylation of two entire chromosomes and two fully sequenced centromeres in rice shoots and cultured cells. Most transposable elements have highly methylated DNA but no H3K4 methylation whereas over half of protein-coding genes have both methylated DNA and di- and/or tri-methylated H3K4. Methylation of DNA but not H3K4 correlates with suppressed transcription. In contrast, if both DNA and H3K4 are methylated transcription is only slightly reduced. Transcriptional activity was positively correlated with H3K4Me3/H3K4Me2 within the gene body: genes with predominantly H3K4Me3 were actively transcribed whereas genes with predominantly H3K4Me2 were transcribed at moderate levels. Both H3K4Me2 and H3K4Me3 exhibited two peaks in long rice genes; a minor peak correlated with putative transcription start sites and a major peak downstream, implying possible roles in transcriptional initiation and elongation. Keywords: whole genome profiling, DNA methylation, ChIP 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 28°C with continuous white light for 7 days, and the entire shoots were harvested. Suspension cultured cells were derived from the same rice strain and maintained as described previously (Su et al., 2007). Isolation of epigenetically modified genomic DNA fragments Methylated DNA was isolated from total genomic DNA prepared using the Plant DNeasy Mini Kit (QIAGEN) by the McrBC-digestion method (Lippman et al., 2004). DNA bearing modified was isolated by chromatin immunoprecipitation (ChIP) with antibodies that specifically recognize H3K4Me2, H3K4Me3 and CenH3. Tiling Microarray Design, Hybridization, Scanning and Data analysis Tiling probes were selected by the NASA Oligonucleotide Selection Algorithm (NOPSA) (Stolc et al., 2005). Microarrays were hybridized with Cy3 or Cy5 labeled DNA for 16-20 hours at 50°C, then washed each 10 min at room temperature. Hybridization images were generated by a GenePix 4200A scanner (Axon). Raw data was sequentially processed by LOESS normalization and Quantile normalization.