IC4R015-RNA-Seq-2010-20086188

Project Title

Global Epigenetic and Transcriptional Trends among Two Rice Subspecies and Their Reciprocal Hybrids

The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. In this project, the researchers reported highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice (Oryza sativa) subspecies and their reciprocal hybrids.

Rice cultivar Nipponbare (Oryza sativa ssp japonica) and 93-11 (O. sativa ssp indica) and their reciprocal F1 hybrids (Nipponbare/93-11 and 93-11/ Nipponbare) were used for all experiments in this study. Seeds were grown in soil under 16-h-light/8-h-dark conditions at 288C in a greenhouse. After 4 weeks, seedling shoots at the four-leaf stage were harvested, frozen in liquid nitrogen, and stored at –808C for DNA and total RNA isolation or processed directly after harvesting for ChIP assay.

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions and was treated with RNase-free DNase I (New England Biolabs) to remove any contaminating genomic DNA. mRNA was extracted from total RNA using Dynabeads oligo(dT) (Invitrogen Dynal) following the manufacturer’s directions. First- and secondstrand cDNA were generated using Superscript II reverse transcriptase (Invitrogen) and random hexamer primers. Double-stranded cDNA was fragmented by nebulization and used for mRNA library construction following the standard Illumina protocol. Small RNAs were gel isolated from total RNA and were used to create libraries for Illumina sequencing as described previously. Genomic DNA extraction, methylated genomic DNA enrichment, and construction of Illumina sequencing libraries were performed as described previously. Chromatin from seedling shoots was immunoprecipitated with antibodies against H3K4me3 (Upstate), H3K9ac (Upstate), or H3K27me3 (Upstate) as described previously. The eluted ChIP DNA was used to generate Illumina sequencing libraries following the manufacturer’s protocol.

The researchers found a high frequency of concurrence between gene expression and two activating histone modifications, H3K4me3 and H3K9ac. Interestingly, we also detected a significant frequency of concurrence between gene expression and the repressive mark H3K27me3. The researchers found that most genes with DNA methylation (84.3%) were transcriptionally silent, indicating a strong repressive effect of this epigenetic modification on gene activity. Further inspection revealed that gene expression levels were positively correlated with the ratio of H3K4me3/H3K27me3 and were negatively correlated with the ratio of H3K27me3/H3K4me3, thereby facilitating our understanding of the repressive role of H3K27me3 on gene activity. These analyses suggest that in rice, DNA methylation plays a role in dictating the transcriptional activity of genes and that the combinatorial interactions of histone modifications provide a finetuning of their expression levels.

Peking-Yale Joint Center of Plant Molecular Genetics and Agrobiotechnology, National Laboratory of Protein Engineering and

Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China

National Institute of Biological Sciences, Beijing 102206, China
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Department of Botany, College of Life Sciences, Hunan Normal University, Changsha 410081, China