Genome-wide analysis of histone modifications: H3K4me2, H3K4me3, H3K9ac, and H3K27ac in Oryza sativa L. Japonica

Abstract

While previous studies have shown that histone modifications could influence plant growth and development by regulating gene transcription, knowledge about the relationships between these modifications and gene expression is still limited. This study used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), to investigate the genome-wide distribution of four histone modifications: di and trimethylation of H3K4 (H3K4me2 and H3K4me3) and acylation of H3K9 and H3K27 (H3K9ac and H3K27ac) in Oryza sativa L. japonica. By analyzing published DNase-Seq data, this study explored DNase-Hypersensitive (DH) sites along the rice genome. The histone marks appeared mainly in generic regions and were enriched around the transcription start sites (TSSs) of genes. This analysis demonstrated that the four histone modifications and the DH sites were all associated with active transcription. Furthermore, the four histone modifications were highly concurrent with transcript regions-a promising feature that was used to predict missing genes in the rice gene annotation. The predictions were further validated by experimentally confirming the transcription of two predicted missing genes. Moreover, a sequence motif analysis was constructed in order to identify the DH sites and many putative transcription factor binding sites.

Keywords: bioinformatics; chromatin structure and remodeling; epigenetics; gene regulation; genomics; rice..

Figure 1.

The Distribution of Histone Modifications and DH Sites within Different Regions of the Rice Genome.

Figure 2.

The Association between Histone Modifications and DH Sites with Rice TE and Non-TE Genes. (A) Distribution of histone marks and DH sites along rice non-TE genes. A meta-gene profile was generated using the normalized sequencing density of the DH sites and four histone modifications: H3K4me3, H3K4me2, H3K9ac, and H3K27me3. The gene body was converted into a percentage in aim to standardize genes of different lengths. The 1-kb upstream and downstream regions of the gene are included. (B) A histogram displaying the percentage of non-TE and TE genes associated with histone modifications or DH sites in rice. The number of genes in each category is shown at the top of each column. (C) Representative genes associated with histone modifications and DH sites.

Figure 3.

Heatmaps for Transcription Activity, Histone Modifications, and DH Sites around the TSSs of Non-TE Genes. The genes were sorted according to their expression level, as determined by mRNA-Seq. For each gene, the histone modification intensities are displayed along –1-kb to 1-kb regions around the TSSs. Each color represents one histone modification, DH sites, or transcription activity.

Figure 4.

Concurrence Frequencies for Histone Modifications, DH Sites, and Transcript Regions. The percentage number indicates the possibility that a histone modification peak, DH site, or transcript region on the x-axis exists in a given histone modification peak, DH site, or transcript region on the y-axis. A transcript region is defined as 1 kb up or downstream of the TSS.

Figure 5.

Sequence Logo of Identified Motifs within DH Sites. (A) The top two motifs have more hits in the promoter-associated DH sites compared to hits in the non-promoter-associated DH sites. (B) These two motifs have more hits in the non-promoter-associated DH sites than in the promoter-associated DH sites. For each motif, the numbers of motif hits within the promoter-associated DH sites and the non-promoter-associated DH sites are shown on the upper right of each figure.

Figure 6.

Experimental Validation of Two Unannotated Genes in the TIGR/MSU Annotation. (A) The gene model for Os02g0510400 (B) PCR analysis of Os02g0510400 under normal conditions and under methyl viologen (MV) treatment. (C) The gene model for Os11g0564800 (D) PCR analysis of Os11g0564800 under normal conditions and under MV treatment. Primers designed for the PCR experiments are highlighted by the black arrows.