Systematic Analysis of Differential H3K27me3 and H3K4me3 Deposition in Callus and Seedling Reveals the Epigenetic Regulatory Mechanisms Involved in Callus Formation in Rice

Abstract

Plant growth and development occurs through meristematic cell activity, and cell fate transition is accompanied by epigenetic modifications. Callus with cell pluripotency exhibits the ability to undergo continued cell division, and is ideal for studying plant meristematic differentiation. By comparing the differential epigenetic modifications between callus and seedling, the changes in chromatin state and effects of various epigenetic modifications on the growth and development of plants can be revealed, and the key genes related to plant growth and development can be identified, providing novel insights into the regulation of plant growth and development. In this study, we performed ChIP assays using various antibodies in rice seed-induced callus and seedlings grown for about 15 days to examine the differential deposition of H3K27me3 and H3K4me3. Furthermore, data for DNase I-hypersensitive sites in the corresponding tissues were downloaded from National Center for Biotechnology Information. We analyzed 4,562 callus H3K27me3-decreased genes especially those encoding transcription factors in callus, and found that most of the transcription factors, including AP2-ERREBP, NAC, and HB gene families, were related to growth and development. Genes related to meristemization, such as OsWOX9, OsWOX11, OsPLT4, OsPLT5, and OsSHR, were also included. In contrast, H3K4me3 positively regulated callus characteristics through its higher deposition in the callus than in the seedling. We further performed transcriptomic analysis on 45 sets of Affymetrix GeneChip arrays and identified 1,565 genes preferentially expressed in the callus. Callus development and root development in rice were found to share a common regulatory mechanism. We found that these genes, which are associated with meristems, require the removal of H3K27me3 and the deposition of H3K4me3, and DNase I-hypersensitive sites to maintain a relatively active state in the callus than in the seedling. The present study provides novel data about the epigenetic mechanisms involved in callus formation and additional resources for the study of cell division and differentiation in plants.

Keywords: ChIP-seq analysis; H3K27me3; H3K4me3; callus; rice; seedling.

FIGURE 1

The characteristics of H3K27me3 in rice callus and seedling tissue. (A) The genomic distribution of H3K27me3 peaks within different regions in rice callus and seedling tissues. The rice genome was characterized into six classes that included five genic regions [promoter, 5′ untranslated region (UTR), 3′ UTR, coding exon, and intron] and intergenic regions. (B) The distribution of H3K27me3 along all the rice genes in the callus and seedling (from 1-kb upstream regions to 1-kb downstream regions). (C) The number of genes with differential deposition of H3K27me3 between callus and seedling. (D) GO enrichment analysis of 4,562 H3K27me3-decreased genes in callus by agriGO and REVIGO. The scatterplot shows the cluster representatives in a two-dimensional space derived by applying multidimensional scaling to a matrix of significant GO terms with semantic similarities. Bubble color and size indicate the log10^FDR.

FIGURE 2

Transcription factor analysis of genes deposited by H3K27me3 in callus and seeding, respectively. (A) Venn diagrams for 2,683 TFs and H3K27me3-targeted genes between callus and seedling. (B) Transcription factor (TF) families enriched in preferentially deposited H3K27me3 in callus and seedling, respectively. (C) Genes in the AP2/EREBP family are associated with H3K27me3 according to data in the UCSC genome browser. (D) Genes in the NAC family are associated with H3K27me3 according to the UCSC genome browser. (E) Genes in the HB family are associated with H3K27me3 according to the UCSC genome browser.

FIGURE 3

Integrated analysis of H3K27me3-targeted and differentially expressed genes. (A) Venn diagrams for H3K27me3-targeted genes and differentially expressed genes between callus and seedling. (B) Heatmap of H3K27me3 deposition in 1,217 genes in (A) that show lower deposition of H3K27me3 and are highly expressed in the callus. (C) The gene expression values are shown for the group of 1,217 genes in (A). (D) GO enrichment analysis of 581 and 1,217 genes with higher deposition of H3K27me3 and lower expression levels in callus and seedling, respectively.

FIGURE 4

Genome-wide profiling of H3K4me3 in rice callus and seedling. (A) Genomic distribution of H3K4me3 peaks within different regions in rice callus and seedling tissues. The rice genome was characterized into six classes that included five genic regions [promoter, 5′ untranslated region (UTR), 3′ UTR, coding exon, and intron] and intergenic regions. (B) The distribution of H3K4me3 along all the rice genes in the callus and seedling (from 1-kb upstream regions to 1-kb downstream regions). (C) The number of genes with differential deposition of H3K4me3 between callus and seedling. (D) GO enrichment analysis of H3K4me3-increased genes in callus by agriGO and REVIGO. The scatterplot shows the cluster representatives in a two-dimensional space derived by applying multidimensional scaling to a matrix of significant GO terms with semantic similarities. Bubble color and size indicate the log10^FDR.

FIGURE 5

Combination analysis of H3K27me3, H3K4me3, and DHSs near the callus-preferential genes. (A) Heatmap of H3K27me3, H3K4me3, and DHSs around the TSSs of 1,587 callus-preferential genes (from 1 kb upstream to 1 kb downstream regions of TSSs). All 1,587 genes were clustered on the basis of their enrichment, using the k-means method. (B) Z-scores (callus vs. other tissues) of 1,587 callus-preferential genes from five clusters in (A). (C) TF enrichment analysis of five clusters using the hypergeometric distribution method. (D) TF family enrichment analysis of all 175 TFs preferentially expressed in the callus, and TFs with histone mark deposition, using the hypergeometric distribution method. (E) UCSC genome browser-based visualization for OsPLT4, OsPLT5, and OsSHR.

FIGURE 6

Gene Ontology analysis of 1,565 highly expressed genes with lower deposition of H3K27me3 but higher deposition of H3K4me3 and DHSs in the callus.

FIGURE 7

The active, bivalent, and repressed states in callus and seedling. (A) The average gene profiles of H3K4me3 and H3K27me3 around TSS (from upstream 3 kb to downstream 3 kb) in the “active,” “bivalent,” and “repressed” states. (B) The gene expression profiles of the genes under the three states mentioned in (A). (C) The H3K4me3 and H3K27me3 signals around TSS (upstream 5 kb and downstream 5 kb) of the genes in the three aforementioned states in the callus. (D) The 7 state transitions of genes preferentially expressed in the callus: “bivalent-active,” genes that are in the bivalent state in the callus, but in the active state in the seedling; “active-bivalent,” genes that are in the active state in the callus, but in the bivalent state in the seedling; “repressed-bivalent,” “bivalent-repressed,” “active-repressed,” “repressed-active,” and “bivalent-bivalent” are the same as defined above. The significance was evaluated by hypergeometric distribution (*P < 0.01, ***P < 0.001).