Genome-wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings

Abstract

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome-wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP-tagged CLF/SWN under their respective native promoters and used them for ChIP-seq analyses to profile the genome-wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild-type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA-like and Telo-box-like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

Keywords: Arabidopsis; CURLY LEAF; H3K27me3; SWINGER; gene repression; genome‐wide occupancy.

Figure 1

The transgenic lines expressing GFP‐tagged CLF or SWN. (a) Plant photos showing complementation of the clf‐29 and swn‐4 flowering phenotypes by the CLF‐ and SWN‐GFP fusion genes driven by their native promoters (pCLF::CLF‐GFP and pSWN::SWN‐GFP), respectively. Scale bar: 1.0 cm. (b) Rosette leaf number at bolting of plants in different genetic backgrounds at 22°C under long‐day condition. Error bars indicate standard deviation from at least 30 plants. Lowercase letters indicate significant differences between genetic backgrounds, as determined by Post‐hoc Tukey's HSD test. (c) GFP signals detected by confocal microscopy in 4‐day‐old clf‐29 pCLF::CLF‐GFP and swn‐4 pSWN::SWN‐GFP roots. Scale bar: 50.0 μm. (d) Western blot analysis of nuclear extracts from 2‐week‐old clf‐29 pCLF::CLF‐GFP and swn‐4 pSWN::SWN‐GFP seedlings. Antibodies used: GFP (anti‐GFP; top) and H4 (anti‐histone H4; bottom). WT: wild type.

Figure 2

Genome‐wide occupancy of CLF and SWN. (a) Table showing the numbers of CLF and SWN binding sites and target genes. (b) Pie charts showing the distribution of CLF and SWN at annotated genic and intergenic regions in the genome. (c) Mean density of CLF/SWN occupancy at all target genes. Plotting regions were scaled to the same length as follows: 5′ ends (−2 kb to transcription starting site (TSS)) and 3′ ends (transcription stop site (TTS) to downstream 2 kb) were not scaled, and the gene body was scaled to 3 kb. (d) Venn diagram showing the overlap between the genes occupied by CLF and those by SWN. (e, f) ChIP‐seq genome browser views of CLF and SWN co‐occupancy (e) and SWN unique occupancy at selected genes (f). Gene structures are shown underneath each panel. (g) Two motifs enriched in CLF and SWN peaks. The number and percent of peaks containing the motifs are shown.

Figure 3

Functional categorization of CLF and SWN target genes. (a) Gene Ontology (GO) analysis of CLF and SWN co‐target genes. (b) Table showing the transcription factor gene families that are co‐targeted by CLF and SWN. The transcription factor gene families can be found at the Arabidopsis genome resources website ( http://arabidopsis.med.ohio-state.edu/AtTFDB/). (c) GO analysis of the SWN unique target genes. (d‐g) ChIP‐seq genome browser views of CLF and SWN occupancy at selected loci; p35S::GFP transgenic plants as the negative control. Gene structures are shown underneath each panel.

Figure 4

Genome‐wide profiling of H3K27me3 in clf‐29, swn‐4, and clf‐29 swn‐4. (a) Mean density of H3K27me3 occupancy at all target genes in WT. Plotting regions were scaled to the same length as follows: 5′ ends (−2 kb to transcription starting site (TSS)) and 3′ ends (transcription stop site (TTS) to downstream 2 kb) were not scaled, and the gene body was scaled to 3 kb. (b) The width ranges for CLF, SWN, and WT_K27 (WT_H3K27me3) peaks. The x axis shows the width of peaks within the ranges (e.g. 1 kb: width <1 kb; 1.5 kb: 1 kb ≤ width ≤ 1.5 kb). The y axis represents the percent of peaks in each range. The REF6 peaks were used as representative “narrow peaks” (Li et al., 2016). (c) Venn diagram showing the overlap among the genes marked with H3K27me3 in WT (WT_K27) and those occupied by CLF or SWN. (d) Mean density of H3K27me3 levels in WT, clf‐29, swn‐4, and clf‐29 swn‐4. Inputs from all backgrounds are shown in gray. The average signal within 2 kb genomic regions flanking the center of the H3K27me3 peaks in WT is shown. (e) Heat maps representing the co‐occupancy of CLF and SWN in the genome (blue, left), and the H3K27me3 levels in WT, clf‐29, swn‐4, and clf‐29 swn‐4 (green, right). Each horizontal line represents a CLF/SWN binding peak or H3K27me3 peak. Columns show the genomic region surrounding each peak summit. Signal intensities are indicated by the shade of blue or green. (f) Venn diagrams showing the overlaps between the genes occupied by CLF and the genes with decreased (clf_K27 down) and increased H3K27me3 levels (clf_K27 up) in clf‐29 (left); and between the genes occupied by SWN and the genes with decreased (swn_K27 down) and increased H3K27me3 levels (swn_K27 up) in swn‐4 (right). (g) ChIP‐seq signals at representative genomic loci showing three distinct types of H3K27me3 reduction pattern in clf‐29 compared to WT. Gene structures are shown underneath the panel. (h) ChIP‐seq genome browser views of selected genes showing the distribution of H3K27me3 in WT (WT_K27), and the occupancy of CLF, SWN, and BRM (Li et al., 2016). BRM ChIP‐seq input signals at these genes are also shown as the negative control. Gene structures are shown underneath the panel. WT: wild type.

Figure 5

Transcriptome profiling of clf‐29, swn‐4, and clf‐29 swn‐4. (a) Volcano plots displaying significantly up‐regulated and down‐regulated genes in clf‐29, swn‐4, and clf‐29 swn‐4 compared to WT (red dots, p < 0.05, fold change > 1.5), respectively. The x axis represents the Log2 value of fragments per kilobase per million (FPKM) mapped reads in each mutant/WT, and the y axis is the −Log10 of the p value for the significance of differential expression. (b) Box plots representing the average expression level (FPKM) of CLF and SWN co‐targets in clf‐29, swn‐4, and clf‐29 swn‐4. Lowercase letters indicate significant differences between genetic backgrounds, one‐way ANOVA. (c) Table showing the percent of overlaps for genes bound by CLF/SWN with differential expression in clf‐29, swn‐4, and clf‐29 swn‐4 compared to these in WT. (d) Heat maps illustrating the ChIP‐seq density in WT and mutants (clf‐29, swn‐4, and clf‐29 swn‐4), ranked by H3K27me3 read intensity within ±2 kb of peak summits in WT (green, left), and the RNA‐seq intensity for the H3K27me3‐marked loci in WT with the same order. Each horizontal line represents an H3K27me3 peak. Columns show the genomic region surrounding each peak summit. Signal intensities are indicated by the shade of green. The expression intensity is measured by Log10 (FC), FC = fold change (mutant vs. WT FPKM). WT: wild type.