ATX3, ATX4, and ATX5 Encode Putative H3K4 Methyltransferases and Are Critical for Plant Development

Abstract

Methylation of Lys residues in the tail of the H3 histone is a key regulator of chromatin state and gene expression, conferred by a large family of enzymes containing an evolutionarily conserved SET domain. One of the main types of SET domain proteins are those controlling H3K4 di- and trimethylation. The genome of Arabidopsis (Arabidopsis thaliana) encodes 12 such proteins, including five ARABIDOPSIS TRITHORAX (ATX) proteins and seven ATX-Related proteins. Here, we examined three until-now-unexplored ATX proteins, ATX3, ATX4, and ATX5. We found that they exhibit similar domain structures and expression patterns and are redundantly required for vegetative and reproductive development. Concurrent disruption of the ATX3, ATX4, and ATX5 genes caused marked reduction in H3K4me2 and H3K4me3 levels genome-wide and resulted in thousands of genes expressed ectopically. Furthermore, atx3/atx4/atx5 triple mutants resulted in exaggerated phenotypes when combined with the atx2 mutant but not with atx1 Together, we conclude that ATX3, ATX4, and ATX5 are redundantly required for H3K4 di- and trimethylation at thousands of sites located across the genome, and genomic features associated with targeted regions are different from the ATXR3/SDG2-controlled sites in Arabidopsis.

Figure 1.

Phylogenetic relationship, protein domain structures, and expression patterns of Arabidopsis ATX3, ATX4, and ATX5 genes. A, Bayesian phylogeny reconstruction of ATX homologs in Arabidopsis, maize, rice, and Physcomitrella patens. Numbers next to branches indicate posterior probability values. The scale indicates number of substitutions per site. Protein sequences were aligned using ClustalX (Jeanmougin et al., 1998), and phylogeny reconstruction was conducted using MrBayes (Huelsenbeck et al., 2001). B, Domain organization of Arabidopsis TRX proteins. Protein structure was analyzed using NCBI-CD and SMART searches. Abbreviations: PWWP, Domain named after a conserved Pro-Trp-Trp-Pro motif; FYRN, F/Y-rich N terminus; FYRC, F/Y-rich C terminus; PHD, plant homeodomain. C, Analysis of spatial and temporal expression patterns of ATX3 (top), ATX4 (middle), and ATX5 (bottom) using promoter-GUS fusion. Bars = 1 mm. D, RT-qPCR analysis of ATX3, ATX4, and ATX5 expression. Expression levels relative to the expression level of the ACTIN2 gene are presented with ses. Values are means of three independent experiments.

Figure 2.

Phenotypes of atx3/4/5 triple mutant plants. A, Diagram of genomic structures of the ATX3, ATX4, and ATX5 genes with T-DNA insertion sites marked with triangles. Bars indicate exons and lines represent introns. Scale bar = 1 kb. B to E, Phenotype comparison of 1-week-old (B), 2-week-old (C), 3-week-old (D), and 6-week-old (E) in wild-type (WT; left) and atx3/4/5 mutant (right) plants. Bars = 1 cm in B to D and 2 cm in E. F and G, Phenotype comparison of siliques of wild-type and atx3/4/5 mutant plants. Bars = 1 mm. H, Flower development at stages 12 to 16 (from left to right) in wild-type (top) and atx3/4/5 mutant plants (bottom). Bars = 1 mm. I, Aniline blue staining of pollen tubes in pollinated gynoecium at stage 15 of flower development in wild-type (left) and atx3/4/5 mutant plants (right). Bars = 200 μm.

Figure 3.

ATX3/4/5-affected H3K4me2/3 sites. A representative western blot analysis of H3K4me1, H3K4me2, H3K4me3, H3K9me2, H3K27me3, and H3K36me3 levels in wild-type (WT) and atx3/4/5 mutant plants. For each antibody, the same amount of total protein was loaded. B, Relative intensity of protein levels in atx3/4/5 versus wild-type. The extent of protein was determined by first normalizing the band intensity of specific antibodies relative to H3 antibody. Data were shown as means ± se from three independent biological replicates. *Significant at P ≤ 0.01. C and D, Distribution of H3K4me2 (C) and H3K4me3 (D) around TSSs. Total numbers of ChIP-seq reads were normalized and quantified in 50-bp windows in regions 2 kb up- and downstream of TSS. E, Venn diagram showing the overlap of ATX3/4/5-affected H3K4me2 and H3K4me3 sites. F, Lack of correlation between a decrease in H3K4me2/3 levels and gene transcript levels. Genes showing a coordinated decrease in ATX3/4/5-affected H3K4me2/3 levels and transcript levels in the atx3/4/5 triple mutant relative to wild type are represented by green bars. Genes whose transcript levels are up-regulated despite a decrease in H3K4me2/3 levels are orange. Genes showing no change in transcript levels while decrease in H3K4me2/3 levels are blue.

Figure 4.

Functional overlap between ATX3/4/5 versus ATX1 and ATX2. A, Comparison of phenotypes of 4-week-old plants. 1, wild type; 2, atx1; 3, atx2; 4, atx3/atx4/atx5; 5, atx1/atx3/atx4/atx5; 6, atx2/atx3/atx4/atx5. Bars = 2 cm. B, Zoom in of triple and quadruple mutants shown in the white box in A. Bars = 1 cm. C, Levels of H3K4me3 in atx1 mutants (orange) and H3K4me2 in atx2 mutants (green) at six representative genomic sites examined by ChIP-qPCR. y axis: Fold enrichment of H3K4me2/3 in wild type (WT) relative to atx1 and atx2 mutant. Enrichment was first normalized using the TA3 retrotransposon locus (AT1G37113).

Figure 5.

ATX3/4/5 function in a different pathway from SDG2. A, Venn diagram showing that the majority of ATX3/4/5-affected H3K4me3 sites do not overlap with SDG2-affected H3K4me3 sites. B, Metagene plots of DNA methylation across gene bodies in three contexts (CG, CHG, and CHH) associated with ATX3/4/5- and SDG2-affected H3K4me3 sites. C, Metagene plots of H3K27me3 across gene bodies associated with ATX3/4/5- and SDG2-affected H3K4me3 sites. D, Boxplot of ChIP-seq peak width of ATX3/4/5- and SDG2-affected H3K4me3 sites. E, Boxplot of ChIP-seq peak height of ATX3/4/5- and SDG2-affected H3K4me3 sites. F, Boxplot of ChIP-seq peak intensity of ATX3/4/5- and SDG2-affected H3K4me3 sites. Peak intensity is defined as the number of reads in a peak normalized by peak width (reads/kb). G, Boxplot of expression levels of genes overlapping ATX3/4/5- and SDG2-affected H3K4me3 sites. H, Phenotype of atx3/atx4/atx5/sgd2 quadruple mutants in 4-week-old (top) and 6-week-old (bottom) plants. 1, Wild type; 2, atx3/atx4/atx5 triple mutant; 3, sdg2; 4, atx3/atx3 atx4/atx4 atx5/atx5 sdg2/+. Bars = 2 cm.