ARABIDOPSIS TRITHORAX-RELATED7 is required for methylation of lysine 4 of histone H3 and for transcriptional activation of FLOWERING LOCUS C

Abstract

In the winter-annual accessions of Arabidopsis thaliana, presence of an active allele of FRIGIDA (FRI) elevates expression of FLOWERING LOCUS C (FLC), a repressor of flowering, and thus confers a vernalization requirement. FLC activation by FRI involves methylation of Lys 4 of histone H3 (H3K4) at FLC chromatin. Many multicellular organisms that have been examined contain two classes of H3K4 methylases, a yeast (Saccharomyces cerevisiae) Set1 class and a class related to Drosophila melanogaster Trithorax. In this work, we demonstrate that ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7), a putative Set1 class H3K4 methylase, is required for proper FLC expression. The atxr7 mutation partially suppresses the delayed flowering of a FRI-containing line. The rapid flowering of atxr7 is associated with reduced FLC expression and is accompanied by decreased H3K4 methylation and increased H3K27 methylation at FLC. Thus, ATXR7 is required for the proper levels of these histone modifications that set the level of FLC expression to create a vernalization requirement in winter-annual accessions. Previously, it has been reported that lesions in ATX1, which encodes a Trithorax class H3K4 methylase, partially suppress the delayed flowering of winter-annual Arabidopsis. We show that the flowering phenotype of atx1 atxr7 double mutants is additive relative to those of single mutants. Therefore, both classes of H3K4 methylases appear to be required for proper regulation of FLC expression.

Figure 1.

ATXR7 Gene Structure and Flowering Phenotype of atxr7. (A) ATXR7 gene structure and T-DNA insertion sites. Thick and thin lines indicate exons and introns, respectively. atxr7-1 and atxr7-2 are in the Col background. (B) and (C) Representative plants of Col, atxr7-1, and atx1-2 atxr7-1 grown in long days ([B], 4 weeks old) or in short days ([C], 10 weeks old). (D) and (E) Primary leaf number at flowering of Col, atxr7-1, and atx1-2 atxr7-1 grown in long days (D) or in short days (E). Closed and open bars indicate rosette and cauline leaves, respectively. The averages of the results from at least 10 plants are shown. Bars indicate sd. [See online article for color version of this figure.]

Figure 2.

Expression Level of FLC and FLC-Related Family Genes in H3K4 Methylase Mutants. Real-time PCR was performed to analyze expression levels. Because there were no significant differences between the results of atxr7-1 and atxr7-2, data from atxr7-1 are presented. The averages of the results from three different biological replicates are shown. Each experiment was normalized to ACTIN2 expression. Bars indicate the se. Asterisks above the bars indicate the significant differences in the expression levels of the genes between Col and mutants (P < 0.05). Triangles indicate the significant difference in the FLM expression level between atx1-2 and atx1-2 atxr7-1 (P = 0.017).

Figure 3.

Phenotypes of atxr7 in a FRI or Autonomous Pathway Mutant Backgrounds. (A) and (C) Representative plants of atx mutants in a FRI ([A], 7 weeks old) or autonomous pathway mutant backgrounds ([C], 9 weeks old) grown in long days. (B) and (D) Primary leaf number at flowering of atx mutants in a FRI (B) or autonomous pathway mutant backgrounds (D) grown in long days. Closed and open bars indicate rosette and cauline leaves, respectively. The averages of the results from 9 to 20 plants are shown. Bars indicate sd. Results of t test between single autonomous pathway mutant and the double mutant with atxr7-1 are P < 0.001. [See online article for color version of this figure.]

Figure 4.

Phenotype of atx Double Mutants in a FRI Background. (A) Representative plants of FRI-Col (6 weeks old), FRI atx1-2 atxr7-2, and Col (5 weeks old) grown in long days. (B) Primary leaf number at flowering of atx1-2 atx2-3 and atx1-2 atxr7-2 double mutants in a FRI background grown in long days. Closed and open bars indicate rosette and cauline leaves, respectively. The averages of the results from at least nine plants are shown. Bars indicate sd. (C) Real-time PCR was performed to analyze FLC expression in Col, FRI-Col, and the FRI atx1-2 atxr7-2 mutant. The averages of the results from three different biological replicates are shown. Each experiment was normalized to ACTIN2 expression. Bars indicate the se. [See online article for color version of this figure.]

Figure 5.

Expression Pattern of ATXR7pro:ATXR7-GUS in the FRI atxr7-1 Mutant. GUS expression pattern in a representative 12-d-old ATXR7pro:ATXR7-GUS transformant. Bars = 1 mm. (A) Entire seedling. (B) Rosette leaf. (C) Main and lateral roots. [See online article for color version of this figure.]

Figure 6.

Enrichment of ATXR7-GFP at the FLC Locus. (A) Schematic model of primer positions at the FLC locus. (B) Levels of the ATXR7-GFP protein at FLC. Homozygous T3 seedlings of two independent ATXR7pro:ATXR7-GFP lines (10 and 18) were used as samples. ChIP using GFP antibody and real-time PCR were performed to evaluate the occupancy of the ATXR7-GFP protein at FLC. The x axis and y axis indicate the primer number and the relative level of ATXR7-GFP enrichment, respectively. The averages of the results from three different biological replicates are shown. Each experiment was normalized to Ta3, in which mono-, di-, and trimethylation of H3K4 are not detected (Zhang et al., 2009). Bars indicate the se.

Figure 7.

H3K4 Methylation and H3K27 Trimethylation at FLC in FRI-Col, FRI atxr7-1, and FRI atx1-2 atxr7-2 Mutants. Levels of H3K4me3 (A), H3K4me2 (B), H3K4me1 (C), and H3K27me3 (D) at FLC. The x axis and y axis indicate the primer number described in Figure 6A and relative levels of modifications, respectively. The averages of the results from two different biological replicates are shown. Each experiment was normalized to total Histone H3 ChIP. Bars indicate the se.

Figure 8.

The Phenotypes of the Double Mutants among atxr7-1, elf7-2, and efs-3. (A), (C), and (E) Representative 8-week-old plants of Col, atxr7-1, efs-3, and atxr7-1 efs-3 (A), Col, atxr7-1, elf7-2, and atxr7-1 elf7-2 (C), and Col, efs-3, elf7-2, and elf7-2 efs-3 (E) grown in short days. (B), (D), and (F) Primary leaf number at flowering of Col, atxr7-1, efs-3, and atxr7-1 efs-3 (B), Col, atxr7-1, elf7-2, and atxr7-1 elf7-2 (D), and Col, efs-3, elf7-2, and elf7-2 efs-3 (F) grown in short days. Closed and open bars indicate rosette and cauline leaves, respectively. The averages of the results from at least nine plants are shown except for atxr7-1 elf7-2 (n = 7). Bars indicate sd. The numbers above the bars indicate the percentages of the primary leaf number at flowering compared with that of Col. [See online article for color version of this figure.]

Figure 9.

Schematic Model of Transcriptional Activation through Histone Modifications. Summary of the relationship among the factors involved in the transcriptional activation of the yeast GAL1 gene ([A]; from Hampsey and Reinberg, 2003) and the FLC gene (B). Circles, histone octamers; K4, Lys 4 of histone H3 (H3K4); K36, Lys 36 of histone H3 (H3K36); me, methyl groups on Lys residues. (A) In yeast, the Paf1 complex is required for the recruitment of both Set1 and Set2, which are H3K4 and H3K36 methylases, respectively. Both trimethylation of H3K4 (H3K4me3) and dimethylation of H3K36 (H3K36me2) are involved in the transcriptional activation of certain genes including GAL1. (B) In Arabidopsis, H3K4me3 and H3K36me2 are also essential for the transcriptional activation of FLC. Both Set1-class (ATXR7) and Trx-class (ATX1/2) H3K4 methylases are required for full H3K4 methylation and, thus, FLC activation. The Paf1 complex (including ELF7 and VIP4) is required for the function of ATXR7 and possibly ATX1/2, but not EFS (a Set2 ortholog). [See online article for color version of this figure.]