Repression of FLOWERING LOCUS T chromatin by functionally redundant histone H3 lysine 4 demethylases in Arabidopsis

Abstract

FLOWERING LOCUS T (FT) plays a key role as a mobile floral induction signal that initiates the floral transition. Therefore, precise control of FT expression is critical for the reproductive success of flowering plants. Coexistence of bivalent histone H3 lysine 27 trimethylation (H3K27me3) and H3K4me3 marks at the FT locus and the role of H3K27me3 as a strong FT repression mechanism in Arabidopsis have been reported. However, the role of an active mark, H3K4me3, in FT regulation has not been addressed, nor have the components affecting this mark been identified. Mutations in Arabidopsis thaliana Jumonji4 (AtJmj4) and EARLY FLOWERING6 (ELF6), two Arabidopsis genes encoding Jumonji (Jmj) family proteins, caused FT-dependent, additive early flowering correlated with increased expression of FT mRNA and increased H3K4me3 levels within FT chromatin. Purified recombinant AtJmj4 protein possesses specific demethylase activity for mono-, di-, and trimethylated H3K4. Tagged AtJmj4 and ELF6 proteins associate directly with the FT transcription initiation region, a region where the H3K4me3 levels were increased most significantly in the mutants. Thus, our study demonstrates the roles of AtJmj4 and ELF6 as H3K4 demethylases directly repressing FT chromatin and preventing precocious flowering in Arabidopsis.

Figure 1. Early flowering of atjmj4 mutants.

A) Domain organization of AtJmj4. Domains were predicted by SMART (http://smart.embl-heidelberg.de/). Lines indicate interdomain regions. T-DNA insertion sites on the genomic sequence of AtJmj4 in atjmj4-1 and atjmj4-2 are marked on the corresponding positions of their translated protein products. B) Early flowering phenotype of atjmj4-1 mutant plants grown in either LD or SD. C) Flowering time of atjmj4 mutants. Wt Col and atjmj4 mutant plants were grown in either LD or SD and their flowering times were determined as the number of primary rosette and cauline leaves formed at bolting. At least 12 individuals were scored for each genotype. Error bars represent sd.

Figure 2. Increased expression of FT in atjmj4 mutants.

A and B) Expression of flowering genes in atjmj4-1 mutants. Col and atjmj4-1 plants were grown in LD (A) for 10 days (d) or in SD (B) for 15 d, harvested every 4 hours (h) at indicated zeitgeber (ZT; h after light-on) for one d, and used for RT-PCR analyses. Ubiquitin (UBQ) was included as an expression control. Identical results were obtained from two independent experiments, and one of them is shown. White and black bars represent light and dark periods, respectively. C and D) Temporal expression of flowering genes in atjmj4-1 mutants. Col and atjmj4-1 plants were grown for up to 12 days after germination (DAG) in LD (C) or 18 DAG in SD (D). Plants were harvested during the growth period at ZT14 (LD) or ZT8 (SD) of designated DAG and used for RT-PCR analyses because FT mRNA expression peaked at the ZT in each photoperiod. E) Histochemical GUS staining of transgenic plants harboring FT::GUS fusion construct in Col or atjmj4-1 plants. Plants were grown for 16 d either in LD or SD before GUS staining.

Figure 3. Genetic interaction between Atjmj4 and other flowering time genes.

A and B) Flowering time of atjmj4-1 flc-3 double mutants. C) Genetic interaction between AtJmj4 and FLC regulators. D to F) Genetic interaction between AtJmj4 and photoperiod-pathway genes. G and H) Genetic interaction between AtJmj4 and LHP1. Flowering times were determined in LD (A and C to G) or SD (B and H). At least 12 individuals were scored for each genotype (A to H). Error bars represent sd (A to H).

Figure 4. AtJmj4 expression.

A) mRNA expression of AtJmj4 in SD and LD. Wt Col plants were grown in SD for 12 d or in LD for 8 d and used for RT-PCR analyses. UBQ was used as an expression control. Number of PCR cycles used for AtJmj4 is indicated on the right. B) Genomic complementation of atjmj4-1. Three independent transgenic lines of atjmj4-1 containing AtJmj4::FLAG (see text for details) were grown in LD and their flowering times were determined as the number of rosette and cauline leaves formed at bolting. At least 12 individuals were scored for each genotype. Error bars represent sd. C) Expression of the AtJmj4::FLAG fusion protein in SD and LD. Plants of atjmj4-1 and two of the complementation lines shown in (B) were grown for 12 d in SD or 8 d in LD, harvested at ZT12, and used for Western blot analyses. Upper panel: Western blot with anti-FLAG antibody. Lower panel: Silver stained gel image of rubisco subunits. D to J) Histochemical GUS staining of transgenic Col plants harboring AtJmj4::GUS. Plants grown in LD (D and I) or SD (E, F, G, H, and J) were used for GUS staining. (D) In 4 d-old seedling. (E) In 6-d old seedling. (F) In trichomes. (G) In leaf. (H) In root tip. (I) In floral organs. (J) In shoot apex. K) Subcellular localization of AtJmj4 in Arabidopsis mesophyll protoplast. From left to right; bright-field image, LHP1::RFP fusion protein, AtJmj4::GFP fusion protein, merged image of the left three images.

Figure 5. Additive effect of elf6 and atjmj4 mutations on FT-dependent early flowering.

A) Early flowering phenotype of elf6-4 atjmj4-1 double mutant. All plants were grown in SD for 63 d before taken picture. B and C) Flowering time of wt Col, elf6-4, atjmj4-1, and elf6-4 atjmj4-1 double mutants in LD and SD as determined by number of leaves formed at bolting. At least 15 individuals were scored for each genotype. Error bars represent sd. D) Expression of flowering genes in elf6-4 atjmj4 double mutants. Plants of each genotype were grown in SD for 57 d and harvested at ZT4 or ZT11 for RT-PCR analyses. Actin1 was included as an expression control. Identical results were obtained from two independent experiments and one of them is shown. E) qPCR analysis of FT expression. The same RNAs used in (D) were evaluated. The wt Col levels were set to 1 after normalization by Actin1 for qPCR analysis. Error bars represent sd.

Figure 6. Increased trimethylation of H3K4 at FT locus by elf6 and atjmj4 mutations.

A) Schematic of FT locus showing regions (F, G, I, EX1, and N) amplified by the primers used for ChIP analysis. The front and the rear black boxes indicate 5′ and 3′ UTRs, respectively. White boxes indicate exons, while lines indicate introns and intergenic regions. B and C) ChIP assay of FT chromatin with antibody against H3K4me3 or H3K27me3. Plants of each genotype were grown in SD for 57 d and harvested for ChIP assay. ‘Input’ indicates chromatins before immunoprecipitation. ‘Mock’ refers to control samples lacking antibody. Actin1 was used as an internal control. D) qPCR analysis of the ChIP assay for H3K4me3 described in (B and C). The wt Col levels were set to 1 after normalization by input. Error bars represent sd. E) Coomassie-blue stained 6His-AtJmj4 protein purified from sf9 cells (left), and in vitro histone demethylation activity assay using the purified protein (right). Assays were performed without (−) or with either two (+) or four (++) µg of purified 6His-AtJmj4 protein. Mr (K), molecular mass in kilo-daltons.

Figure 7. Direct association of ELF6 and AtJmj4 with FT chromatin.

A) FT regions tested for ChIP assay. Schematic is as described in Figure 6A except for the region Ia, which was added in assays in (C). B) ELF6 binding to FT chromatin. LD grown 16 d-old wt Col and ELF6::GUS–containing transgenic elf6-4 plants were harvested and used for ChIP assay using GUS-specific antibody. Amount of immunoprecipitated chromatin was measured by qPCR (B and C). Actin1 and CO were used as internal controls, and the level of Actin1 in each sample was set to 1 for normalization (B and C). Error bars represent se of three independent biological replicates (B and C). C) AtJmj4 binding to FT chromatin. LD grown 16 d-old wt Col and AtJmj4::FLAG–containing atjmj4-1 plants were harvested and used for ChIP assay using FLAG-specific antibody.