SKB1-mediated symmetric dimethylation of histone H4R3 controls flowering time in Arabidopsis

Abstract

Plant flowering is a crucial developmental transition from the vegetative to reproductive phase and is properly timed by a number of intrinsic and environmental cues. Genetic studies have identified that chromatin modification influences the expression of FLOWERING LOCUS C (FLC), a MADS-box transcription factor that controls flowering time. Histone deacetylation and methylation at H3K9 and H3K27 are associated with repression of FLC; in contrast, methylation at H3K4 and H3K36 activates FLC expression. However, little is known about the functions of histone arginine methylation in plants. Here, we report that Arabidopsis Shk1 binding protein 1 (SKB1) catalyzes histone H4R3 symmetric dimethylation (H4R3sme2). SKB1 lesion results in upregulation of FLC and late flowering under both long and short days, but late flowering is reversed by vernalization and gibberellin treatments. An skb1-1flc-3 double mutant blocks late-flowering phenotype, which suggests that SKB1 promotes flowering by suppressing FLC transcription. SKB1 binds to the FLC promoter, and disruption of SKB1 results in reduced H4R3sme2, especially in the promoter of FLC chromatin. Thus, SKB1-mediated H4R3sme2 is a novel histone mark required for repression of FLC expression and flowering time control.

Figure 1

Structure of the SKB1 gene and identification of skb1 mutants. (A) Structure of the SKB1 gene and a diagram of the SKB1 protein. Exons are indicated as boxes and introns as lines. T-DNA insertions in skb1 mutants are indicated by arrowheads. The SKB1 methyltransferase regions I, post I, II, III, and THW loop are shown in black. (B) A 72-kDa 6 × His-tagged full-length SKB1 protein purified from Escherichia coli. (C) Western blot detection of Arabidopsis endogenous SKB1 protein with the use of polyclonal anti-SKB1 antiserum. (D) RT–PCR analysis of the expression of genes upstream and downstream of skb1-1. (E) RT–PCR (upper panel) and Western blot (lower panel) analyses of SKB1 expression in wild-type Col and skb1-1 and skb1-2 plants 20 days after sowing; ACTIN7 serves as an internal control; total proteins stained with Coomassie blue showed equal loading.

Figure 2

Phenotype of skb1 mutants and SKB1 overexpression. (A) Wild-type Col (Col) and skb1 mutations grown under long-day (LD) conditions for 35 days after germination (DAG). (B) Col and skb1-1 mutant at the flowering stage grown under LD. (C) Flowering times of Col, skb1 mutants and transgenic plants under LD. (D) Col and skb1-1 mutant at 14 DAG under LD. (E) Col- and SKB1-overexpressing (S1) plants at 24 DAG under LD. (F) Abundance of SKB1 protein levels in homozygous transgenic seedlings (12 DAG under LD) determined by Western blot with anti SKB1 antibody. (G) Flowering times of the transgenic plants in (F) under LD. Bars represent means±s.d. of rosette leaf number at bolting. For each line, 20 plants were scored.

Figure 3

Analysis of skb1 mutant response to environmental cues and target genes. (A) Flowering times of skb1-1 under LD, short-day (SD), vernalization (V) and GA treatment. Bars represent means±s.d. For each line, 30–50 plants were scored. (B) Col and skb1-1 mutant plants germinated and grown at 2–4°C for 6 weeks and moved to LD conditions for 26 days. (C) Plants grown at 98 DAG in SD. (D) Plants treated with GA₃ and grown at 37 DAG under LD. (E) RT–PCR analysis of flowering control genes expression in wild-type and skb1-1 mutant plants. RNA was isolated from 20-day-old seedlings grown under LD. ACTIN7 was used as a loading control. Signal intensities were normalized relative to ACTIN7 with the use of ImageQuant and shown by bar graphs at the right from three independent experiments. (F) Flowering time of Col, skb1-1, flc-3 and skb1-1 flc-3. Ten plants were scored and grown under LD. (G) The phenotypes of wild-type Col, skb1-1, flc-3 and skb1-1 flc-3 mutant grown under LD.

Figure 4

Analysis of SKB1 expression pattern and methyltransferase activity. (A, B) Immunoblot analyses of SKB1 with the use of polyclonal anti-SKB1 antiserum. (A) Total protein extracts from different tissues of the wild type at 38 days. R, root; S, stem; L, leaf; F, flower; Sa, silique. Control shows equal loading. (B) Expression of SKB1 protein at different developmental stages. Col was grown under LD and whole parts were harvested at the indicated growth stages for total protein isolation. (C–E) RNA in situ hybridization analysis of SKB1 expression in Col grown under LD for 10 (C), 21 (D) and 26 (E) days. AFM: axillary flower meristem; SAM, shoot apical meristem; FM, floral meristem; YL, young leaf. Scale bar, 100 μm. (F) In vitro methylation of histones (10 μg) and myelin basic protein (MBP, 10 μg) by GST-SKB1 (2 μg) purified from E. coli. GST (3 μg) was a negative control; left, Coomassie blue-stained gel; right, autoradiograph of ³H-labelled proteins produced by in vitro methylation; methylated H4 is indicated by an arrow. (G) SKB1-mediated H4R3sme2 was analyzed by Western blot with specific antibodies. (H) Western blot analysis of modification of histone H3 and H4. Histone-enriched protein extracts from 20-day-old Col and skb1 mutant plants grown under LD were probed with antibodies that specifically recognize the indicated forms of histone H3 and H4. (I) Levels of SKB1 and H4R3sme2 at different developmental stages in Col. Total soluble proteins isolated from seedlings grown in SD were exposed to antibody against SKB1. Histone-enriched proteins were extracted from plants grown under SD or LD and immunoblotted with antibodies against H4R3sme2 and H3. (J) The level of H4R3sme2 in specific tissues of Col grown under LD. YL: young leaves plus apex from seedlings at 5 DAG; OL: old leaves from plants at 35 DAG. (K) Abundance of H4R3sme2 in Col, fld-4 and fve-3. Proteins were isolated from seedlings grown at 20 DAG under LD.

Figure 5

ChIP assays of wild type and skb1-1 mutant at the FLC locus. (A) A diagram of the FLC gene structure, with bars representing the A–E regions examined by ChIP. (B, C) ChIP results with antibodies against SKB1, H4R3sme2, H3K4me2, H3K9me2, H4R3me2 and acetylated H3K14 (H3K14ac). Samples were from wild-type and skb1-1 plants in LD for 20 days. The input is chromatin before immunoprecipitation. ‘No AB': the control sample lacks an antibody. ChIP assays involved at least three independent experiments; data represent results of one experiment.