SET DOMAIN GROUP25 encodes a histone methyltransferase and is involved in FLOWERING LOCUS C activation and repression of flowering

Abstract

Covalent modifications of histone lysine residues by methylation play key roles in the regulation of chromatin structure and function. In contrast to H3K9 and H3K27 methylations that mark repressive states of transcription and are absent in some lower eukaryotes, H3K4 and H3K36 methylations are considered as active marks of transcription and are highly conserved in all eukaryotes from yeast (Saccharomyces cerevisiae) to Homo sapiens. Paradoxically, protein complexes catalyzing H3K4 and H3K36 methylations are less-extensively characterized in higher eukaryotes, particularly in plants. Arabidopsis (Arabidopsis thaliana) contains 12 SET DOMAIN GROUP (SDG) proteins phylogenetic classified to Trithorax Group (TrxG) and thus potentially involved in H3K4 and H3K36 methylations. So far only some genes of this family had been functionally characterized. Here we report on the genetic and molecular characterization of SDG25, a previously uncharacterized member of the Arabidopsis TrxG family. We show that the loss-of-function mutant sdg25-1 has an early flowering phenotype associated with suppression of FLOWERING LOCUS C (FLC) expression. Recombinant SDG25 proteins could methylate histone H3 from oligonucleosomes and mutant sdg25-1 plants showed weakly reduced levels of H3K36 dimethylation at FLC chromatin. Interestingly, sdg25-1 transcriptome shared a highly significant number of differentially expressed genes with that of sdg26-1, a previously characterized mutant exhibiting late-flowering phenotype and elevated FLC expression. Taken together, our results provide, to our knowledge, the first demonstration for a biological function of SDG25 and reveal additional layers of complexity of overlap and nonoverlap functions of the TrxG family genes in Arabidopsis.

Figure 1.

SDG25 is a member of the TrxG family. A, Phylogenic analysis of 12 Arabidopsis SDG proteins (At, Arabidopsis), nine rice SDG proteins (Os, rice), together with H3K4 and H3K36 methyltransferases so far found in fungi (Sp, Schizosaccharomyces pombe; Sc, yeast; Nc, Neurospora crassa), Drosophila melanogaster (Dm), and Homo sapiens (Hs). The plant proteins fall in two groups: Group 1 contains proteins of other organisms involved primarily in H3K36 methylation whereas group 2 contains proteins of other organisms involved primarily in H3K4 methylation. B, Comparison of structural domains of ATX1, ATX2, and SDG25. The SET domain is the most highly conserved, and the post-SET Cys-rich region C is also well conserved in all three proteins. The other domains (PWWP, FYR, and PHD) found in ATX1 and ATX2 are not conserved in SDG25. Instead, SDG25 contains a GYF domain. C, Sequence alignment of the C-terminal last 133 amino acids from SDG25, SpSET1, and HsSET1. This highly conserved region contains the SET domain and the post-SET Cys-rich region.

Figure 2.

Loss of SDG25 function causes early flowering. A, Exon-intron structure of the SDG25 gene. The T-DNA insertion site in the sdg25-1 mutant is indicated by a triangle. B, RT-PCR analysis of SDG25 expression in 15-d-old plants of wild-type Col and mutant sdg25-1. ACTIN serves as an internal control. C, Wild-type Col and mutant sdg25-1 plants 28 d after germination grown under long-day (16-h light and 8-h dark) conditions. D and E, Flowering time of wild-type Col and mutant sdg25-1 plants measured by days to bolting and total number of rosette leaves at flowering. M, Medium day (12-h light and 12-h dark); L, long day (16-h light and 8-h dark); −V, without vernalization; +V, with vernalization (40 d of 4°C exposure). The mean value from 10 to 15 plants is shown. Vertical bars represent sds. F, Number of juvenile leaves at flowering of wild-type Col and mutant sdg25-1 plants. The measurement was performed on the same plants shown in E.

Figure 3.

Rescue of the sdg25-1 mutant phenotype by transformation with SDG25 gene. A, Diagram of genomic organization at the SDG25 gene region. The fragment of about 10 kb in length containing the SDG25 gene with promoter and terminator (pSDG25) is used to rescue the SDG25-1 mutant. B, Total number of rosette leaves at flowering of the wild-type Col, the mutant sdg25-1, and the pSDG25-rescued mutant sdg25-1 plants. M, Medium day (12-h light and 12-h dark); L, long day (16-h light and 8-h dark); −V, without vernalization. The mean value from 10 to 15 plants is shown. Vertical bars represent sds.

Figure 4.

Expression of the flowering repressor FLC but not MAF genes is down-regulated in sdg25-1 mutant. RT-PCR analysis was performed on plants of 10-d-old (A) and 20-d-old (B), grown under medium-day (12-h light and 12-h dark) conditions. ACTIN serves as an internal control.

Figure 5.

ChIP analysis of H3K4 and H3K36 methylations at FLC chromatin in wild-type Col and mutant sdg25-1 plants. A, Diagram representing FLC gene structure and different regions examined by ChIP analysis. Black boxes represent exons; lines represent promoter and introns; bars labeled a to g represent regions amplified by PCR. B, Fifteen-day-old plants grown under medium-day (12-h light and 12-h dark) conditions were ChIP analyzed with antibodies that specifically recognize dimethyl-H3K4, trimethyl-H3K4, or dimethyl-H3K36. Input and no antibody (−) are shown as positive and negative controls, respectively. ACTIN serves as an internal control.

Figure 6.

Analysis of expression, subcellular localization, and enzyme activity of SDG25. A, RT-PCR analysis of SDG25 expression in different types of plant materials. ACTIN serves as an internal control. B, Localization of GFP:SDG25 in a cortex cell of the transgenic Arabidopsis root. Fluorescence confocal image is shown together with differential interference contrast image. Note that fluorescence is distributed in the nucleus (indicated by arrowhead) as well as in the cytoplasm. A similar subcellular localization in the nucleus and the cytoplasm was also observed in other types of cells in roots, stems, and leaves. C, In vitro methylation assay with recombinant SDG25 fragments. The top section shows Coomassie Blue-stained gel and the bottom section shows corresponding autoradiography. The tested recombinant proteins as enzymes are indicated on top of lanes and substrates used in the test are indicated at bottom of lanes. Positions of nucleosome core histones H2A, H2B, H3, and H4 are indicated.

Figure 7.

Comparison of differentially expressed genes in sdg25-1, sdg26-1, and sdg8-1 mutants. A, The circles represent the genes that were down-regulated (left) or up-regulated (right) in sdg25-1 mutant upon transcriptome analysis. The total number of genes is shown in brackets. The percentages of these genes that were also down- or up-regulated in sdg26-1, in both sdg26-1 and sdg8-1, or in sdg8-1 are shown. *, Statistically significant overlap: P = 3.9 × 10⁻⁶⁹ (left) and P = 7.1 × 10⁻⁸¹ (right). B, RT-PCR analysis of expression of some genes perturbed in both sdg25-1 and sdg26-1 mutants. ACTIN serves as an internal control.