Figure 1.
(A) T-DNA insertion and RNAi locations within the JMJ703 gene. (B) Comparison of the main panicle internodes between wild type and mutant plants. Lengths of the uppermost internodes were measured. (C) Comparison of stem epidermal cell lengths between wild type and mutant plants. No significant difference of cell lengths between wild type and mutant plants is detected by t-tests (p = 0.02). Bar = 50 µm. (D) Relative expression, H3K4me2 and H3K4me3 levels of cytokinin oxidase (CKX) genes in wild type and mutant plants. Expression levels in wild type are set as 1.
Figure 2.
JMJ703 is a histone H3K4 demethylase.
(A) Schematic presentation of JMJ703 protein structure. Fragment spanning amino acids 139–498 was obtained for crystal growth and structural determination. The region between amino acids 113–700 was used for enzymatic assays. (B) In vivo histone H3K4 demethylase activity of JMJ703. Constructs containing the JMJ703 fragment tagged by FLAG-HA (FA-J3NCZ) were transfected into tobacco leaf cells. Nuclei expressing FA-J3NCZ (stained with anti-HA) were examined for H3K4 methylation by anti-H3K4me1/2/3 and anti-H3K27me3 (indicated by arrows). At least 30 nuclei expressing JMJ703 per transfection were observed and imaged. Bar = 25 µm. (C) In vitro demethylase activity of JMJ703. Bulk histones were incubated with three quantities of FA-J3NCZ and analyzed by Western blots using antibodies of H3K4me1/2/3. The same blots were analyzed by anti-H3 to control loadings. FA-J3NCZ levels were revealed by anti-HA. (D) Characterization of c-JMJ703 binding to H3K4me0/1/2/3, H3K9me0 and H3K36me0 peptides using surface plasmon resonance. Curves for different concentration of peptide are differentially colored and labeled.
Figure 3.
Overall structure of c-JMJ703.
(A) Cartoon representation of the overall structure of c-JMJ703. Separate domains are colored differently and labeled. (B–E) Structural comparison of JmjN domain (B), long-beta sheet domain (C), mixed domain (D), and JmjC domain (E) among c-JMJ703 (deep salmon), c-JMJD2A (tv green), and c-Rph1 (slate).
Figure 4.
H3K4 demethylase-specific residues within the JmjC domain.
(A) Sequence alignment of the JmjC domain of JMJ703, plant JMJ proteins (in black) and representative human/yeast JMJ proteins (in blue). Residues conserved in and specific to H3K4 demethylases are boxed in red and labeled above the alignment. (B) Structure of JmjC domain of JMJ703 showing the locations of H3K4 demethylase-specific residues. H3K4me3 peptide and H3K4 demethylase-specific residues are shown in yellow sticks and purple sticks respectively. A proposed extension path of H3K4me3 peptide toward its C-terminal is shown in dashed red line.
Table 1.
Summary of H3K4 demethylation assays in tobacco cells of substitution mutants of key residues of JMJ703 catalytic domain.
Figure 5.
The interaction network of NOG (colored green), Fe(II) (colored brown), and c-JMJ703 residues (colored purple) involved in the interactions are shown. The H3K4me3 peptide is colored yellow. The secondary structure surrounding the active site is shown as a purple cartoon.
Figure 6.
Electron density of bound H3K4me3.
The H3K4me3 residues are shown in yellow sticks and labeled in red. Residues surrounding the peptide binding pocket are shown as cartoon and sticks and are colored as in Figure 3. The H3K4me3 peptide is covered by electron density (2Fo-Fc map at 1.0σ).
Figure 7.
Comparison of the substrate peptide binding modes of c-JMJ703 and c-JMJD2A.
(A) Binding of H3K4me3 peptide by c-JMJ703. (B) Binding of H3K9me3 by c-JMJD2A. (C) Binding of H3K36me3 by c-JMJD2A. c-JMJ703 and c-JMJD2A are shown as electrostatic surface and the substrate peptides are shown as yellow sticks and the residues are labeled in red. NOG and α-KG are shown as green sticks and labeled.
Figure 8.
Conformational shifts of α-KG/NOG interacting residues of c-JMJ703 and other members of JMJD2A.
Residues interacting with (A) α-KG and (B) NOG in c-JMJ703 structures are represented as blue and green sticks, respectively. Dashed lines denote hydrogen bonds, including the solvent-mediated interactions formed by residues of c-JMJ703 and solvent molecules and bound α-KG/NOG. Fe(II) and solvent molecules are shown as deep red and red spheres, respectively. (C) Comparison of residues involved in binding to Fe(II) and α-KG/NOG in c-JMJ703, c-JMJD2A, and c-Rph1. Residues of c-JMJ703-α-KG, c-JMJ703-NOG-H3K4me3, c-JMJD2A, and c-Rph1 are colored in blue, green, light pink, and purple, respectively. Numbers for each residue in c-JMJ703, c-JMJD2A, and c-Rph1 are shown with black, light pink, and purple, respectively. Solvent molecules are not included in this panel. N404 and Y321, which adopt significant conformation shifts, are framed out by red squares and a circle.