Identification of a novel inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1)-mediated methylation of histone H3 Arg-17

Abstract

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.

FIGURE 1.

TBBD is a specific inhibitor of the arginine methyltransferase CARM1. A, structure of TBBD. Structural formula representation (panel I) and ball and stick model (panel II). B, filter binding assay for inhibition of histone modification. The HMTase assay was performed with CARM1 and G9a, and the HAT assay was performed with p300, p300/CBP-associated factor (PCAF) in the presence or absence of TBBD by using highly purified HeLa core histones and processed for filter binding assay. Lane 1, core histones without enzyme; lane 2, histones with enzyme in the presence of DMSO; lanes 3–6, histones with enzyme in the presence of 10, 25, 50, or 100 μm TBBD. Error bars represent mean ± S.D. of duplicate reactions. C, HMTase assay with CARM1 using core histones as substrate in the presence or absence of TBBD, processed for immunoblotting analysis. Lane 1, core histones without enzyme; lane 2, histones with enzyme in the presence of DMSO; lanes 3–6, histones with enzyme in the presence of 10, 25, 50, or 100 μm TBBD. Panel I represents Coomassie staining; panel II represents immunoblotting with dimethylated H3R17 antibody. Panel III represents the histone loading control using histone H3 antibody.

FIGURE 2.

TBBD is an uncompetitive inhibitor of CARM1 with preferential affinity to the substrate histone H3. A, Lineweaver-Burk plot representation of the effect of TBBD on CARM1 activity at a fixed concentration of [³H]AdoMet (0.88 μm) and increasing concentrations of histone H3 in the presence (10, 20, or 50 μm) or absence of TBBD. The results were plotted using GraphPad Prism software. B, Lineweaver-Burk plot representation of the effect of TBBD on CARM1 activity at a fixed concentration of histone H3 (1.467 μm) and increasing concentrations of [³H]AdoMet in the presence (10, 20, or 50 μm) or absence of TBBD. The results were plotted using GraphPad Prism software. C, ITC was carried out by titrating histone H3 (7 μm) against the ligand TBBD (140 μm) at 25 °C. The one-site binding model of the isotherm is shown. D, ITC was carried out by titrating CARM1 (7 μm) against the ligand TBBD (140 μm) at 25 °C.

FIGURE 3.

TBBD is a site-specific inhibitor of CARM1-mediated methylation of histone H3. A, the possible sites of acetylation and arginine methylation on histone H3 tail sequence are indicated. B, HeLa cells were treated as indicated for 24 h: histones isolated from untreated cells (lane 1); DMSO-treated cells (lane 2); and TBBD-treated cells (lanes 3–5). Histone modifications were probed by Western blotting using the indicated site-specific antibodies.

FIGURE 4.

Molecular modeling studies of TBBD with histone H3. A, the H3 (PRL) peptide has already been modeled into CARM1 substrate binding site. The H3 pentapeptide KAPRK has been modeled in the substrate binding site of CARM1. The figure shows the H3 pentapeptide near the active site residues of CARM1 (TYR417, HIS415, ASP166, TYR262). B, TBBD is modeled into this docked complex of CARM1 and H3 pentapeptide. Green, CARM1; magenta, H3 pentapeptide; red, TBBD. C, the docked conformation of TBBD near the KAPRK motif of histone H3. The free energy of binding for this ligand conformation is ΔG = −5.36 kcal/mol. D, the docked conformation of TBBD near the KAARK motif of histone H3. The free energy of binding for this ligand conformation is ΔG = −3.72 kcal/mol, which is higher than the free energy of binding at the KAPRK motif.

FIGURE 5.

The proline residue (Pro-16) of the histone H3 tail is responsible for TBBD-mediated inhibition of arginine methyltransferase activity. A, sequence details of the histone H3 proteins used for the assay. B, in vitro histone methyltransferase assays were performed with CARM1 in the presence or absence of TBBD using histone H3 (lanes 1–4), mutant A25P histone H3 (lanes 5–8), and mutant P16A histone H3 (lanes 9–12) as substrates. Histone H3 in the absence of enzyme (lane 1), presence of enzyme and DMSO (lane 2), or in the presence of 25 μm and 100 μm TBBD (lanes 3 and 4). Histone H3 was probed with antibodies against dimethylated H3R17 (panel I) or dimethylated H3R26 (panel II). As a loading control, histones were probed with antibody against histone H3 (panel III). C, ITC was carried out by titrating histone H3 mutant A25P (7 μm) against the ligand TBBD (140 μm) at 25 °C. The one-site model with two binding sites is shown. D, ITC was carried out by titrating histone H3 mutant P16A (7 μm) against the ligand TBBD (140 μm) at 25 °C. No interaction was observed.

FIGURE 6.

Histone H3R17 methylation regulates p53-responsive gene expression. A, scheme representing the experimental procedure. HEK293T cells were treated as indicated and processed for either ChIP or gene expression analysis. B, state of histone H3 Arg-17/H3 Arg-26 methylation in the p53-responsive element (p53 RE) of the p21 promoter analyzed by ChIP. ChIP by the input ratio in cells treated with DMSO (lane 1) or TBBD (lane 2); n = 3, p < 0.005. C, repression in p21 expression was observed on TBBD treatment in the HEK293T cell line. Reverse transcriptase-PCR amplification of p21 expression in cells treated with DMSO (lane 1) or TBBD (lane 2). D, the expression of p21 is repressed on TBBD treatment. Real time PCR quantification of fold-change in p21 gene expression in cells treated with DMSO (lane 1) and treated with TBBD (lane 2) (n = 3, p < 0.001).