Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor

Abstract

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 A-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-pi contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignancies, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

Figure 1. The ING1 PHD finger recognizes H3K4me3

(a) Peptide microarrays containing the indicated histone peptides were probed with GST-ING1 PHD. Red spots indicate binding. me, methylation; ac, acetylation; ph, phosphorylation; s, symmetric; a, asymmetric. (b) Structure of the ING1 PHD finger in complex with the histone H3K4me3 peptide. The PHD finger is shown as solid surface. The histone peptide is depicted as ball-and-stick model with C, O and N atoms colored green, red and blue, respectively. (c) The PHD finger is shown as a ribbon with residues mutated in human cancers colored brown. (d) Interactions of the GST-fusion wild type and mutant ING1 PHD fingers with biotinylated histone peptides examined by western blot experiments.

Figure 2. The function of ING1 requires its H3K4me3 binding activity

(a) Assessment of the repair rate of UV-damaged DNA by luciferase reporter assay in MMRU cells cotransfected with undamaged or damaged pRL-CMV luciferase plasmid and pcDNA3-vector (control), wild-type ING1 expression vectors and ING1W235A mutant. Forty hours after transfection, luciferase activity was measured with a luminometer. Columns, mean from triplicates; bars, SD. The experiment was repeated twice with similar results. (b) Stimulation of apoptosis by overexpressed full-length wild type ING1 and W235A mutant. Cell death was measured in HT1080 cells transfected with 1 µg of the indicated expression vectors. The cells were first treated with Doxorubicin and then trypsinized and stained with trypan blue. The error bars represent the mean ± s.e.m. from three independent experiments.

Figure 3. The cancer specific residues of ING1 are involved in the interaction with H3K4me3

(a) Six superimposed ¹H,¹⁵N heteronuclear single quantum coherence (HSQC) spectra of PHD (0.2 mM), collected during titration of a H3K4me3 peptide, are color-coded according to the ligand concentration (inset). (b) The histogram displays normalized ¹H,¹⁵N chemical shift changes observed in the corresponding (a) spectra of the PHD finger. The normalized chemical shift change was calculated using equation [(ΔδH)² + (ΔδN/5)²]^0.5, where δ is the chemical shift in parts per million (ppm). Colored bars indicate a significant change being greater than average plus one half standard deviation.

Figure 4

The PHD-H3K4me3 binding stabilizes ING1 at the methylated chromatin. Intracellular localization of the wild type (WT) and mutant EGFP-ING1 PHD fingers (green) in HeLa cells. The cells were stained with anti-H3K4me3 antibodies (red) and DAPI (blue). The ratio of the nuclear vs. cytoplasmic EGFP [2.28 (wild type), 1.92 (N216S), 1.75 (V218I) and 1.84 (G221V)] was obtained by averaging the fluorescence intensity in ten or more cells.

Figure 5. The cancer specific mutations disrupt the DNA repair and apoptotic functions of ING1

(a) Evaluation of the DNA repair efficiency of ING1wt (48.9%; P = 0.01, t test), N216S (44.7%; P = 0.2, t test), V218I (33.1%; P < 0.01, t test), and G221V (30.8%; P < 0.01, t test) by luciferase reporter assay in MMRU cells. (b) Stimulation of apoptosis by overexpressed full-length N216S (17.5%; P = 0.06, ttest), V218I (13.5%; P= 0.03, t test) and G221V (16.4%; P < 0.01, t test) mutants of ING1 in HT1080 cells.

Figure 6

A model of the ING1 functioning. Binding of the C-terminal PHD finger and the N-terminal SAID domain of ING1 to H3K4me3 and a SAP30 subunit of the Sin3a/HDAC1/2, respectively, tethers the histone modifying complex to the nucleosome for subsequent deacetylation of acetylated lysine residues of histone H3.