Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2

Abstract

Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 A resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.

Figure 1. Structure of ING2 PHD finger in complex with a histone H3 peptide trimethylated at Lys 4

a, The PHD finger is shown as a solid surface with the binding site residues coloured and labelled. The Lys 4 and Arg 2 binding grooves are in brown and yellow, respectively. The histone peptide is shown as a ball-and-stick model with C, O and N atoms coloured green, red and blue, respectively. b, Ribbon diagram of the structure. Dashed lines represent intermolecular hydrogen bonds.

Figure 2. ING2 PHD finger recognizes H3K4me3

a, Six superimposed ¹H,¹⁵N heteronuclear single quantum coherence (HSQC) spectra of PHD (0.2 mM) collected during titration of H3K4me3 peptide are colour-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 the equation [(ΔδH)² + (ΔδN/5)²]^0.5, where δ is the chemical shift in parts per million (p.p.m.). Coloured bars indicate significant change being greater than average plus one-half standard deviation.

Figure 3. ING2 function requires its H3K4me3 binding activity

a, Interactions of the GST fusion wild-type and mutant ING2 PHD fingers with biotinylated histone peptides examined by western blot analysis. b, Stimulation of apoptosis by overexpressed full-length wild-type and mutant ING2. Cell death was measured in HT1080 cells transfected with 10μg of the indicated plasmids. The error bars represent the mean ± s.e.m. from at least three independent experiments. c, A model of the association of HAT/HDAC chromatin-modifying complexes with the nucleosome through the binding of PHD fingers of ING proteins to H3K4me3.