Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF

Abstract

Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.

Figure 1. NMR, calorimetry and surface plasmon resonance-based studies of the binding of the BPTF PHD finger by H3(1–15)K4 peptides as a function of methylation state

a, Sequences of the PHD finger of human BPTF and histone H3(1–20). b, Left panel: superposition of ¹H,¹⁵N-HSQC NMR spectra showing BPTF PHD finger amide resonances in the absence (black) and presence (red) of 5 equivalents of H3(1–15)K4me3 peptide. Right panel: plots of chemical shift changes of the Y17 amide resonance of the BPTF PHD domain as a function of peptide concentration for different methylation states of H3K4. c, Isothermal titration calorimetry enthalpy plots for the binding of the BPTF PHD finger by H3(1–15)K4me3 (left panel) and H3(1–15)K4me2 (right panel) peptides. d, Surface plasmon resonance-based binding curves for the BPTF PHD finger with biotin-labelled H3(1–20) peptides as a function of K4 methylation state.

Figure 2. Crystal structures of human BPTF PHD finger-linker-bromodomain in the free state and bound to H3(1–15)K4me3

a, Domain architecture of BPTF bromodomain and proximal PHD finger. b, Ribbon representation of the crystal structure of the BPTF PHD finger-linker-bromodomain in the free state. Two bound Zn ions within the PHD fold are shown as balls. c, Crystal structure of the H3(1–15)K4me3-bound complex, with bound peptide shown in a space-filling representation. d, Structure of the PHD finger complex with the 2F_o − F_c omit electron density (0.8j level) highlighted for the bound H3(1–15)K4me3 peptide. e, Positioning of the H3(1–15)K4me3 peptide (space-filling representation) on the surface of the PHD finger portion (electrostatic surface representation with red as negatively charged and blue as positively charged surface) of the complex. f, Different view emphasizing the positioning of R2 and K4me3 in adjacent channels.

Figure 3. Details of the intermolecular contacts in the H3(1–15)K4me3 peptide–BPTF PHD finger complex and comparison with its H3(1–15)K4me2-bound counterpart

a, Intermolecular backbone interactions between the A1–T6 segment of bound H3(1–15)K4me3 peptide and the PHD finger in the complex. b, Intermolecular hydrogen-bonding interactions involving the guanidinium group of R2 in the complex. c, Superposition of free (coloured green) and H3(1–15)K4me3-bound complex (coloured yellow) of the BPTF PHD finger. d, e, Positioning of the trimethylated lysine of the H3(1–15)K4me3 peptide (d) and the dimethylated lysine of the H3(1–15)K4me2 peptide (e) within a four-aromatic-amino-acid cage of the BPTF PHD finger. Two bridging water molecules link the NH of K4me2 to the carboxylate of D6, as indicated by dashed lines.

Figure 4. NMR-based structures of free and H3(1–15)K4me3-bound BPTF PHD finger, together with binding studies of PHD finger mutants with H3(1–15)K4me3 peptide

a, b, Backbone superposition of 20 NMR-based structures of the free BPTF PHD finger (residues 8–58) (a) and the H3(1–15)K4me3-bound BPTF PHD finger in solution (b). c, d, Superposition of structures centred about a cluster of aromatic residues (Y10, Y17, Y23 and W32) in the free BPTF PHD finger (c) and when bound to H3(1–15)K4me3 (d). e, f, Fluorescence polarization-based binding curves for fluorescein-labelled H3(1–15)K4me3 peptide (e) and surface plasmon resonance-based binding curves for biotin-labelled H3(1–20)K4me3 peptide (f) with BPTF PHD finger mutants that cluster about either the K4-binding aromatic-amino-acid-lined cage (left panels) or the R2-binding pocket (right panels).