PHF14_PZP is an unmodified histone H3_(1–34) reader. (A) Schematic diagram showing the domain architecture of human PHF14. (B) PZP motif based-evolutionary analyses of PHF14 homologs using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method and are drawn to scale in the diagram. The numbers next to each node were given as percentages and represent bootstrap support for the node (500 replicates). Evolutionary analysis was conducted in MEGA7 (37–40). (C–E) ITC fitting curves of zebrafish PHF14_PZP (C), BRPF1_PZP (D) and AF10_PZP (E) titrated with histone H3 peptides in different frames. N.D. = not detected.

Crystal structure of PHF14_PZP in a peptide-free state. (A) Wall-eye stereo view of the overall structure of free PHF14_PZP. PHD1 (cyan), ZnK (light blue), PHD2 (light pink), and α-helix (white) are shown as ribbons. Zinc ions are depicted as spheres. Red dotted lines indicate the invisible insertion loop. Key secondary structural elements are labeled. (B) Structural alignment of PHF14_PZP (magenta), AF10_PZP (green) and BRPF1_PZP (white). (C) Interactions between the C-terminal α-helix with PHD1 (left) and PHD2 (right). Key residues are shown as sticks. Hydrogen bonds are shown as green dashes with distances labeled in Å. (D) Structural alignment of PHF14_PHD1 (cyan) and PHF14_PHD2 (light pink). Note that the insertion loop is topologically equivalent to the β-hairpin tower.

Molecular details for H3_(1–9) readout by PHF14_PZP. (A) 2Fo−Fc omit map (blue meshes) around the H3 peptide (yellow sticks) is contoured at the 0.7σ level. PHD1 finger is shown as cyan ribbon. (B) Overall structure of the H3_(1–25)-PHF14_PZP complex. PHD1 finger (cyan), ZnK (light blue), PHD2 finger (light pink), and H3 peptide (yellow) are shown as ribbons. Zinc ions are depicted as white spheres. (C) Electrostatic surface view of PHF14_PZP bound to H3 peptides. Electrostatic potential is shown as a spectrum ranging from − 5 kT/e (red) to + 5 kT/e (blue). (D) Topology diagram of the H3_(1–9)-PHF14_PZP complex. Helices and strands are labelled and rainbow-colored from the N- (blue) to C- (red) termini. H3 is depicted in yellow and the insertion loop is highlighted in magenta. (E) Binding details of PHF14_PZP with H3 peptide. The PHD1 finger of PHF14_PZP is shown as a cyan and magenta (loop) ribbon. The residues of H3 peptide are depicted as yellow sticks. Key residues of PHF14_PZP are depicted as purple or cyan sticks. Green dashes and red dashes represent the hydrogen bonds and ionic bonds, respectively. (F) ITC fitting curves of PHF14_PZP mutants titrated with the H3_(1–15) peptide. N.D. = not detected. (G) ITC fitting curves of PHF14_PZP titrated with the H3_(1–15) peptide bearing different modifications. N.D. = not detected.

A novel recognition model for H3 peptide readout by PHF14_PZP. (A) A summary of different recognition modes of PHD fingers. PHD fingers and their ligands are depicted by cyan and yellow ribbons, respectively. Zinc ions are shown as white spheres and the corresponding secondary structure are labelled. (B) Structural alignment of H3 (yellow)-PHF14 (pale cyan), H3 (light pink)-BHC80 (blue white) and H3 (cyan)-BPTF (white) complexes in wall-eye stereo view. H3 R2, K4, and R8 are depicted as sticks. (C) Sequence alignment of different PHD finger proteins: BHC80 (2PUY), PYGO (2VP7), ING2 (2G6Q), BPTF (2F6J), UHRF1 (3SOX), TAF (5WXG), MLL4PHD6 (6O7G), AIRE (2KFT), MOZ (5B78), BRPF1 (6U04), TRIM24 (3O37), AF10 (5DAH), ORC1b (5HH7).

Molecular basis for H3_(14–34) recognition by PHF14_PZP and HDX-MS analyses of H3_(1–34) recognition by PHF14_PZP. (A) Overall structure of H3(1–13)-PHF14_PZP. PHF14_PZP is shown as a light blue ribbon. Histone H3 peptide is shown as yellow sticks. Zinc ions are depicted as white spheres. Blue meshes, 2Fo−Fc omit map of histone H3 peptide contoured at 0.7 σ level. (B) ITC fitting curves of H3(1–13)-PHF14_PZP and PHF14_PZP titrated with H3.1/3_(14–34) peptides. (C) Thermodynamic parameters of PHF14_PZP and H3(1–13)-PHF14_PZP titrated with H3.3_(14–34) peptides. (D) Mapping H3_(1–34) binding regions on PHF14_PZP based on the HDX-MS data. (E) Structure of PHF14_PZP is shown in surface view with the corresponding H3(1–34) binding regions color coded as light pink, blue, yellow, and green corresponding to panel (D). Histone H3-Nter and H3-middle are displayed in cyan ribbon and dashes, respectively. (F) The structural alignment of H3-AF10_PZP and H3-PHF14_PZP complexes. PHF14_PZP and AF10_PZP are shown as grey and cyan ribbons, respectively. H3 peptides are show as lime (H3-PHF14) and yellow (H3-AF10) ribbons, respectively. The corresponding key elements forming the ‘αZP’ groove are marked. (G) Sequence alignment of PHF14_PZP and AF10_PZP. Magenta stars represent the key residues for H3_(20–28) recognition in AF10_PZP.

Molecular basis for H3_(14–34) recognition by PHF14_PZP. (A) Structural alignment of PHF14_PZP (magenta), H3(1–13)-PHF14_PZP (pale cyan), H3_(1–25)-PHF14_PZP (light pink) and H3-AF10_PZP (yellow-grey). Close-up views are a comparison of a ‘closed’ (i) and an ‘open’ (ii) conformation of the ZP linker. Key residues of ZP linker and H3 are shown as sticks and labelled as indicated. The red cross denotes a clash between F383 and a lysine residue of H3; hydrophobic residues are also shown as dotted spheres in cyan. (B) The interaction of insertion loop between ZP-linker region. Key residues are shown as sticks (left). ITC fitting curves of indicated H3(1–13)-PHF14_PZP wild type, mutants and PHF14_PZP, titrated with H3.3_(14–34) peptides (right). P14 is short for PHF14_PZP. (C) ITC fitting curves of indicated H3(1–13)-PHF14_PZP mutants, PHF14_PZP-Δloop and PHF14-I386M V378M titrated with H3.3_(14–34) or H3.3_(1–34) peptides. P14 is short for PHF14_PZP. (D) Co-immunoprecipitation results from HEK293T cells with 3×Flag-PHF14-WT, 3×Flag-PHF14-ΔPZP, 3×Flag-PHF14-Δloop, and 3× Flag-PHF14-I386M overexpression showing bipartite recognition of the H3 tail by PHF14_PZP.

Modification crosstalk and hierarchical switch of H3-PHF14_PZP engagement. (A,B) ITC fitting curves of H3(1–13)-PHF14_PZP (A) and AF10_PZP (B) titrated with H3_(14–34) peptides with indicated modifications. N.D. = not detected. (C) A model illustrating modification switch features of H3_(1–34)-PHF14_PZP engagement. H3-Nter and H3-middle are colored magenta and orange, respectively. Indicated modifications are shown as color-coded spheres. Strictly sensitive, mildly sensitive, and tolerated modifications are denoted by red cross, half-tick, and tick signs, respectively. (D) A model highlighting hierarchical modification switch features for downregulation of the H3_(1–34)-PHF14_PZP complex formation. H3-Nter and H3-middle are colored magenta and orange, respectively. Hexagonal stars of different colors indicate protein mutations at insertion loop region, mutation at ‘α-ZP’ surface, histone modifications in H3-Nter, and histone modification in H3-middle region, respectively.