Selective recognition of histone crotonylation by double PHD fingers of MOZ and DPF2

Abstract

Recognition of histone covalent modifications by 'reader' modules constitutes a major mechanism for epigenetic regulation. A recent upsurge of newly discovered histone lysine acylations, such as crotonylation (Kcr), butyrylation (Kbu), and propionylation (Kpr), greatly expands the coding potential of histone lysine modifications. Here we demonstrate that the histone acetylation-binding double PHD finger (DPF) domains of human MOZ (also known as KAT6A) and DPF2 (also known as BAF45d) accommodate a wide range of histone lysine acylations with the strongest preference for Kcr. Crystal structures of the DPF domain of MOZ in complex with H3K14cr, H3K14bu, and H3K14pr peptides reveal that these non-acetyl acylations are anchored in a hydrophobic 'dead-end' pocket with selectivity for crotonylation arising from intimate encapsulation and an amide-sensing hydrogen bonding network. Immunofluorescence and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) showed that MOZ and H3K14cr colocalize in a DPF-dependent manner. Our studies call attention to a new regulatory mechanism centered on histone crotonylation readout by DPF family members.

Figure 1. Identification of MOZ_DPF and DPF2_DPF as favorable readers of Kcr

(a) Chemical structures of known histone lysine acylations (abbreviations and numbering correspond to data shown in (c and d) and are used throughout text with emphasis on Kcr). (b) The domain architectures of MOZ/MORF and DPF1/2/3 proteins (top). The sequence of H3_1–25 tail with K14 colored in red (bottom). (c and d) ITC fitting curves of MOZ_DPF (c) and DPF2_DPF (d) titrated with a series of H3_1–25 peptides containing different K14 acylations shown in (a). (e and f) MOZ_DPF (e) and DPF2_DPF (f) have a greater affinity for nucleosomes bearing Kcr than those bearing Kac. Direct Blue staining of the membrane shows comparable inputs. The streptavidin monomer co-migrates with histone H4. Nucleosome IP was done in two replicates. The blotting was confirmed by 3 independent blots. Full images of the gels in (e) and (f) are shown in Supplementary Fig. 6a–d.

Figure 2. Molecular details for H3K14cr readout by MOZ_DPF

(a) Overall structure of MOZ_DPF bound to H3_1–25K14cr (yellow). The PHD1 and PHD2 subdomains are colored blue and pink, respectively; Key H3 residues are shown as sticks; 1 and 2 denote two core β-strands; N and C denote amino- and carboxyl-termini. Grey spheres, zinc ions. (b) Electrostatic surface view of the MOZ_DPF-H3K14cr complex. Electrostatic potential is expressed as a spectrum ranging from -10 kT/e (red) to +10 kT/e (blue). (c) Hydrophobic contacts between helix H3_17–25 and the “I208–L213” saddle pair. (d) Insertion of K14cr into the MOZ_DPF reader pocket. K14cr is depicted as space-filling spheres with yellow for carbon, blue for nitrogen and red for oxygen. (e) Cut-away view of K14cr in the MOZ_DPF reader pocket. K14cr is represented as yellow stick with the crotonylamide group shown as cyan surface. (f) Composition of the K14cr-binding β2 pocket. K14cr is shown as yellow stick with its extended two-hydrocarbon group highlighted as green dots; small red balls, water molecules; orange spheres, zinc ions; Cyan dashes, hydrogen bonds. (g) Sequence alignment of the β2 motif among different histone-binding PHD fingers, the zinc-coordinating residues are shaded blue. Key pocket-forming glycine (G) residues are shaded orange. (b) Structural alignment of MOZ PHD1 (orange) and BPTF PHD (blue) domains. Key residues are shown as sticks. Arrow head points to the position of MOZ G237 and BPTF Y2885. Note the bulky feature of Y2885 side chain that occupies the K14cr pocket.

Figure 3. Recognition of different histone H3K14 acylations by MOZ_DPF

(a) Insertion of K14ac (pink), K14pr (green), K14bu (cyan) and K14cr (yellow) into the MOZ_DPF reader pocket. Water molecules are shown as red balls, and hydrogen bonding interactions are indicated by red dashes. (b) Stereo view of H3K14 acylation readout by MOZ_DPF reader pockets. H3K14ac (pink), H3K14pr (green), H3K14bu (cyan) and H3K14cr (yellow) peptides are superimposed for comparison. Note the outwards shift of key Kcr-binding residues (yellow sticks and grey ribbons). For clarity, key pocket residues of Kac and Kcr complexes are shown here. (c) Hydrogen bonding network involving H3K14 acylations and MOZ_DPF reader pocket. H3K14 acylations and water molecules are covered by the simulated annealing Fo-Fc omit map countered at 2.5 σ level. Key residues are shown as sticks.

Figure 4. Size-selective recognition of H3K14cr by MOZ_DPF

(a) Comparison of lysine crotonyllysine (Kcr, yellow) and butyryllysine (Kbu, cyan) side chains. Hydrogen, nitrogen, and oxygen atoms are colored grey, blue and red, respectively. Half-transparent surfaces are shown to highlight a slim feature of Kcr and a bulgy feature of Kbu. (b) The MOZ_DPF β2 pocket is highly size-selective for K14cr. Crotonyl- (yellow), butyryl- (cyan) and Propionyl- (green) amide groups are showed as space-filling dots. The plates (highlighted by arrow heads) indicate steric clashes. Key residues are highlighted with sticks with hydrogen atoms colored grey. (c) Superimposition of K14pr, K14bu and K14cr in the MOZ_DPF reader pocket. Key residues and zinc ions are showed as sticks and spheres. K14pr, K14bu and K14cr complexes are colored green, cyan and yellow, respectively. (d) Thermodynamic parameters of MOZ_DPF titrated with the annotated H3K14 acylation peptides. Detailed fitting values are listed in Supplementary Table 2.

Figure 5. Recognition of H3K14cr by DPF2-mimicking MOZ_DPF

(a) Structural comparison of the free DPF2_DPF (green) with MOZ_DPF (pink) bound to H3K14cr (grey) peptide. The two amide-sensing residues are shown as sticks and labelled in the close-up window. (b) ITC fitting curves of MOZ_DPF and MOZ_DPF^DM (S210D/N235R) (top), and of wild type DPFDPF and DPF_DPF^DM (D274S/R300N) (bottom) titrated with H3_1–25 K14cr peptides. (c) Crystal structure of MOZ_DPF^DM bound to H3K14cr (yellow) peptide. The wild type MOZ_DPF-H3K14cr complex (grey) is overlaid for comparison. Key residues are shown in stick representation. Small red balls, water molecules; cyan dashes, hydrogen bonds.

Figure 6. Mutagenesis and colocalization studies

(a) ITC fitting curves comparing wild type and mutant MOZ_DPF titrated with H3_1–25K14cr peptides. (b) IF of 293T cells reacted with antibodies against FLAG (green) or H3K14cr (red). FLAG-tagged WT, S210M, and D282A mutant full-length MOZ were transformed into 293T cell for IF imaging (left). Scale bar: 3 μm. (c) Quantification of the FLAG signal colocalized with H3K14cr (counts are based on 50 cells) (right). ****P<0.0001 by unpaired t-test with Welch’s correction, compared to FLAG-tagged WT-K14cr group. Standard derivation of every sample group < 0.1. (d) ChIP-qPCR analysis using anti-FLAG antibody and IgG control to probe the enrichment of FLAG-tagged WT or mutant MOZ in the promoter regions of indicated HOX genes in 293T cells. A Western blot with anti-FLAG and anti-tubulin antibodies shows that all of the constructs are expressed at appropriate levels. Full image of the gel is shown in Supplementary Fig. 6e. Standard derivation < 0.3. (e) ChIP-qPCR analysis using anti-H3K14cr antibody and IgG control to probe the enrichment of H3K14cr in the promoter regions of indicated HOX genes in 293T cells (w/o MOZ overexpression). standard derivation < 0.1. In (d) and (e), an average of three real-time PCR replicates of a representative experiment is displayed as a function of percentage of input signal.