Structural insights into the recognition of histone H3Q5 serotonylation by WDR5

Abstract

Serotonylation of histone H3Q5 (H3Q5ser) is a recently identified posttranslational modification of histones that acts as a permissive marker for gene activation in synergy with H3K4me3 during neuronal cell differentiation. However, any proteins that specifically recognize H3Q5ser remain unknown. Here, we found that WDR5 interacts with the N-terminal tail of histone H3 and functions as a "reader" for H3Q5ser. Crystal structures of WDR5 in complex with H3Q5ser and H3K4me3Q5ser peptides revealed that the serotonyl group is accommodated in a shallow surface pocket of WDR5. Experiments in neuroblastoma cells demonstrate that H3K4me3 modification is hampered upon disruption of WDR5-H3Q5ser interaction. WDR5 colocalizes with H3Q5ser in the promoter regions of cancer-promoting genes in neuroblastoma cells, where it promotes gene transcription to induce cell proliferation. Thus, beyond revealing a previously unknown mechanism through which WDR5 reads H3Q5ser to activate transcription, our study suggests that this WDR5-H3Q5ser-mediated epigenetic regulation apparently promotes tumorigenesis.

Fig. 1. Serotonylation of H3Q5 facilitates WDR5 binding to H3.

(A) Chemical structure of serotonyl group covalently attached to the Q5 residue of histone H3. The histone octamer core and the wrapped DNA of the nucleosome are respectively shown as a solid yellow cycle and a blue line. The N-terminal 1 to 14 residues of H3 are indicated by their single-letter codes, with Q5 being colored red and the others colored black. Trimethylation on the K4 residue is indicated by “me3.” The chemical structure of the serotonyl group on Q5 is shown in red. (B) Streptavidin pull-down assay of recombinant WDR5^22–334 (upper left) or full length WDR5^FL (middle left), with various biotinylated H3_1–14 variants, with or without K4 trimethylation or Q5 serotonylation as indicated. The relative intensities of WDR5^22–334 and WDR5^FL bands pulled down by various peptides are quantitated by densitometry and expressed as the fold of the protein level pulled down by H3 peptide. The numbers below the bands are the average of three independent experiments, and the means ± SD are also presented as the bar plots (the right). *P < 0.05; **P < 0.01. FL, full length. (C) ITC analysis to measure the binding affinity of WDR5^22–334 with an unmodified H3 (gray) ligand peptide or H3 variant peptides bearing modifications including H3K4me3 (blue), H3Q5ser (red), or dual H3K4me3Q5ser (green).

Fig. 2. Crystal structures of WDR5^22–334 in complex with either H3Q5ser or H3K4me3Q5ser peptide.

(A) Overall structures of WDR5^22–334 in complex with either H3Q5ser or H3K4me3Q5ser peptide. The structures of WDR5^22–334 are shown in a cartoon diagram; WDR5^22–334/H3Q5ser is colored in wheat and WDR5^22–334/H3K4me3Q5ser is colored in gray. The H3Q5ser (green) and H3K4me3Q5ser (yellow) peptides are shown in a cartoon diagram, with the serotonyl group on Q5 shown as sticks. (B and C) Omit Fo – Fc electron density map of the H3Q5ser (B) and H3K4me3Q5ser (C) peptides, contoured at the 2.5σ level. The peptides are shown as sticks and colored green and yellow [following color scheme from (A)]. Trimethyl and serotonyl groups are indicated with red labels. (D) Structural comparison of complexes including WDR5^22–334/H3 (cyan; PDB code: 2H9M), WDR5^23–334/H3K4me3 (orange; PDB code: 2H6Q), WDR5^22–334/H3Q5ser (green), and WDR5^22–334/H3K4me3Q5ser (yellow). Enlarged view in the right panel shows binding-induced conformational changes in the peptides. (E) Electrostatic potential surface view of WDR5 in complex with the H3Q5ser peptide. The peptide is shown as sticks. (F) The interaction of H3Q5ser (green) and H3K4me3Q5ser (yellow) peptides with WDR5^22–334. Amino acid residues of WDR5 involved in the peptide interaction are shown as sticks; these are colored wheat in WDR5^22–334/H3Q5ser and colored gray in WDR5^22–334/H3K4me3Q5ser. The enlarged views in the left and right panels show the detailed interactions between the H3 peptides and WDR5. Hydrogen bonds are indicated as dashed lines.

Fig. 3. Recognition of serotonyl group by WDR5.

(A and B) Serotonyl group of H3Q5ser (A) and H3K4me3Q5ser (B) peptides bound to WDR5^22–334. The hydrogen bonds are shown as dashed lines. Amino acid residues involved in serotonyl group binding are shown as sticks and are labeled in black. (C) Streptavidin pull-down assays of WT and five putative binding-deficient mutant variants of WDR5^22–334 with the aforementioned biotinylated H3 peptides as ligands, as indicated. The relative intensities of WT or mutant variants of WDR5 pulled down by each peptide are quantitated by densitometry and expressed as the fold of the WT WDR5 level pulled down by the same type of peptide. The numbers below the bands are the average of four independent experiments. (D and E) The binding of WDR5^22–334 N130A (D) and WDR5^22–334 Y131A (E) to various H3 peptides was evaluated via ITC. Peptides were titrated into sample cell containing WDR5^22–334 N130A (D) or WDR5^22–334 Y131A (E) proteins.

Fig. 4. Interaction of WDR5 with H3Q5ser affects H3K4me3.

(A) Immunoprecipitation-western blot (IP-WB) analysis to detect the existence of H3Q5ser (top) in human neuroblastoma SK-N-SH cells, both in directly loaded cell lysates and in anti-histone H3 antibody enriched precipitates. Histone H3 (bottom) was visualized using an anti-histone H3 antibody (as a control). (B) 3× FLAG-tagged WDR5 (WT or with N130A mutation) were complemented in WDR5 knockout (WDR5^−/−) SK-N-SH cells. Colocalization of WT WDR5 or N130A mutant (green, anti-FLAG tag antibody) with H3Q5ser (red, anti-H3K4me3Q5ser antibody) in the SK-N-SH cell nuclei was detected by immunofluorescence microscopy. (C) Disruption of WDR5-H3Q5ser interaction significantly decreased the level of H3K4me3 modification. Cellular levels of WDR5, H3K4me3, and H3Q5ser in various cell lines were visualized by Western blotting after staining with anti-WDR5, anti-H3K4me3, and anti-H3Q5ser antibodies, respectively. The relative intensity of each band is quantitated by densitometry after normalization to β-actin and then expressed as the fold of that in the control cells. *P < 0.05; ***P < 0.001; ****P < 0.0001; n.s., not significant.

Fig. 5. WDR5 regulates neuroblastoma cell proliferation by recognizing H3Q5ser.

(A) WDR5^−/− significantly decreased the proliferation of SK-N-SH cells assessed by BrdU assays. The decreased cell proliferation phenotype was completely rescued upon complementation of the WDR5^−/− cells with WT but not N130A mutant WDR5. (B) mRNA expression levels of three cell proliferation-related genes in WDR5^−/− cells or WDR5^−/−cells complemented with WT or N130A mutant WDR5, as assessed by qPCR. (C and D) ChIP was performed with an anti-histone H3K4me3 or anti-histone H3K4me3Q5ser antibody in SK-N-SH cells (C) or with an anti-FLAG antibody [(D) for WDR5] in WDR5^−/− cells complemented with 3× FLAG-tagged WT WDR5 or N130A variant. qPCR analysis of the ChIP precipitates was performed to assess the co-occurrence of H3K4me3, H3K4me3Q5ser, and WDR5 at the promoters of PDCD6, GPX1, and C-MYC genes shown to regulate tumor cell proliferation. The data are presented as the means ± SD calculated from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.