Fig. 2: H3Q5his antagonizes H3K4 methyltransferase activities and WDR5 binding.
From: Bidirectional histone monoaminylation dynamics regulate neural rhythmicity
a, H3K4 methyltransferase complexes. b, MALDI-TOF analysis of MLL1-mediated H3K4 methylation on peptides. n = 1 per peptide per timepoint. Q5un, unmodified Gln5. c, LC–MS/MS analysis of MLL–SETD1-mediated H3K4 methylation on peptides. n = 3 per peptide per complex. **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical details are provided in Extended Data Fig. 6b–g. Unmod, unmodified. d, Peptide IPs against WDR5. Streptavidin was used to visualize peptides after IP (used for normalization). n = 4 per peptide. Statistical analysis was performed using one-way analysis of variance (ANOVA) (P < 0.0001, F2,9 = 57.34) with Tukey’s multiple-comparison test; significant comparisons are noted (H3un versus H3Q5his (*P = 0.0243); H3un versus H3Q5ser (***P = 0.0001); H3Q5his versus H3Q5ser (****P < 0.0001)). e, Peptide IPs against WDR5. Streptavidin was used to visualize peptides after IP (used for normalization). n = 4 per peptide. Statistical analysis was performed using one-way ANOVA (P = 0.0492, F2,9 = 4.287) with Tukey’s multiple-comparison test; significant comparisons are noted (H3K4me3Q5his versus H3K4me3Q5ser (*P = 0.0456)); #P = 0.0177 indicates a significant difference as determined using an a posteriori unpaired Student’s t-test (t6 = 3.237). f,g, Electron density map (f) and electrostatic potential surface view (g) of the WDR5-WD40–H3Q5his complex. The peptide is shown as sticks. h,i, Electrostatic potential surface view (h) and amino acid orientation (i) of alignments between WDR5-WD40–H3Q5un versus WDR5-WD40–H3Q5ser versus WDR5-WD40–H3Q5his. j, ITC assessments of WDR5-WD40 (left), WDR5-WD40(K259A) (middle) or WDR5-WD40(K259E) (right) peptide binding. k, 3×Flag-tagged WT versus K259A WDR5 chromatin binding in HeLa cells. n = 3 biological replicates per construct. Statistical analysis was performed using unpaired Student’s t-tests (t4 = 4.163); *P = 0.0141. l, Quantitative PCR (qPCR) analysis (right) of H3Q5his-enriched target genes in HeLa cells (IGV browser tracks of H3Q5his versus IgG enrichment at H3Q5his targets are shown on the left) after transfection with WDR5 WT versus WDR5(K259A). Statistical analysis was performed using unpaired Student’s t-tests for each gene (MYC: **P = 0.0052, t10 = 3.559, n = 6 per group; HOXB6: **P = 0.0098, t10 = 3.182, n = 6 per group; TP53: *P = 0.0186, t16 = 2.620, n = 9 biological replicates per group). Uncropped blots are shown in Supplementary Fig. 1. Data are mean ± s.e.m. a.u., arbitrary units, all normalized to the respective controls.
