Extended Data Fig. 6: MLL and SETD1 enzymatic assay quantifications.
From: Bidirectional histone monoaminylation dynamics regulate neural rhythmicity
a, LC-MS/MS quantification of H3K4 methylation states (H3K4me0 vs. H3K4me1 vs. H3K4me2; H3K4me3 signal was negligible and was thus omitted) on H3 (1-21) unmodified vs. H3Q5ser vs. H3Q5his peptides, titrating the concentration of MLL1 complex in the system. b–g, Enzymatic quantifications related to Fig. 2c: LC-MS/MS quantification of H3K4 methylation states (H3K4me0 vs. H3K4me1 vs. H3K4me2 vs. H3K4me3) on H3 (1-21) unmodified vs. H3Q5his peptides for (b) MLL1, (c) MLL2, (d) MLL3, (e) MLL4, (f) SETD1A and (g) SETD1B complexes (n = 3 per peptide/complex). Two-way ANOVA (main effects of interaction: MLL1 – p < 0.0001, F3,16 = 105.5; MLL2 – p = 0.0002, F3,16 = 12.29; MLL3 – p < 0.0001, F3,16 = 86.82; MLL4 – p = 0.0003, F3,16 = 11.26; SETD1A – p < 0.0001, F3,16 = 18.86; SETD1B – p < 0.0001, F3,16 = 41.49), Sidak’s MC tests; significant post hoc comparisons are noted [MLL1: **p = 0.0017 (H3K4me0), ****p < 0.0001 (H3K4me1/2); MLL2: **p = 0.0036 (H3K4me0), **p = 0.0014 (H3K4me1); MLL3: ****p < 0.0001 (H3K4me0 and H3K4me1); MLL4: ***p = 0.0001 (H3K4me1); SETD1A: ***p = 0.0006 (H3K4me0), ***p = 0.0001 (H3K4me1); SETD1B: ****p < 0.0001 (H3K4me0/1)]. h, H3 (1-21) unmodified peptide IPs against recombinant core members of the MLL1 complex (ASH2L, RBBP5, DPY30, MLL1SET and, WDR5) demonstrating that only WDR5 interacts with the unmodified H3 tail in monomeric form. i, H3 (1-21) unmodified vs. H3Q5his vs. H3Q5ser peptide IPs (Streptavidin) against the recombinant MLL1 complex, followed by western blotting for WDR5 (n = 3 per peptide). Streptavidin was used to visualize the peptides post-IP, which were used for normalization. One-way ANOVA (p = 0.0014, F2,6 = 24.03), Tukey’s MC test: significant comparisons are noted (H3 unmodified vs. H3Q5ser, p = 0.0065; H3Q5his vs. H3Q5ser, p = 0.0013). #p = 0.0462 indicates a significant difference via an a posteriori unpaired Student’s t-test (t4 = 2.854). Data presented as mean +/– SEM. A.U., arbitrary units, normalized to respective controls (e.g., H3 unmodified peptide). Supplementary Fig. 1 = uncropped blots. Source data provided as a Source Data file.
