a, Peptide dot blot titrations testing the ability of the H3K4me3 antibody to detect the mark in the absence or presence of Q5ser; note that Q5ser does not occlude antibody recognition of the mark. Results confirmed in at least two independent experiments. b, Peptide competition qChIP validation of H3K4me3Q5ser enrichment at two target loci (Actb and Vim) identified via ChIP-seq in RN46A-B14 cells (undifferentiated, n = 1 per group). c, Overlap of differential enrichment events at protein-coding genes for H3K4me3 and H3K4me3Q5ser versus differential gene expression in response to RN46A-B14 cell differentiation indicates positive correlations between H3K4me3Q5ser gene expression in the absence of significant changes in H3K4me3 enrichment (FDR <0.05, FC >1.2 cut-offs applied after adjusting for multiple comparisons. n = 3 independent biological replicates per differentiation state per antibody). d, Recombinant methylation assays (MLL1-complex-mediated) on wild-type H3 versus H3(Q5A), followed by western blot for H3K4me3, indicating that the Q5A mutation does not affect methylation capacity at K4. Direct blue was used to control for protein loading. Results confirmed in at least two independent experiments. e, Odds-ratio analysis (using Fisher’s exact tests) of overlapping genes displaying differential H3K4me3Q5ser enrichment (or not) versus differential gene expression (DEx, n = 5 (H3.3(WT)) versus n = 6 (H3.3(Q5A)) independent biological replicates per virus), FDR <0.05 cut-off applied after adjusting for multiple comparisons) comparing differentiated RN46A-B14 cells expressing either H3.3(WT) or H3.3(Q5A). Insert numbers indicate respective P values for associations, followed by the number of protein-coding genes overlapping per significant category. f, Heat map of RNA-seq data comparing undifferentiated and differentiated (n = 5 H3.3(WT) versus n = 6 H3.3(Q5A)-expressing) RN46A-B14 cells using normalized RNA expression values (averaged between replicates) to generate z-scores for each row. The genes that displayed differential enrichment during differentiation are represented, along with altered gene expression (see Fig. 3), and along with opposing regulation in the context of H3.3(Q5A). These genes were found to significantly enrich for pathways associated with axon guidance signalling via KEGG analysis (see Supplementary Table 28). g, RT–qPCR analysis of candidate gene expression in RN46A-B14 cells (n = 5 DMSO versus n = 6 LDN 27219/Tgm2i; one-tailed Student’s t-test; Iws1: t 9 = 2.559, *P = 0.0154; Sema3e: t 8 = 3.982, **P = 0.0020; Robo1: t 9 = 3.344, **P = 0.0043; Srgap1: t 8 = 3.312, **P = 0.0053; Nrp1: t 9 = 2.452, *P = 0.0183; Zmat3: t 9 = 3.820, **P = 0.0020; Actg1: t 8 = 7.836, ****P < 0.0001; Reln: t 9 = 2.209, *P = 0.0273; Qser1: t 8 = 2.513, *P = 0.0181; Ppp3ca: t 8 = 2.418, *P = 0.0210; Arid5b: t 9 = 1.754, *P = 0.0567; Pten: t 9 = 1.936, *P = 0.0424). Actb was used as a normalization control. h, Immunofluorescence of RN46A-B14 cells infected during differentiation with lentiviruses expressing either wild-type H3.3–HA or H3.3(Q5A)–HA. Results confirmed at least three independent coverslips per viral treatment. Scale bars, 20 μm. i, Neurite outgrowth analysis examining RN46A-B14 cellular length post-differentiation (n = 54 H3.3(WT) versus n = 44 H3.3(Q5A)-expressing cells; two-tailed Student’s t-test, t 96 = 4.664, ****P < 0.0001). Data are mean ± s.e.m.