Fig. 5: EZH1 LOF and GOF variants alter hPSC-derived cortical neuron differentiation.

a EZH1 and EZH2 expression levels (RPKM) during pre- and postnatal cerebral cortex development. Data mined from mRNA-sequencing datasets in the Brainspan. b Images of EdU labeling and TUJ1 immunostainings at day 2 (D2) and 5 (D5) of NPC differentiation into neurons showing more EdU⁺ cells in EZH1^−/−. Graph shows mean ± SEM of proliferation rates relative to D0 from n = 3 independent neuronal differentiations of hPSCs carrying EZH1^+/+(+/+), EZH1^−/− (−/−) or EZH^+/A678G (+/G). ns = non-significant. Two-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. c Representative flow cytometry contour plots of SOX2⁺ and Ki67⁺ cell populations at D0 and D5 of NPC differentiation into neurons. Graph shows mean ± SEM of D5 vs D0 ratio of SOX2⁺Ki67⁺ cell percentages from n = 3 independent differentiations. ns = non-significant. Two-sided paired t test with Holm–Sidak post hoc analysis test for multiple comparisons. d Representative flow cytometry contour plots of HuC/D⁺ and Ki67⁺ cell populations at D0 and D5 of NPC differentiation into neurons. Graph shows mean ± SEM of HuC/D⁺ cell percentage at D5 vs D0, for n = 3 independent differentiations. ns = non-significant. Two-sided paired t test with Holm–Sidak post hoc analysis test for multiple comparisons. Plots illustrating the gating strategy used for flow cytometry analysis in (c, d) is shown in Supplementary Fig. 7a. e Images of TUJ1 immunostaining at day 2 of NPC differentiation into neurons show shorter neurites in EZH1^−/−. Bottom panel shows traces of TUJ1 neurites. Violin plots show neurite length quantifications or n = 353 +/+, n = 228 −/− and n = 179 +/G cells recorded from 3 independent differentiations. One-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. f Volcano plots showing differentially expressed genes in EZH1^−/− (-/-) and EZH1^+/A678G (+/G) compared to EZH1^+/+ (+/+) 2-month-old neurons. Dotted vertical lines mark log2FoldChange = 1 and black dots represent genes with statistically significant changes (padj<0.05). Wald test with Benjamini–Hochberg method for multiple comparison. g Gene set enrichment analysis (GSEA) of neural stem cell, early-born and late-born cortical neuron gene sets, showing enrichment of neural stem cell gene set expression in EZH1^−/− (NES = 3.14, padj = 1E−10) and late-born neuron gene set in EZH1^+/A678G (NES = 0.3, padj = 0.00026) compared to controls (EZH1^+/+). Kolmogorov-Smirnov test with Holm method to adjust for multiple comparisons. Source data are provided as a Source Data file.