Fig 1: Loss and gain of EZH1 expression impairs neuronal differentiation during the chick embryo neural tube development.a Images of immunostained transverse neural tube sections showing EZH1 enrichment in the mantle zone (MZ) neurons of HH23 and HH30 chick embryos. Dashed lines highlight ventricular zones (VZ) which are comprised by neural progenitor cells. Note that HH12 neural tubes are exclusively formed by neural progenitor cells. Images represent immunofluorescence results obtained from at least two embryos. b Diagram summarizing the in ovo neural tube electroporation procedure used to introduce DNA plasmids encoding indicated shRNA or cDNAs into neural progenitor cells of the HH14 chick embryos neural tubes. During 48 h after the electroporation, neural progenitor cells undergo proliferative divisions that produce more neural progenitors in the VZ and neurogenic divisions that generate neurons that delaminate to the MZ. Electroporated (EP) cells co-express EGFP and are shown in green. c, Images of transverse neural tube sections immunostained for SOX9 and HuC/D 48 h after the electroporation with DNA plasmids encoding EGFP and either a scramble shRNA (Scrb) or a mix of two EZH1 shRNAs (shEZH1). Right panels show EGFP signal over the VZ area perimeter highlighting predominant VZ localization of shEZH1 electroporated cells. Top graph shows the ratio of EP cells (EGFP+) located in the MZ vs in the VZ. Bottom graph shows HuC/D stained MZ area of the electroporated neural tube sides (EP side) normalized to the non-electroporated side (C side). Data represents the mean ± SD of n = 8 Scrb and n = 7 shEZH1 embryos. Two-sided Mann–Whitney U test. d Images of transverse neural tube sections immunostained for SOX2 and HuC/D 48 h after the electroporation with DNA plasmids encoding EGFP (Ctrl) or EZH1 and EGFP (EZH1). Right panels show EGFP signal over the VZ perimeter highlighting lack of EZH1 electroporated cells in the VZ. Top graph shows the ratio of EP cells (EGFP+) located in the MZ vs in the VZ. Bottom graph shows HuC/D stained MZ area of the EP side normalized to the C side. Data represents the mean ± SD of n = 4 Ctrl and n = 7 EZH1 embryos. ns = non-significant. Two-sided Mann–Whitney U test. Source data are provided as a Source Data file.
Fig 2: Heterozygous missense variants cause EZH1 gain of function leading to hypermethylation of H3K27.a, b Western blot analysis of EZH1, EZH2 (a) and H3K27me3 (b) in ReNcells transiently expressing either wild type or indicated EZH1 missense variants. ACTB or H4 are shown as loading controls. Graph shows mean ± SD of H3K27me3/H4 levels quantified by band densitometry in n = 7 EZH1, n = 7 p.A678G, n = 5 p.Q731E and n = 6 p.L735F independent transductions. ns = non-significant. One-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. c Enrichment plots showing average signal of H3K27me3 in ChIPseq peaks (top) and heatmaps showing normalized H3K27me3 ChIPseq intensities (bottom) ±5 kb around the center of the peak in EZH1 or A678G expressing ReNcells. Plots represent data combined from 3 independent transductions. Two-sided paired t-test of signal in H3K27me3 peaks indicates statistically significant increase of H3K27me3 in A678G (p-value < 2.2e-16). d, e Western blot analysis of EZH1, EZH2 (d), and H3K27me3 (e) in 4-week old neurons derived from hPSCs carrying EZH1 p.A678G or p.Q731E variants in heterozygosity (EZH1+/A678G (+/G), EZH1+/Q731E, (+/E)) or their isogenic controls (EZH1+/+). ACTB or H4 are shown as loading controls. Graph shows mean ± SD of relative H3K27me3/H4 levels quantified by band densitometry in n = 3 independent differentiations. ns = non-significant. Two-sided paired t test. f, g Autoradiography and Coomassie stains of HMT assay reactions using two increasing concentrations of PRC2 complexes and unmethylated nucleosomes (f) or nucleosomes with dimethylated H3K27 (H3K27me2) as substrate (g). Graphs show mean ± SD of relative methylation levels quantified by band densitometry in n = 3 (f) or n = 2 (g) independent assays. ns = non-significant. Two-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons for main variant effect (f). Statistical comparisons are not shown for graph (g) due to small sample size. Source data are provided as a Source Data file.
Fig 3: Forebrain organoids derived from EZH1 LOF and GOF hPSCs show defects in neurogenesis.a Images of 35-day old forebrain cortical organoids immunostained for SOX2. Dashed lines highlight ventricular zones (VZ), which are thicker in EZH1-/- organoids. Graph shows mean ± SEM of VZ thickness in 30 (n = 40 +/+, n = 25 -/- and n = 25+/G), 35 (n = 37 +/+, n = 24 -/- and n = 23 +/G) and 40 (n = 43 +/+, n = 26 -/- and n = 23 +/G) day old organoids collected from two independent batches. ns = non-significant. Two-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. b Images of 60-day old forebrain cortical organoids immunostained for SOX2 and CTIP2 (early-born cortical neuron marker) or SATB2 (late-born cortical neuron marker), showing less neurons in EZH1-/- organoids and more SATB2+ neurons in EZH1+/A678G organoids. Graphs show mean ± SEM of the number of CTIP2+ (left) and SATB2+ cells (right) over SOX2+ cells in n = 13 +/+, n = 10 -/- and n = 12 +/G organoids collected from two independent batches. ns = non-significant. Two-sided unpaired t test with Holm–Sidak post hoc analysis test for multiple comparisons. Source data are provided as a Source Data file.
Fig 4: 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.
Fig 5: Biallelic EZH1 variants cause loss of function.a–c Pedigree and Sanger sequencing showing segregation of variants with the diseases in consanguineous families with (a), 2 affected children (p10-11) harboring the homozygous nonsense EZH1 c.772C>T; p.R258X variant (note that the youngest brother in the family was not included in the study) (b), 4 affected children (P13-16) harboring the homozygous nonsense EZH1 c.1453G>T: p.E485X variant. and (c), a child (P18) from an unrelated family with shared haplotype and EZH1 c.1453G>T: p.E485X variant. “F” indicates father, “M” mother and “U” unaffected family members. d Western blot analysis of EZH1, EZH2 and ACTB showing significant loss of EZH1 in hPSCs harboring the homozygous EZH1 p.E485X variant compared to isogenic controls. Graph shows mean ± SD of relative levels quantified by band densitometry in n = 3 independent clones. Two-sided unpaired t test. e Schematic representation of EZH1 exon 7-13, indicating the location of deletion and splice variants in P12 and the primers used for RT-PCR with green arrowheads (top). RT-PCR results showing undetectable EZH1 exon 10 containing transcripts in P12 lymphoblastoid cells compared to two unrelated wild type lymphoblastoid cells (WT(F) = females; WT(M) = male) and GAPDH as loading control (bottom). f Western blot analysis showing undetectable EZH1 levels and intact EZH2 in P12 cells compared to unrelated wild type (WT) cells. ACTB is shown as loading control. Graphs show mean relative EZH1/ACTB and EZH2/ACTB levels quantified by band densitometry in n = 3 WT cell lines and n = 2 P12 independent clonal cell lines. Statistical comparisons are not shown due to small sample size. Source data are provided as a Source Data file.
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