Fig 2: Transcriptome analysis and EB assay for CHD8-KO ESCs.A Heatmap showing representative genes of pluripotency and lineage specification in the RNA-seq analysis of CHD8-KO#1, KO#2 and WT ESCs. B The GSEA showing the cell cycle related genes in WT and CHD8-KO ESCs. C Venn diagram showing the number and P- value of overlap DEGs between KO#1 and KO#2 compared with WT. D, E GO terms of down- and up-regulated genes in CHD8-KO ESCs. F Schematic diagram showing the EB formation assay and bright-field images of WT and CHD8-KO EBs at day 7 (n = 3). G RT-qPCR of selected genes during EB formation (n = 3). The error bars represented the mean ± SD and the significance level was calculated by Student’s t-test (two-tailed, equal variance) (ns means not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 3: CHD8-KO blocks the activation of neural genes and results in derepression of apoptosis-related genes during neuroectoderm differentiation.A Heatmap showing CHD8-KO affected genes during neuroectoderm differentiation. Go terms of these genes (i.e., class I and class II genes) were also shown. B The GSEA showing the expression pattern of apoptosis-related genes in WT and CHD8-KO NPCs. C The PCA correlation map of WT and CHD8-KO in ESCs and NPCs and the GO terms of top PC2 genes. D Western blot analysis showing the PARP1, ROCK1, and P53 protein expression in WT and CHD8-KO NPCs. cPARP1 represents the cleaved PARP1. E The cartoon showing the different roles of CHD8 in ESCs and NPCs.

Fig 4: Depletion of CHD8 in hNSCs significantly affects ASD risk genes.(a) Mean differential expression P values for ASD risk genes from Liu et al. or Willsey et al. bound by CHD8 versus other genes bound by CHD8 but not in the respective ASD risk gene list. The significance of differences between mean differential expression P-values across gene sets was assessed using Wilcoxon rank tests. Note that CHD8 targets in Liu et al. are significantly dysregulated compared with other CHD8 targets in both knockdowns, whereas CHD8 targets in Willsey et al. are significantly dysregulated compared with other targets only in knockdown C. (b) Scatterplot of log2 fold change gene expression values and log2 read counts per million (CPM) for genes strongly dysregulated in hNSCs transfected with shRNA target C, as compared with scrambled control (EdgeR Poisson P value<1.68 × 10-6 and absolute log2 fold change>0.1, and log2(CPM) between 2 and 10). CHD8 is indicated by a red circle. ASD risk genes from Liu et al. are indicated by purple dots.

Fig 5: CHD8 targets in neurodevelopment are enriched for ASD risk genes.(a) Reproducible CHD8-binding sites identified in two biological replicates of hNSC and midfetal human brain. The number of reproducible sites in each tissue and the subset identified in both tissues are indicated in each section of the Venn diagram. The number of ASD risk genes from Liu et al. bound by CHD8 in each subset is noted in parentheses. (b) Histogram showing the results of ASD risk gene label permutations (n=10,000, green bars) assessing enrichment of ASD risk genes reported by Liu et al. within targets of CHD8 shared in hNSCs and midfetal human brain. The observed number of ASD risk genes identified is indicated by a vertical red line. (c) List of ASD risk genes identified by Liu et al. with shared CHD8 binding between hNSCs and midfetal human brain (n=47). (d) Representative ChIP-Seq signal tracks for H3K27ac and CHD8 from hNSCs at the high-confidence ASD gene POGZ. CHD8 peak calls from hNSCs and midfetal human brain are indicated by horizontal bars. CHD8 binding is coincident with strong H3K27ac signal surrounding the transcription start site in hNSCs.