The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

study the role of CHD8 in human neurons, we engineered the CHD8 locus in pluripotent stem cells to produce heterozygous and homozygous conditional knockout (cKO) cells. The heterozygous cKO allele was constructed by surrounding exon four with two loxP sites (Figs. 1a and 2a). Deletion of exon four is predicted to produce a frameshift and early termination mutation. We produced two correctly targeted embryonic stem (ES) cell clones (C1&2) and one correctly targeted induced pluripotent (iPS) cell line (C3, see also Fig. 2b,c and characterization of the subclones in Fig. 2d-f). To generate a homozygous cKO of CHD8, we used CRISPR/Cas9 to introduce an indel mutation in the non-targeted wild-type allele of each of the three heterozygous cKO clones resulting in clones CR1-3 (Figs. 1b and 3a,b). The CR1 clone had a 2 bp deletion, CR2 had a ten bp insertion, and CR3 had a 7 bp deletion at non-conditional allele (Fig. 3c). Immunofluorescence and western blot analysis of neurons differentiated from human ESC clones revealed protein depletion in heterozygous and homozygous cKO cells (Figs. 1c, d and 2g, the full-length blots of technical replicates provided in Fig. 3d-left). Unlike in mouse embryos, lack of CHD8 in human neurons did not impact cell viability, enabling us to characterize the loss of function in differentiated, functional human neurons ( Fig. 3d-right) 28 .

CHD8 knockout neurons exhibit intact basic neuronal and synaptic function. Since single-cell
RNA sequencing data from the middle temporal gyrus of the human adult cortex showed that CHD8 is predominantly expressed in excitatory neurons, we sought to analyze the conditional CHD8 mutations in differentiated excitatory neurons from human pluripotent stem cells (Fig. 4a) 36,37 . To that end, we differentiated conditional cells to excitatory neurons and infected the cells with lentiviral vectors encoding Cre recombinase or ΔCre (inactive enzyme). Electrophysiological characterization of differentiated neurons revealed that intrinsic and active membrane properties of heterozygote and homozygote CHD8-mutant neurons were robust but not altered compared to control neurons ( Fig. 5a,b,e,f). Additionally, mutant neurons formed functional synapses and the frequency and amplitude of spontaneous miniature EPSCs in CHD8 heterozygous cKO cells were not statistically different from control neurons (Fig. 5d). We further found that evoked excitatory postsynaptic currents (EPSCs) were unchanged in heterozygous and homozygous mutant cells (Fig. 5c,g). Thus, loss of CHD8 did not affect the gross intrinsic physiological and synaptic properties using standard electrophysiology in cultured human neurons suggesting that more subtle functional phenotypes may lead to neuronal dysfunction in CHD8-mutant patients' brains.
CHD8 regulates actively transcribed genes. Next, we evaluated the transcriptional effects of CHD8 depletion in neurons. RNA sequencing showed that heterozygous CHD8 mutant cells exhibit only subtle transcriptional changes compared to WT cells, confirming previous reports (Fig. 4b) 26,38 . Homozygous knockout neurons, on the other hand, showed pronounced transcriptional changes, with substantially more downregulated genes than upregulated genes (Fig. 4c). The transcriptional change in genes which were downregulated in both heterozygous and homozygous KO cells was more pronounced than that observed in overlapping upregulated genes (Fig. 4d). These results suggest that CHD8 is a transcriptional activator in human neurons.
Gene ontology (GO) and pathway enrichment analysis of heterozygous and homozygous CHD8 reveals CHD8 regulates ASD genes knockout cell transcriptomes demonstrated that CHD8 uniformly affects genes involved in neuronal function (axon and synapse development) and signaling pathways related to MAPK/ERK signaling (Fig. 4e). Furthermore, disease pathway enrichment and disease-associated Gene Set Enrichment Analysis (GSEA) of DE genes reveal that CHD8 depletion influences gene signatures commonly associated with neurodevelopmental disease (Figs. 6a and 7a-left). Given its genetic association with autism we interrogated the expression changes of established autism-risk genes and indeed found an overrepresentation of these genes among all CHD8-regulated genes (Figs. 6b and 7a-right) 46 .

CHD8 binds at the promoters of actively transcribed genes in neurons.
To map chromatin binding targets of CHD8 in human neurons, we generated a human embryonic stem (ES) cell line endogenously tagged at the C-terminus of the CHD8 gene with the FLAG-HA epitope sequence (Figs. 8a and 9a-d). Western blotting demonstrated that the tagged protein ran at the expected size of CHD8 (Fig. 8b, the full-length blot is provided in Fig. 9e). Next, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) on differentiated human ESC-derived neurons using antibodies for both the HA-tag and the N-terminus of the  26 (Fig. 8c). The binding profiles of CHD8 pulled down by the two antibodies were highly correlated (Pearson r 2 = 0.80, Fig. 10a,b). Weak correlation of ChIP-seq signal with published ChIPseq dataset from neural progenitors suggests that CHD8 binding is cell context-dependent (Pearson r 2 = 0.25) 40 . We found a pronounced enrichment of promoter sequences among CHD8 binding sites (80% of CHD8 peaks) and a survey of various histone marks from the ENCODE repository revealed enrichment of active histone marks at neuronal promoters overlapping CHD8 peaks (Figs. 11a,c and 12a,b).Ontology terms of genes significantly associated with CHD8-bound promoters are related to chromatin biology, transcription, and translation ( Fig. 10c) 41 . These observations suggest that the genomic binding of CHD8 is generally related to pathways governing gene regulation.
The ETS motif is enriched among CHD8 binding sites. Motif enrichment analysis showed that CHD8 binding in human neurons is enriched for the ETS and YY1 motifs (Fig. 11b). The odds ratio of ETS motifs enrichment was significantly higher in the strong binding sites of CHD8 than weaker binding sites (Fig. 12c) 44 . Indeed, motif discovery analysis across multiple databases revealed that the top 30 enriched motifs within CHD8 binding sites were specific to ETS factors (Fig. 12d, ETS motifs highlighted in red). Classification of strong CHD8 peaks in neurons revealed a distinct enrichment around the proximal promoters and little binding at distal regulatory and enhancer sites (Fig. 12e) 45 .  rons, we next investigated the functional consequences on local chromatin and transcription at CHD8 target sites in response to CHD8 depletion. RNA-sequencing between control and CHD8 KO cells in the cumulative distribution of CHD8-bound and unbound genes showed that the loss of CHD8 leads to downregulation of its target genes, suggesting that CHD8 primarily acts as a transcriptional activator of its direct target genes (Fig. 13a). The observation that CHD8 binding is stronger at promoters of downregulated genes than of upregulated genes and an overall gene downregulation in CHD8 KO neurons supports this conclusion (Fig. 14a).
The expression analysis of overlapping autism genes revealed that CHD8 is required for their active transcription; therefore, majority of these genes are upregulated in CHD8 KO (Figs. 13b,c and 14b). Intriguingly, a subset of these autism genes that upregulated show distinct correlation of gene expression in the human fetal cortex ( Fig. 13d; group of four genes in Fig. 13c also cluster in Fig. 13d) 37 .
CHD8 promotes chromatin accessibility. Next, we characterized the chromatin remodeling activity of CHD8 in human neurons, utilizing Assay of Transposase Accessible Chromatin (ATAC)-seq 50,51 . Differential accessibility analysis of CHD8 heterozygous and homozygous knockout neurons revealed that many more sites lost accessibility (1,481 peaks in homozygous KO) than gained accessibility (106 peaks in homozygous KO) (Figs. 15a,b and 16a). These findings align with our previous results showing that CHD8 acts primarily as a transcriptional activator in neurons. The ontology enrichment analysis for genes with a significantly changed ATAC-peak signal at their promoter vicinity (± 5 Kb) revealed transcriptional and RNA processing pathways (Fig. 15c). Motif enrichment showed significant overrepresentation of CAAT (an RNA polymerase II binding sequence) and the GGAA (the ETS factor motif) at sites that lost accessibility in CHD8 knockout neurons (Fig. 15d). The CAAT-box enrichment commonly found at core promoters, whereas the ETS motif is not specific to promoter regions 52 . Thus, this finding shows CHD8 play distinct role in activating ETS-containing sites, not in general promoter regions. The ATACseq peaks in the heterozygous knockout experiment at sites with moderate change (no statistical significance) also revealed similar results for ETS motif enrichment, suggesting mild but specific effect of CHD8 mutation on chromatin accessibility at ETS motif regions (Fig. 16b,c).
The enrichment of an ETS motif was intriguing since we had already identified it among CHD8 binding targets in neurons. We therefore next investigated chromatin accessibility changes at CHD8 target sites in homozygous knockout neurons (Fig. 17a). Co-analysis of ATAC-seq and CHD8 ChIP-seq sites revealed that CHD8 binding is stronger at regions with increased chromatin accessibility (Fig. 18a). Conversely, in CHD8-KO cells, CHD8 target sites lost accessibility (Fig. 17b,d). Analysis of all overlapping ATAC-seq and CHD8 binding sites showed a significant loss of chromatin accessibility and transcription at CHD8 peak regions and associated target gene promoters (± 5 Kb); ASD-related genes were among the most highly changed genes. (Figs. 17c and 18b). Next, we analyzed CHD8 binding at the promoters of a distinct group of genes with strong differential expression and whose promoters change in chromatin accessibility upon CHD8 knockout. The results revealed that CHD8 binding is enriched at closing promoters in downregulating genes, providing further evidence that CHD8 may promote chromatin accessibility and induce gene expression (Fig. 18c).
CHD8 targets harbor epigenetic signatures denoting actively transcribed genes. We next applied a multivariate hidden Markov model (HMM) to annotate the genome-wide chromatin state of CHD8 target regions using publicly available datasets for chromatin modifications from human H9-derived neurons 53,54 . First, we validated that our model accurately described the expected chromatin state at a group of actively transcribed promoters in neurons (n = 500) (Fig. 19a). Next, we analyzed the enrichment of CHD8 targets, including the sites of CHD8 binding and the ATAC-Seq peaks at annotated genome. Enrichment analysis revealed that CHD8 regulates chromatin accessibility at regions of the genome with an active chromatin state and with no preference for a distinct classification or mapping to a particular genomic annotation (e.g., promoters or enhancers). In contrast, CHD8 binding displayed a strong preference for proximal promoters (Fig. 18d).
CHD8 affects chromatin accessibility ETS motif-containing sites. Next, we wondered whether our observed enrichment of ETS motifs at CHD8 targets may indicate a functional cooperation of ETS motif binding factors and CHD8. To this end, we plotted the ATAC seq peaks at CHD8 target regions with or without ETS  Fig. 1a with the inclusion of the 'screening' primers). Deletion of exon four is predicted to create a frameshift mutation with early truncation. (b) Screening PCR using external primers designed for outside the homology arm towards inside the targeting vector identified two subclones from the hESC line (C1 & C2) and one subclone from the iPSC line (C3) that were positive for the insertion of the targeting vector. (c) Sanger sequencing is spanning the targeting arms' transition into endogenous sequences, demonstrating correct targeting of the construct into the CHD8 locus (clones C1, C2, and C3). (d, e) Excision of exon four after infection with LV-Cre and screening with the primers around the loxP sites (primer 30 and primer 31) resulted in a single band from heterozygous KO compared to two bands in WT cells as expected. (f) Quantitative reverse transcription PCR (RT-qPCR) using the probes for three exons of CHD8 gene shows the levels of mRNA decreases in heterozygous KO neurons. (g) Immunofluorescence analysis of heterozygous KO and WT neurons for Map2 and CHD8. The nuclear staining signal intensity significantly decreases in heterozygous mutant neurons. www.nature.com/scientificreports/ motifs. We observed a much more pronounced loss of ATAC-seq signal at the ETS motif-containing sites than in ETS motif-free regions (Fig. 20a). Combined with ETS motif enrichments in CHD8 binding sites and CHD8dependent ATAC-Seq sites, these results suggested a functional interaction between an ETS factor and CHD8 in regulating chromatin accessibility.
To further characterize ETS motif-dependent CHD8 activity, we implemented a cross-correlation analysis of the ATAC-seq signal to infer nucleosome density at transcriptional start sites (TSS) with ETS motifs but not at TSS lacking ETS motifs (Fig. 20b, c) 51 . We found the altered density of the + 1 nucleosome intriguing, prompting us to investigate the symmetry of CHD8 binding at promoters with and without ETS motifs. Indeed, the average CHD8 ChIP-seq signal (100 bp binned) upstream of promoters of ETS motif-containing genes actively transcribed in neurons was stronger than downstream of the promoters, on the other hand, sites not containing ETS motifs did not show such a relationship (Fig. 20d).

CHD8 is recruited to ETS motif-containing sites by ELK1.
To investigate which ETS factor may functionally interact with CHD8, we first turned to our gene expression data from wild-type neurons. We found that ELK1 is the highest expressed ETS factor in human neurons ( Fig. 21a) 37,55 . Next, we analyzed the expression of ETS factors in the human prefrontal cortex. Clustering analysis of single-cell RNA-seq data from the human prefrontal cortex revealed a correlative gene expression pattern between ELK1 and CHD8 but no other ETS factors (Fig. 21b). Furthermore, immunoprecipitation revealed that endogenous ELK1 co-immunoprecipitates with endogenous CHD8 in human neurons (one experimental replicate shown in Fig. 22a, and two replicates shown in Fig. 23a).
To examine the potential functional cooperativity between ELK1 and CHD8 in targeting chromatin, we sought to investigate whether ELK1 may affect CHD8 binding. To this end, we constructed lentiviral vectors with short hairpin RNA (shRNA) targeting ELK1 and ELF4 as a control. Quantitative qPCR and western blotting confirmed a robust decrease in mRNA and the protein after infecting the neurons with two hairpins against ELK1 and one hairpin against ELF4 (Fig. 24a,b). First, we validated the specificity of our selected ChIP-seq peaks for CHD8 binding by ChIP-qPCR in three independent pull-down experiments, which showed a complete absence of CHD8 binding in KO neurons ( Fig. 25a-c). Next, we measured the binding of CHD8 at a series of CHD8 binding and ETS motif-enriched peak regions. Knockdown of the control factor (ELF4) did not change CHD8 binding at selected peaks (Fig. 22b). In contrast, CHD8 binding was specifically lost at ETS motif-containing CHD8 peaks upon ELK1 knockdown (Fig. 22c).
To further validate these findings, we treated neurons with a peptide inhibitor of ELK1 56,57 , and we observed similar results as we found with the shRNA experiments ( Fig. 25d). Finally, overexpression of ELK1 did not significantly affect CHD8 binding, suggesting that ELK1 recruitment may be saturated or subject to posttranslational regulation such as phosphorylation that could not be captured by over expressing ELK1 (Fig. 25e). These results indicate that CHD8 and ELK1 interact and functionally cooperate in regulating chromatin accessibility and gene expression regulation in human neurons, and ELK1 might be guiding CHD8 to its motif sites at promoters.

Discussion
The chromatin remodeling factor CHD8 is associated with developmental brain disorders, but its molecular function in mature human neurons is largely unknown 58 . To fill this knowledge gap, we report that CHD8 is responsible for maintaining an open chromatin configuration in promoters and overall transcriptional activation in human neurons. Our finding aligns with previous observations that showed CHD8 plays a role in the functional maturation of human oligodendrocyte precursors cells (OPCs) in a mechanism that involves recruiting histone methyltransferase complexes to target gene promoters 59 . We found that CHD8 regulates gene expression at active promoters enriched in the H3K4 methylation mark. Indeed, previous reports showed that CHD8 regulates Pol III-dependent promoters associated with H3K4 methylation 60 .
Our data also revealed functional cooperativity of CHD8 and ELK1 (the effector of MAPK/ERK) in chromatin regulation through a distinct model of directional activity oriented around the ELK1 motif.
Intriguingly, ELK1 plays a role in developing psychiatric disorders affecting the neural circuitry of the adult brain, and it is a prominent therapeutic target for treating depression and addiction 56,61,62 .
Finally, we revealed CHD8 regulates a distinct group of autism genes positively correlated in expression patterns in the developing human cortex, suggesting a conserved and developmentally regulated transcriptional connectivity between CHD8 and its targets. Our results indicate that MAPK/ERK/ELK1 may play a functional Figure 3. Targeting conditional homozygous CHD8 knockout stem cell lines. (a) Introduction of an indel mutation by CRISPR-CAS9 to non-conditional exon 4 of CHD8 gene, to generate a conditional homozygous knock out cells. (b) Validation of the genotype by PCR around the loxP sequence (spanning the gRNA targeting region) and amplification of two bands; one allele is 32 bp smaller than the other allele. Therefore, the top band corresponds to the floxed allele, and the bottom band is the non-conditional allele, a candidate for carrying an indel mutation. Each band is cut and gel-purified, TOPO cloned, and sequenced using M13 forward and M13 reverse primers (CR1, CR2; both hESC and CR3, is an iPSC subclone, confirmed to carry an indel mutation in non-floxed allele). (c) Sanger sequencing of floxed and non-floxed alleles identified three subclones that carry frameshift indel mutations in the non-conditional allele with an un-altered floxed allele. Note that the conditional exon is shown only once as the representative sequence for all three subclones. (d) Western blotting of heterozygous and homozygous CHD8 knockout neurons reveals a decrease in total protein compared to the control condition (shows two independent replicates). The Right is the phase-contrast images of day 5 neurons and shows cultures of KO and control cells are indistinguishable.       66 . In summary, about ten million the HEK293T cells were transfected with the packaging and the lentiviral vectors using Calcium-phosphate precipitation in a 10 cm petri dish. The cells were washed from transfection reagents the next day, and the supernatant was collected approximately 48 h after the transfection.

Production of adeno-associated virus (AAV). Recombinant adeno-associated virus (rAAV-DJ) is used
to deliver the targeting vector to pluripotent stem cells. To produce rAAV, we co-transfected three plasmids: 25 µg of pAAV 67 , 25 µg of a helper plasmid (pAd5), and 20 µg of the capsid (AAV-DJ), into one T75 flask with 80% confluent HEK293T cells (ATCC) by calcium phosphate transfection method 67,68 . Two days after transfection, trypsin harvested cells for 10 min and lysed by three rounds of freeze and thawing in dry ice and a water bath (37 °C). The rAAV virus was collected from the supernatant by spinning the whole lysate and removing the pellet. The virus was aliquoted in small volumes to freeze at − 80 °C. Before using every 100 µl of supernatant, ten units of Benzonase endonuclease (EMD Chemical Inc, Merck 1.01695.002) were added at (37 °C) for 5 min to digest DNA from HEK cells; the capsid protects AAV DNA from digestion.

Generation of human induced excitatory neurons (iN). Human excitatory neurons differentiated
from pluripotent stem cells by over-expression of lineage-specific transcription factor-Neurogenin 2 (Ngn2) as described before 36 . In summary, one day before conversion, we dissociated stem cells into single cells with Accutase (Innovative Cell Technologies) and seeded at ~ 40 K cells into one 24 well plate pre-coated with Matrigel (BD Biosciences) in a medium supplemented with Thiazovivin (5 µM) (STEMCELL Technologies) and doxycycline (2 mg/ml, Clontech). After 6 h, we infected the cells with lentivirus containing Ngn2, RTTA, and Cre recombinase or ΔCre (truncated form of Cre, which is not functional and is used as control). The next day, we replaced the medium with neuronal medium N2/DMEM/F12/NEAA (Invitrogen) containing doxycycline (2 mg/ml, Clontech). We kept the cells in this medium for five days, and on day six, we added ~ 10 K mouse glia cells into each 24 well and replaced the culture medium with a serum-containing medium. We analyzed the cultures approximately 3-5 weeks after induction. To generate homozygous knockout neurons, we infected the neurons with LV-Cre or ΔCre one day after induction of the Ngn2 transcription factor.

Western blotting and immunoprecipitation (IP). Human stem cells and neurons were lysed with
RIPA lysis buffer supplemented with 5 mM EDTA and protease inhibitor (Roche), for 5 min at room temperature and 10 min on ice. After the lysis, sample buffer (4 × Laemmli buffer containing 4% SDS, 10% 2-mecaptaneol, 20% glycerol, 0.004% 4-Bromophenol blue, 0.125 M Tris HCl, pH 6.8) was added, and the samples were either directly loaded on 4-12% SDS-PAGE gel or froze in -80 for further analysis. Approximately 20 to 30 µg of protein was separated on an SDS-PAGE gel for all the immunoblots. Antibodies used in this manuscript used with these dilutions: CHD8 antibody (Rabbit-Behtyl lab-A301-224A) used as 1:4,000, ELK1 (Rabbit, Bethyl lab-A303-529A) used as 1:1,000, HA (Rabbit-Sigma-H6908) used as 1:1,000, β-actin antibody (Rabbit, Abcam-ab8227) used as 1:20,000. Fluorescently labeled secondary antibodies visualized all blots on Odyssey CLx Infrared Imager with Odyssey software (LI-COR Biosciences). We lysed approximately 50 million human neurons grown in glia-free conditions for immunoprecipitation, differentiated from stem cells for ten days. We used five ugr anti-CHD8 antibodies to prepare protein G agarose beads blocked in serum-containing buffer overnight. We incubated cell lysate with an antibody-bound bead for three hours and washed the unbound fraction with house-made washing buffer containing 10% Triton -x-100, 0.5 M EGTA, 0.5 M PH8 EDTA, 5 M NaCl, and 1 M HEPES. We eluted the bound protein with elution buffer at 60 °C. The blots are generally presented as full-length blot, and whenever there is cropping, it is due to cutting the membrane prior to hybridization with antibody. For heavily cropped blots we have provided additional technical replicates as full-length blot.

RNA-sequencing. RNA was obtained from 3 weeks-old cultures of iN cells by adding Trizol LS (Thermo
Fisher Scientific) directly into the cell culture well. Total 500 ng RNA processed for library preparation using "TruSeq" RNA sample preparation-V2 kit and "Ribo-Zero" rRNA removal kit (Illumina) according to manufacturer's instruction. The sequencing ran on Illumina`s NextSeq 550 system with a 1 × 75-bp cycle run.

RNA-seq data analysis.
FastQ files were run on FastQC to obtain high-quality (trimmed and cleaned) reads. The reads were aligned to the human reference genome sequence (hg19) and assembled with TopHat/ Bowtie (version 2.1.1) 69 for transcriptome analysis. Since we generated the library from a mix of mouse and human RNA, the resulting reads were also from a mixture of both species. We, therefore, aligned our reads to the human genome with stringent criteria (zero mismatches allowed). The aligned sequences were randomly sampled and re-aligned to the other species' genome (the mouse, mm9 genome) to ensure that cross-species DNA alignment is not happening. Note that ~ 1% of the reads aligned to human and mouse genomes were discarded from SAM files with SAMtools 70 . The Refseq hg19 GTF file of transcriptome annotation was downloaded from Ensembl (https:// uswest. ensem bl. org/ index. html) and used as a reference annotation file in the TopHat alignment run command to increase the speed and the sensitivity of alignments to splice junctions. Duplicate reads (from PCR step during library preparation) were removed with SAMtools. Pre-built indexes of bowtie were downloaded from the "Bowtie" webpage (http:// bowtie-bio. sourc eforge. net/ tutor ial. shtml). All SAMtools subcommands were used to convert SAM files to BAM files (Bindery Alignment Map). Additionally, SAMtools were    71 . These raw counts were used as input for DESeq2 to perform differential expression analysis and to generate summarizing plots 72 .
The single-cell RNA data of the human brain and the bulk RNA-seq of the developing human cortex were obtained from the Allen Human Brain Atlas, and the Image credit in Fig. 1 (with some modification) is the Allen Institute 37 . We used Seurat V3 with default parameters for each function 73 .

Figure 16. Comparison of ATAC-Seq results between heterozygous CHD8 knockout and WT neurons. (a)
Heatmaps depicts normalized ATAC-seq signal across the overlapping peaks in wild type and heterozygous CHD8 knockout neurons. (b) Boxplots of chromatin accessibility at ETS motif factor sites in wild type and heterozygous CHD8 knockout neurons (ATAC peaks with more than one ETS motifs within each peak included in this analysis). The p-value is calculated with a non-parametric two-sided KS-test to compare the distribution of signals across the ATAC-seq peaks between the samples. (c) The enrichment plot of ATAC-seq signal from wild type and heterozygous CHD8 knockout neurons at selected sites that overlap with CHD8 binding in neurons and also carrying ETS factor motif. www.nature.com/scientificreports/ ChIP-seq and data analysis. ChIP-seq was performed with modifications from a published protocol 74 .
In summary, ten confluent 10 cm plates of iN cells (approximately 10 × 10 6 neurons in total) 10 days after differentiation were used for chromatin extraction. Cultures were crosslinked with 1% Formaldehyde (Sigma) for 10 min at RT. Glycine (125 mM) was added to quench and terminate the cross-linking reaction, and after washing with PBS, cells were scraped off the dishes and collected into a 50 mL tube. DNA samples were subjected to sonication to obtain an average fragment size of 200-600 bp, using Covaris (S220-Focused Ultrasonicator). After sonication, the pellets were cleared from debris by centrifugation at 4 °C. The supernatant was collected for further analysis of DNA fragment size (column-purified DNA ran in 2% agarose gel to determine the size) and for DNA/protein concentration analysis. For input calculation, approximately 0.5% of cross-linked chromatin was separated and saved before the addition of IP antibodies. For immunoprecipitation (IP), 1.5 µg anti-CHD8 The next day, the antibody-bound chromatin was added to protein G and spun 5 h in 4 °C. The immunoprecipitated material was washed, and the IP material was eluted from beads with elution buffer (50 mM NaCl, Tris-HCl; pH 7.5) by vortexing at 37 °C for 30 min. The eluted DNA was separated from beads by spinning. For reverse cross-linking, the IP and input material were incubated at 65 °C/shaking, RNaseA (10 ug/ul), and 5 M NaCl plus proteinase K (20 ug/ul). DNA was purified on a column (Zymo Research) and processed for library preparation. NEBNext ChIP-seq library prep kit was used for library preparation. Sequencing was performed on Illumina`s NextSeq 550 system with a 1 × 75-bp cycle run. We obtained 18 to 20 million total reads per sample in one sequencing run.
ATAC-seq experiment. We followed the Pi-ATAC-seq protocol to transpose homozygous knockout and control neurons 75 . The cells were fixed in culture for 5 min with 1% PFA, detached from the plate with EDTA, and stained for GFP, allowing us to sort the Cre-GFP positive cells. After that, the transposition proceeded as standard ATAC-seq protocol with slight modification (extra step of reverse cross-linking performed overnight in 65C o ). Note that for the heterozygous knockout and wild-type transposition, we followed the original ATACseq protocol in which un-fixed nuclei are permeabilized and subjected to transposition 76 . was used to obtain significant peaks 41,53 . For motif discovery, we used HOMER (v4.10) (http:// homer. ucsd. edu/ homer/). For clustering analysis, we used Cluster 3.0 77 . Heatmaps were generated using the R program: ComplexHeatmap 78 . For ontology analysis, we used DAVID analytical tool 79 . To obtain estimated counts within the region of interest in the ATAC-seq experiment we used FeatureCounts-a general-purpose read count tool from Rsubread package 80 and a custom GTF file with the coordinates of the overlapping ATAC-seq peak in all the samples used as input for the program. For library normalization and differential accessibility analysis, we used DESeq2 72 . Differential accessible sites (opening and closing regions) were manually examined in UCSC Genome Browser with the 2019 update (http:// genome. ucsc. edu). For enrichment analysis and generating normalized heatmaps and signal intensity plots, we used "deepTools, " using normalized bigWig files as input and bin size of 10 for almost all the heatmaps 81 .
NucleoATAC analysis. We employed python package and the below code to obtain nucleosome position and occupancy (occ) from ATAC-seq files: ***** Calling nucleosomes nucleoatac run -bed input-chip.bed -bam corresponding-input-chip.bam -fasta hg19.fa -out occ-out-file ***** To obtain nucleosome position and normalized signal nucleoatac nuc -bed Encode-outout.narrowPeak.gz -vmat out.VMat -bam ATAC.bam -out nucleoatac-nuc-files ***** To convert bedgraph to bigwig file observed in genome browser or generate normalized density plot with the output we employed ./bedGraphToBigWig nucleoatac_signal.smooth.bedgraph hg19.chrom.sizes nucleatac-_signal.smooth.bigwig ChromHMM analysis. We used the ChromHMM algorithm to characterize neuronal chromatin state and the functional chromatin domains at CHD8 targets. We obtained histone mark ChIP-seq data of H9 derived neurons from ENCODE portal 65 . The histone signals are binarized across the genome to build a multivariate hidden Markov model and learn histone modification's combinatorial and spatial pattern at CHD8 target regions 41 . An example code for running one file: ***** Binarizing input file: java -mx1200M -jar ChromHMM.jar BinarizeBed -b 500 -peaks hg19.chromsizes cellmarkfiletable.txt/ output-binarized ***** Learn model  Gene expression of CHD8 and ETS factor analysis in human developing cortex and unsupervised clustering of expression levels (Alan brain data) 37 . ChIP quantitative PCR (ChIP-qPCR) experiment. Total 5-10 ng ChIP DNA and the input were used to perform a quantitative PCR experiment and measure the enrichment levels. All primers used are listed in "ChIP-seq-peaks.xlsx" file (attached to GSE141085), along with the relevant information, including the closest gene and the number of the motif on the peak. Three independent technical replicates (independent IP experiments) were used for qPCR analysis for each peak site. We normalized the ChIP signal over the input signal, less than 0.5% for total IP material. Analysis of qPCR experiment performed on the light Cycler 480II (Roche).

RNA extraction and RT-qPCR experiment for gene expression.
For RT-qPCR and RNA-seq experiments, we applied similar RNA isolation methods: neurons differentiated on mouse glia cells for ~ 3 weeks Figure 22. CHD8 co-immunoprecipitates with ELK1 in human neurons, and its chromatin binding perturbed in the absence of ELK1. (a) Pull-down assay for CHD8 and western blotting of CHD8 and ELK1 in human neurons (also see two other experimental replicates in Fig. 23a). (b) Knockdown of ELF4 does not affect CHD8 binding on either of ETS or YY1 motif sites, suggesting ELF4 does not influence chromatin binding of CHD8. (c) ChIP-qPCR for CHD8 binding after ELK1 knockdown with two different hairpin RNAs (shRNAs). Control condition is an empty vector. The number of ETS motif at each peak region indicated beneath each peak. There was no change in CHD8 binding at sites without the ETS motif. See also Fig. 25c

AAV-mediated gene targeting.
For the generation of conditional CHD8 heterozygous knockout cell line, we designed a donor vector for homologous recombination that carries two homology arms around the exon 4 of the CHD8 gene and included two loxP sequences in the same direction for frameshifting mutation. A positive selection cassette (neomycin expression to confer resistance to Geneticin) was included for purifying clones that carry the integrated donor cassette. The selection cassette contained a splice acceptor (SA) and a sequence for internal ribosomal entry site (IRES) attached to the Neomycin resistance gene (NEO) and a polyadenylation (PA) signal. The NEO resistant clones were used for screening PCR to verify the correct insertion of the targeting vector in the locus (Fig. 2a-c). The PCR primers are designed to cover the region from outside the homology arm (primers # 1 and #4) to inside the cassette. The drug resistance cassette was flanked with FRT sequence and later removed by transient expression of FlpE recombinase. For HA-FLAG tagging of the CHD8 gene, the tags were inserted into the C-terminus region in the frame before the stop codon of Exon 38, together with the Neomycin resistance gene (see Fig. 2a).  The selected subclones from ES and iPS cells were cultured on MEF and expanded to extract genomic DNA and perform screening PCR. The number of picked colonies was around a hundred and fifty, and the number of positive colonies (positive PCR band for screening PCR assay from both sides of the homology arms) was five. We confirmed the correct genotype for three subclones: C1, C2, and C3.  Table of selected CHD8 ChIP-seq peaks for ChIP-qPCR assessment; the number of motifs, peak score, and the location of each peak relative to TSS is shown. (c) ChIP-qPCR experiment to measure CHD8 binding on CHD8 ChIP-seq peaks in CHD8 knockout neurons. This experiment explicitly validates our ChIP-Seq results for CHD8 binding and provides good sites for downstream assays. (d) ChIP-qPCR for CHD8 binding with ELK1 inhibition in wild-type neurons. (e) ChIP-qPCR for CHD8 binding with ELK1 overexpression in CHD8 heterozygous knockout neurons (both red and pink barographs are CHD8 heterozygous knockout). The p-values are calculated with two-tailed T-test; *p < 0.01, **p < 0.001, ***p < 0.0001. www.nature.com/scientificreports/ CRISPR-Cas9 knockout. To generate CHD8 knockout cells, a frameshift mutation is introduced to non floxed allele by Nucleofection of 5 ugr Cas9 plasmid (lentiCRISPR v2-addgene # 52961) and two ugr guide RNA that cover exon four (spacer sequence: tagcaccatcactcctgtag) transfected into five to six million ES cells with the use of nucleofection method. One day after transfection, the cell culture media changed with a cell medium containing puromycin to select the cells that contain the Cas9-Puro lentiviral construct. The resistant cells were left to grow to form colonies and later picked for DNA extraction and Sanger sequencing. The number of selected colonies was fifty-five. The number of positive colonies (indel occurring in the non-floxed allele) was six, and three subclones out of six carried frameshift mutation in the non-floxed allele. We confirmed the correct genotype for three of the subclones: CR1 (2 bp deletion; ES cell line), CR2 (10 bp insertion; ES cell line), and CR3 (7 bp deletion; iPSC line).

Scientific
Electrophysiology. Electrophysiological recordings in cultured iN cells were performed in the wholecell configuration as described previously 36,84 . Patch pipettes were pulled from borosilicate glass capillary tubes (Warner Instruments) using a PC-10 pipette puller (Narishige). The resistance of pipettes filled with intracellular solution varied between 2 and 4 MOhm. The standard bath solution contained (in mM): 140 NaCl, 5 KCl. 2 CaCl 2 , 2 MgCl 2 , 10 HEPES-NaOH pH 7.4, and 10 glucose; 300-305 mosm/l. Excitatory postsynaptic currents (EPSCs) were pharmacologically isolated with picrotoxin (50 µM) and recorded at − 70 mV holding potential in voltage-clamp mode with a pipette solution containing (in mM): 135 CsCl, 10 HEPES-CsOH pH 7.2, 5 EGTA, 4 MgATP, 0.3 Na 4 GTP, and 5 QX-314; 295-300 mosm/l. Evoked EPSCs were triggered by a 0.5-ms current (100 µA) injection through a local extracellular electrode (FHC concentric bipolar electrode, Catalogue number CBAEC75) placed 100-150 µm from the soma of neurons recorded. The frequency, duration, and magnitude of the extracellular stimulus were controlled with a Model 2100 Isolated Pulse Stimulator (A-M Systems, Inc.) synchronized with the Clampex 9 data acquisition software (Molecular Devices). Spontaneous miniature EPSCs (mEPSCs) were monitored in the presence of tetrodotoxin (TTX, 1 µM). The mEPSC events were analyzed with Clampfit 9.02 (Molecular Devices) using the template matching search and a minimum threshold of 5pA, and each event was visually inspected for inclusion or rejection. Intrinsic action potential (AP) firing properties of iN cells were recorded in current-clamp mode using a pipette solution that contained (in mM): 123 K-gluconate, 10 KCl, 7 NaCl, 1 MgCl 2 , 10 HEPES-KOH pH 7.2, 1 EGTA, 0.1 CaCl 2 , 1.5 MgATP, 0.2 Na 4 GTP and 4 glucose; 295-300 mosm/l. First, minimal currents were introduced to hold membrane potential around − 70 mV, next, the increasing amount of currents (from − 10 to + 60 pA, five pA increments) were injected for 1 s in a stepwise manner to elicit action potentials. Input resistance (R in ) was calculated as the slope of the linear fit of the current-voltage plot generated from a series of small subthreshold current injections. To determine whole-cell membrane capacitance, square wave voltage stimulation was used to produce a pair of decaying exponential current transients that were each analyzed using a least-squares fit technique (Clampfit 9.02). Neuronal excitability recordings were performed using a standard bath solution supplemented with 20 µM CNQX, 50 µM AP5, and 50 µM PTX to block all possible glutamatergic (AMPAR-and NMDAR-mediated) as well as GABAergic synaptic transmission. Drugs were applied to the bath solutions before all recordings. Data were digitized at 10 kHz with a 2 kHz low-pass filter using a Multiclamp 700A amplifier (Molecular Devices). For all electrophysiological experiments, the experimenter was blind to the condition/genotype of the cultures analyzed. All experiments were performed at room temperature.
Quantifications and statistical analysis. All data are shown as means + -SEM and from a minimum of three biological replicates (independent differentiations). GraphPad Prism and R were used for statistical analysis and calculations of significance.