Fig 2: ELK1 perturbations and measurements of CHD8 binding to validated ChIP peaks. (a) Schematic depicts experiment outline and the timing of lentivirus-shRNA infection, dox treatment, and ChIP-qPCR experiment. (b) 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.

Fig 3: CHD8 and ELK1 cooperate to regulate chromatin accessibility. (a) Normalized CHD8 signal and ATAC-seq signal in CHD8-KO and the control samples plotted, and ELK1 motif density plotted as a green enrichment plot. Genome-wide ChIP-seq and ATAC-seq signal divided into three groups: sites with exclusive enrichment for ELK1 motif, sites without ELK1 motif, and the control sites, which are the randomly shuffled peak sets from control samples. For each group, we compared the ATAC signal from KO to the control sample. (b) Cross-correlation analysis of ATAC-seq signal with NucleoATAC to measure nucleosome density. The enrichment plot is a subset of the calculated nucleosome density signal taken from TSS with ± ELK1 (ETS) motif (motif occurrence > 1). (c) Violin plots compare normalized and averaged nucleosome density signal at 100 bp region around the position + 1 of TSS with the presence or absence of ELK1 motif. Statistical analysis of distribution comparison is calculated with the Kolmogorov Smirnov test. (d) CHD8 binding signal taken from 100 bp upstream and downstream of transcription start sites (TSS), with the presence or absence of ELK1 motif.

Fig 4: Pull down (IP) assay and detection of direct interaction between CHD8 and ELK1. (a) Two independent experimental replicates for IP of CHD8 and detection of ELK1 with western blot. Experimental replicate #1 shown in Fig. 24a.

Fig 5: 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 for validation of CHD8 peaks in CHD8-KO neurons.