Extended Data Fig. 5: hGN activity-dependent enhancers and promoters.

hGNs from four independent iPSC donors (4G) were cultured in parallel and H3K27ac ChIPseq was performed in unstimulated cultures, at 15 minutes, and two hours after membrane depolarization for each of them in duplicate (technical replicates). Using each of the four genotypes as biological replicates, differential H3K27ac enrichment analysis was performed to identify promoters and enhancers that underwent histone modification changes in response to neuronal depolarization. a. MA-plots representing all 4G H3K27ac peaks identified with those in red having increased significantly in H3K27ac and those in blue having decreased significantly at 15 minutes or 2 hours. b. The total number of reproducible 4G H3K27ac peaks that were called across the four genotypes of hGNs and the subset that were significantly inducible. As with the 1G H3K27ac peaks, 4G H3K27ac peaks are highly represented in the in vivo reference list, and most inducible peaks were inducible at either 15 min or 2 hours but not at both time points. c. Proportions of H3K27ac peaks that intersect a TSS (promoter regions) or do not intersect a TSS (enhancer regions). As with the 1G H3K27ac peaks, 4G 15 minute inducible H3K27ac peaks are significantly enriched for promoter regions. d. As with the 1G H3K27ac peaks, the CREB binding sequence is the most enriched sequence motif within 15 min 4G inducible H3K27ac regions, and the AP-1 motif is the most enriched within 2 hour 4G inducible H3K27ac regions. e. 1G inducible H3K27ac promoter and enhancer regions were cloned into the pGL4.11 (promoter regions) or NUED2 (enhancer regions) plasmids and transfected into embryonic mouse cortical cultures. Luciferase assays were performed to test for these sequences’ ability to drive luciferase reporter gene expression in response to membrane depolarization. 15/15 constructs that show significant reporter expression induction compared to the corresponding empty control plasmid condition are indicated with an asterisk. n = 2–10 independent replicates (neuronal dissection, plasmid transfection, and luminometer reading); box and whisker plot showing median and interquartile range. f. Pearson correlations showing similar levels of H3K27ac induction between hGNs from independent genetic donors of the 4G data set. g. Hierarchical clustering of H3K27ac induction across all 4G samples.