Fig. 1

Integrative analysis of brain enhancers during fetal development. A Various steps taken in the integrative analysis of this study. See text for details. B Functional enrichment analysis using GREAT [17], for DAEs (upper panel, n = 39,709) and nDAEs (lower panel, n = 162,454), determined using whole genome as a background. X-axis reports the − Log10 p value as determined by GREAT. C Venn diagram showing the overlap between DAEs (upper panel) and nDAEs (lower panel) interacting with protein-coding and lincRNA genes in CP (left) and GZ (right). D Venn diagram showing the overlap between interactions of protein-coding and lincRNA genes with nDAEs (left) and DAEs (right), for protein-coding and lincRNA genes in CP (upper panel) and GZ (lower panel). E Box plots showing gene expression levels as determined by RNA-seq, for genes that interact by HiC with DAEs (light gray) or nDAEs (dark gray) in CP (left) and GZ (right), for fetal (red) or adult (blue) brain samples. Boxes are interquartile range (IQR); line is median; and whiskers extend to 1.5 the IQR. PCW, postconceptional week. FPKM, fragments per kilobase of transcript per million mapped reads. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (wilcox.test). Data obtained from: 12 PCW, Yan et al [18]; 15-17 PCW, De la Torre-Ubieta et al [19]; 17 PCW, Roadmap [10]; 81 years, Roadmap [10]; mean of fetal sources is the mean expression of the first three fetal samples. F Box plots showing RNA-seq gene expression for genes interacting with 1, 2, 3, 4, or 5 or more DAEs in CP (left) and GZ (right). Left y-axis shows gene expression (log2 FPKM), right y-axis, and line plot shows the number of genes per DAE group. * p < 0.05; ***p < 0.001; ****p < 0.0001 (wilcox.test). RNA-seq data from Allen human brain atlas [20]. G Bar plot showing the percentage of GFP+ cells in NSCs (blue) and HEK cells (red), from cell transfection experiments with an enhancer reporter plasmid for 22 tested enhancers and an empty plasmid control. Plotted is the percentage of GFP+ in cells co-transfected with an mCherry expressing plasmid, to correct for transfection efficiency. Bars show the average from two independent experiments, with each enhancer tested each in duplicate. Error bars represent standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (one-way ANOVA test followed by multiple comparison test (Fisher’s LSD test)