Fig. 4 | Nature Communications

Fig. 4

From: Human evolved regulatory elements modulate genes involved in cortical expansion and neurodevelopmental disease susceptibility

Fig. 4

Functional validation of HAR-interacting genes. ac Left, chromatin interaction map of HARs that interact with GLI2 (a), GLI3 (b), and TBR1 (c). Gene Model is based on Gencode v19 and possible target genes are marked in red; Genomic coordinate for HARs is labeled as HAR; −log10 (P-value), significance of the interaction between HARs and each 10 kb bin, gray dotted line for FDR = 0.01; TAD borders in CP and GZ are shown. Right, targeted binding sites for two guide RNAs (gRNAs). The HAR is located in the active enhancer marks (H3K27ac) in fetal brain. Targeting dCas9-VP64 to HARs in primary human neural progenitor cells (phNPCs) results in an 30–80% increase in the expression level of putative target genes predicted by Hi-C. Normalized expression levels of putative target genes (GLI2 (a), GLI3 (b), and TBR1 (c)) relative to control (Ctrl). n = 6 (Ctrl), 8 (gRNA1), 6 (gRNA2) for GLI2; 7 (Ctrl), 6 (gRNA1 and gRNA2) for GLI3; 5 (Ctrl), 6 (gRNA1 and gRNA2) for TBR1. P-values, one-way ANOVA and post hoc Tukey test. Center, median; box = 1st–3rd quartiles (Q); lower whisker, Q1 − 1.5×interquartile range (IQR); upper whisker, Q3 + 1.5 × IQR

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