A map of enhancer regions in primary human neural progenitor cells using capture STARR-seq

Genome-wide association studies (GWAS) and expression analyses implicate noncoding regulatory regions as harboring risk factors for psychiatric disease, but functional characterization of these regions remains limited. We performed capture STARR-sequencing of over 78,000 candidate regions to identify active enhancers in primary human neural progenitor cells (phNPCs). We selected candidate regions by integrating data from NPCs, prefrontal cortex, developmental timepoints, and GWAS. Over 8,000 regions demonstrated enhancer activity in the phNPCs, and we linked these regions to over 2,200 predicted target genes. These genes are involved in neuronal and psychiatric disease-associated pathways, including nervous system development, gliogenesis, and developmental delay. We functionally validated a subset of these enhancers using mutation STARR-sequencing and CRISPR deletions, demonstrating the effects of genetic variation on enhancer activity and enhancer deletion on gene expression. Overall, we identified thousands of highly active enhancers and functionally validated a subset of these enhancers, improving our understanding of regulatory networks underlying brain function and disease. Overall design: We used primary human neural progenitor cells (phNPCs) isolated from the fetal brain at 15-18 weeks post-conception. Experiments were done on a phNPC line isolated from a female fetus of Mexican decent. We investigated over 78,000 regions for enhancer activity using capture STARR-sequencing. We performed two replicates of STARR-sequencing for each of two separate candidate panels. We further validated a subset of the active enhancer regions using mutation STARR-sequencing and CRISPR deletions. For the validation experiments, we compared phNPC cells with specific mutations to those without (mutation STARR-seq) and with deleted enhancer regions (CRISPR deletions) to those without the deletion.