Genetic variants associated with complex traits often reside in distal regulatory elements like enhancers, which orchestrate precise gene expression in a cell type- and developmental context-specific manner. While extensive maps of candidate enhancers have been generated across human tissues including the brain, the function of these candidate regions in individual cell types, along with the genes they regulate, remain to be elucidated.
To bridge this gap, we conducted a functional assessment of nearly a thousand enhancers in primary human astrocytes—the predominant glial cell type in the brain. Leveraging CRISPR inhibition (CRISPRi) coupled with single-cell RNA-seq (scRNA-seq), we uncovered over 150 enhancer-gene regulatory interactions. Through extensive characterisation of functional enhancers and their target genes, we demonstrate that enhancers control the expression of key physiological processes in astrocytes, as well as genes dysregulated in neurodegenerative disorders, particularly in Alzheimer’s disease (AD). We also demonstrate that regulatory interactions identified through CRISPRi screening in astrocytes are not well captured by eQTL data, thus providing essential complementary functional annotation of non-coding variants. The CRISPRi screening data, coupled with enhancer RNA expression profiling through transient-transcriptome sequencing (TT-seq), also enabled us to investigate the interplay between enhancer transcription and function, as well as the genomic characteristics of functional enhancers. This information uncovers regulatory circuitry in astrocytes, and by providing insights from a primary cell type, contributes toward a broader understanding of gene expression regulation beyond transformed cell lines.
Given the important role of astrocytes in neurodegeneration, neuroinflammation and injury response, our data provides critical insights into the biology of these brain disorders, contributing to the interpretation of non-coding genetic variation, and of gene expression changes identified in patient samples. We demonstrate how CRISPRi screening data links genetic variants identified by GWAS to genes dysregulated in brain tissue from Alzheimer’s disease (AD) patients; we also identify regulatory elements that control AD-associated genes in a cell type specific manner, which in turn represent potential targets for therapeutic manipulation by (epi)genome editing. Overall, our results provide a comprehensive functional investigation of the regulatory landscape of a key brain cell type in brain function and disease.