cis-Regulatory Chromatin Contacts in Neural Cells Reveal Contributions of Genetic Variants to Complex Neurological Disorders

Abstract

47Mutations in gene regulatory elements have been associated with a wide range of complex neurological 48 disorders. However, due to their inherent cell type-specificity and difficulties in characterizing their 49 regulatory targets, our ability to identify causal genetic variants has remained limited. To address these 50 constraints, we perform integrative analysis of chromatin interactions using promoter capture Hi-C 51(pcHi-C), open chromatin regions using ATAC-seq, and transcriptomes using RNA-seq in four 52 functionally distinct neural cell types: iPSC-induced excitatory neurons and lower motor neurons, iPSC-53 derived hippocampal dentate gyrus (DG)-like neurons, and primary astrocytes. We identify hundreds of 54 thousands of long-range cis interactions between promoters and distal promoter-interacting regions 55 (PIRs), enabling us to link regulatory elements to their target genes and reveal putative pathways that are 56 dysregulated in disease. We validate several novel PIRs using CRISPR techniques in human excitatory 57 neurons, demonstrating that CDK5RAP3, STRAP, and DRD2 are transcriptionally regulated by 58 physically linked enhancers. Finally, we show that physical chromatin interactions mediate genetic 59 interactions in autism spectrum disorder (ASD). Our study illustrates how characterizing the 3D 60 epigenome elucidates novel regulatory relationships in the central nervous system (CNS), shedding light 61 on previously unknown functions for noncoding variants in complex neurological disorders. 62 63 the FTO gene have been shown not to regulate FTO, but IRX3 in the brain and both IRX3 and IRX5 in 75 adipocytes, respectively 3,4 . The FTO locus in obesity illustrates the potentially intricate and cell type-76 specific manner in which noncoding variants contribute to disease. However, such well-annotated cases 77 are rare, and we still lack systematic mappings of GWAS SNPs to their regulatory targets, especially in 78 the context of complex neurological disorders. 79 80 Previous epigenomic annotations of the germinal zone (GZ) and cortical and subcortical plates (CP) in 81 the human brain revealed the importance of 3D chromatin structure in gene regulation and disease 5,6 . 82However, these studies utilized complex, heterogeneous tissues, limiting their abilities to interpret gene 83 regulation in a cell type-specific manner. Therefore, charting the landscape of epigenomic regulation in 84 well-characterized, physiologically relevant cell types should offer significant advantages for identifying 85 causal variants, deciphering their functions, and enabling novel therapies for previously intractable 86 diseases. Towards this goal, we used wild type human induced pluripotent stem cells (iPSCs) from the 87 WTC11 line 7 to generate three neuronal cell types: excitatory neurons 8 , hippocampal dentate gyrus 88 (DG)-like neurons 9 , and lower motor neurons 10 . GFAP-positive astrocytes from the gastrulating brains 89 of two individuals were also included for their relevance to human brain development and di...