Discovery and validation of genetic variation impacting the gene regulatory landscape during human cortical development

The vast majority of common genetic variation underlying risk for neuropsychiatric disorders resides in poorly annotated non-coding regions of the genome and likely impacts the regulation of gene expression. In order to move from a location in the genome associated with risk to a regulatory mechanism, there are several major gaps in knowledge including: (a) the causal variant(s) within the associated locus, (b) the regulatory elements impacted by those causal variant(s), (c) the cell-type(s) and developmental time period(s) at which the causal variants(s) exert their effects, and (d) the gene(s) impacted by those causal variant(s). In this proposal, we will identify genetic influences on two features of chromatin architecture (enhancer histone marks and their 30 interactions) during human cortical development in order to more completely explain regulatory mechanisms leading to risk for neuropsychiatric disorders. In a large population of post-mortem human developing cortical tissue that has previously undergone genome-wide genotyping and transcriptomic profiling, we will utilize a technique that allows us to simultaneously measure enhancer activity and its interaction profile (H3K27ac HiChIP). We will then identify genetic influences on these two features of chromatin and their co-localization with previous and growing neuropsychiatric disorder genome-wide association (GWAS) risk loci. Psychiatric disorder risk variants may exert their regulatory impact by (1) changing enhancers (H3K27ac QTLs or histone acetylation (ha)QTLs) and/or (2) chromatin interaction (interaction-QTLs). This novel class of QTLs will enhance our understanding of the molecular processes underlying human neurodevelopment and how that development is altered in neuropsychiatric disorders. Further, we will conduct two orthogonal methods to validate the impact of the genetic variants and assess their cell-type specificity. We will perform cell-type specific massively parallel reporter assays (MPRA) to validate the functional impact of haQTLs. In this assay, cloned oligos containing the enhancer associated alleles drive expression of barcoded transcripts that can be used to assess regulatory differences and identify causal variants. We will also apply a haplotype-specific chromatin imaging technique to visualize how regulatory variation impacts chromatin interactions in individual nuclei. This technique paints sections of each chromosome with allele-specific oligos in order to visualize and measure the physical interactions of the 0NA molecule. Completing the aims of this proposal will allow us to identify largely complete regulatory mechanisms impacting human brain development and risk for neuropsychiatric disorders.