PROJECT SUMMARY/ABSTRACT Neuropsychiatric disorders often affect our most distinguishing cognitive and social capabilities, which are thought to have developed as a result of the expansion of the human neocortex. The unique mechanisms orchestrating cortical neurogenesis and differentiation in the developing human neocortex forming the basis of this expansion are beginning to be described. However, although genetic variation in non-coding gene-regulatory regions, rather than in protein coding genes, drives these evolutionary changes, the cis gene regulatory elements (GREs), including promoters and enhancers, and the transcription factors (TFs) governing cortical neurogenesis remain to be characterized. To begin to investigate this understudied mechanism, we and others have leveraged next generation sequencing approaches to profile chromatin accessibility and interaction in parallel with gene expression to create GRE maps of varying levels of spatiotemporal specificity. We previously identified thousands of developmentally dynamic GREs and their putative gene targets by contrasting GRE activity in progenitor versus neuron-enriched laminae of mid-gestation human neocortex, and functionally validated the role of select GREs in cortical neurogenesis using primary human neural progenitor cells. Further, we found that human-gained enhancers (HGEs), a subset of GREs more active in the human than the macaque or mouse neocortex, regulate genes enriched in outer radial glia (oRG), a neural progenitor with prominent roles in cortical gyrification. This work supports the hypothesis that human developmentally dynamic GREs and HGEs direct gene expression programs controlling the proliferation and differentiation of progenitor pools key to cortical expansion. In this proposal, we seek to test this hypothesis and move from a tissue- and gene-level resolution atlas to a cellular- and gene isoform-level resolution atlas. We will perform single nucleus ATAC-seq to identify cell-specific GREs and leverage a novel single-cell isoform sequencing (scIso-seq) technology to investigate a previously understudied mechanism of gene regulation – alternative promoter usage. This new atlas will inform our work to functionally define the GREs impacting cortical neurogenesis at scale using CRISPR interference (CRISPRi) libraries containing capture tags enabling simultaneous reading of transcriptome and sgRNA at the single-cell level. Finally, we will define and characterize the TFs directing the balance of proliferation versus differentiation of progenitors. These results will enable us identify the cellular basis of genomic variation causing risk for neuropsychiatric disease, and influencing cognition and brain structure. Together this work will create a robust single cell-resolution functional annotation of non-coding GREs and TFs acting in developing human neocortex and elucidate evolutionary mechanisms driving cortical expansion. Broadly, this work will provide a blueprint fo...