ABSTRACT Abstract of the funded parent grant: Down syndrome is the most prevalent genetic condition in humans and a major cause of intellectual disability. Although the severity and extent of phenotypes present in Down syndrome partially result from an extra copy of chromosome 21, details regarding the biological mechanisms and the emergence of atypical development are not understood. To address this significant gap in knowledge, we previously created a ‘Developmental Cell Atlas of Down Syndrome’ by using single-cell RNA-sequencing to profile the transcriptomes of over 700,000 cells derived from multiple tissues. This proposal represents our ongoing efforts to characterize the molecular and cellular identity of cellular phenotypes present in the developing brain in Down syndrome. In Aim 1, we will map the Down syndrome cells onto a reference framework of the developing human brain by pooling existing single-cell RNA-sequencing data. In Aim 2, we will use our established human brain functional genomics pipeline to infer cell-type-specific gene regulatory networks altered in Down syndrome. Leveraging existing data, this study will provide critical information about the emergence and regulation of early brain development in Down syndrome that can be used to elucidate the molecular mechanisms underlying early neuropathology and to benchmark model systems for disease relevant neuronal phenotypes. Abstract of the requested supplement: This application is being submitted for PA-20-272 in accordance with NOT-OD-21-076. The goal of this research supplement is to experimentally validate the findings from our cell- type specific gene regulatory network analysis using high resolution chromatin mapping. We propose to use single cell chromatin mapping in control and trisomy 21 developing brain tissue. We will integrate these new data with our existing single-nucleus gene expression data to confirm the gene regulatory networks predicted via our human brain functional genomics pipeline. Successful completion of the supplementary aims will define gene regulatory networks that are perturbed in Down syndrome brain development and form the basis of future studies aimed at elucidating the cellular and molecular consequences that precede clinical phenotypes.

PROJECT NARRATIVE Down syndrome is the most common genetic disorder associated with intellectual disability. Yet the cellular and molecular abnormalities that underlie cognitive impairment have not been deciphered. We will integrate existing single-cell RNA-seq and functional multi-omic datasets derived from human brain to identify the cell- type-specific gene regulatory networks altered in the developing Down syndrome brain.