Project Summary/Abstract We know that genetics plays an essential part in the etiology of Alzheimer’s Disease (AD), yet we do not fully understand the complex fashion in which the many disease-predisposing genetic loci interact. The overall goal of our proposal is the creation of comprehensive and integrated maps of chromatin accessibility, chromosome folding and transcriptional patterns, delineating regulatory regions in the genomes of a key disease relevant anatomical region of adult brains, in brains from patients with AD. These maps will unmask regional or long- range targets of epigenomic regulatory interactions that may also be of great relevance in patients with the same clinical phenotype. We will use comprehensive and highly-resolving epigenomics assays, many of which were recently developed by us, and novel ways to integrate the data for the first time in AD relevant brain tissues. We will generate comprehensive maps of the spectrum of organization and function of regulatory regions by integrating complementary techniques: single-cell ATAC-Seq (scATAC-Seq) to characterize chromatin openness and HiChIP to characterize long-range folding interactions of sorted neuronal and non-neuronal cells, both of which are coupled to single-cell RNA-Seq and long-read RNA-Seq for expression information, further complemented by information about transcription factors through proteomic analysis of nuclear fractions. These maps will then be combined with coding or non-coding/regulatory variants in the genomic sequence in the AD genetic candidate regions, e.g. coming from recent large GWAS and meta-GWAS efforts, and integrated with AD multi-omics data sets that are based on larger numbers of specimens but were generated with less high-resolving methods (e.g. are not cell type specific, such as those generated by the Rush RADC effort), which will allow us to create and validate reference maps for epigenomic marks and interactions, determine aberrations to the reference state in patient tissue, and connect such aberrations to genetic disease loci as well as assemble such loci into disease pathways (and likely discover additional genetic disease loci in the process). This project will not only greatly expand our understanding of regulatory information encoded in the human genome and its impact on human brain function and neurodegeneration, but also produce bioinformatics tools necessary to analyze the complex data being generated in AD multi-omics projects.