R01AG077899

Genome-Wide Identification and Characterization of Alzheimer's Disease-Associated Enhancers - Project Summary/Abstract

The sequencing of many tens of thousands of human genomes has revealed a plethora of sequence differences or variants. Most variants appear to be of no or little functional consequence; however, a small fraction of these variants can alter genome regulation and the susceptibility to and prognosis of particular diseases. Our goal is to develop and use a general and efficient approach to identify and characterize Alzheimer's disease-associated variants (ADAVs) that reside in powerful transcription regulatory elements (TREs) called enhancers, which are distributed across vast non-coding regions of the genome and can map considerable distances from the genes that they regulate. The TREs that contain ADAVs are called here ADAV-TREs.

Our unique approach relies on our recent demonstration that divergent transcription of enhancer RNAs (eRNAs; most sensitively detected by our PRO-CAP assay) is the best mark for precisely defining active enhancers genome-wide (generally to 300 bp or less). We focus on identifying ADAV-TREs associated with AD, the most common cause of dementia, using the exquisitely-controlled differentiation of an induced-pluripotent stem cell line, WTC11, to generate highly homogeneous excitatory neurons and microglia, two of the most relevant cell types in AD.

In Aim 1, we use our PRO-CAP assay to identify and delimit all TREs in this pair of CNS cell types. These TREs that overlap ADAVs, either rare variants from whole genome sequencing or common variants from genome-wide association studies, provide a highly enriched set of variants that are likely relevant to genome regulation and a particular disease, i.e., AD.

In Aim 2, we examine enhancer activity of each ADAV-TRE by high-throughput ESTARR-SEQ assays relative to the reference (WT) allele. Additionally, we will assay synthetic mutations in these TREs that target and cripple specific TF motifs, and features of core promoter pairs that direct divergent enhancer RNA (eRNA) transcription, using high-throughput mutagenesis and ESTARR-SEQ assays.

In Aim 3, to characterize genome-wide effects of those ADAV-TREs and synthetic mutations showing the most robust alteration in enhancer activity, we will use CRISPR to introduce these perturbations at native loci into WTC11 cells and induce these to differentiate to relevant CNS cell types. We will then characterize the effects of these perturbations using: 1) PRO-SEQ to measure changes in genome-wide transcription at base-pair resolution; 2) chromatin conformation capture (4C-SEQ) to examine changes in the 3D enhancer-promoter interaction profiles; 3) ChIP-qPCR to measure alterations in transcription factor (TF) and co-activator binding; and 4) phenotypic assays to reveal disease phenotypes.

Our systematic and molecularly-precise analyses will identify TREs that are altered in their regulatory activity and long-range interactions by variants, as well as the TFs and co-activators whose association with TREs are affected. This information will be hypothesis-generating and critical for modeling genome regulation and for dissecting molecular mechanisms of AD.

Cornell University

TO ENCOURAGE BIOMEDICAL, SOCIAL, AND BEHAVIORAL RESEARCH AND RESEARCH TRAINING DIRECTED TOWARD GREATER UNDERSTANDING OF THE AGING PROCESS AND THE DISEASES, SPECIAL PROBLEMS, AND NEEDS OF PEOPLE AS THEY AGE. THE NATIONAL INSTITUTE ON AGING HAS ESTABLISHED PROGRAMS TO PURSUE THESE GOALS. THE DIVISION OF AGING BIOLOGY EMPHASIZES UNDERSTANDING THE BASIC BIOLOGICAL PROCESSES OF AGING. THE DIVISION OF GERIATRICS AND CLINICAL GERONTOLOGY SUPPORTS RESEARCH TO IMPROVE THE ABILITIES OF HEALTH CARE PRACTITIONERS TO RESPOND TO THE DISEASES AND OTHER CLINICAL PROBLEMS OF OLDER PEOPLE. THE DIVISION OF BEHAVIORAL AND SOCIAL RESEARCH SUPPORTS RESEARCH THAT WILL LEAD TO GREATER UNDERSTANDING OF THE SOCIAL, CULTURAL, ECONOMIC AND PSYCHOLOGICAL FACTORS THAT AFFECT BOTH THE PROCESS OF GROWING OLD AND THE PLACE OF OLDER PEOPLE IN SOCIETY. THE DIVISION OF NEUROSCIENCE FOSTERS RESEARCH CONCERNED WITH THE AGE-RELATED CHANGES IN THE NERVOUS SYSTEM AS WELL AS THE RELATED SENSORY, PERCEPTUAL, AND COGNITIVE PROCESSES ASSOCIATED WITH AGING AND HAS A SPECIAL EMPHASIS ON ALZHEIMER'S DISEASE. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM: TO EXPAND AND IMPROVE THE SBIR PROGRAM, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM: TO STIMULATE AND FOSTER SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION.

93.866 - Aging Research

National Institute on Aging (NIA) [HHS - NIH]

Ithaca, New York 148536007 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 298% from $806,365 to $3,208,396.