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Genome-wide identification and characterization of Alzheimer's Disease-associated enhancers

阿尔茨海默病相关增强子的全基因组鉴定和表征

基本信息

批准号：
10621939
负责人：
Li Gan
金额：
$ 80.61万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10621939
关键词：
3-Dimensional Affect Alleles Alzheimer&apos s Disease Architecture Base Pairing Binding Biological Assay Cell Line Cells Characteristics Chromatin Chromatin Conformation Capture and Sequencing Clustered Regularly Interspaced Short Palindromic Repeats Data Data Set Dementia Development Disease Disease Pathway Disease Progression Distal Elements Enhancers Gene Expression Regulation Gene Mutation Genes Genetic Enhancer Element Genetic Materials Genetic Transcription Genetic Variation Genome Genomics Goals Healthcare Systems Human Genome Maps Measures Microglia Modeling Molecular Mutagenesis Mutation Needles Pathologic Patients Pattern Persons Pharmaceutical Preparations Phenotype Pluripotent Stem Cells Predisposition Prognosis Protocols documentation RNA Regulation Regulator Genes Resolution Sampling Testing Therapeutic Intervention Transcription Coactivator Untranslated RNA Variant aging population cell type chromosome conformation capture disease phenotype excitatory neuron gene regulatory network genome sequencing genome wide association study genome-wide genome-wide analysis genomic locus high throughput screening molecular targeted therapies mutant promoter rare variant synergism targeted treatment transcription factor whole genome

项目摘要

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-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 coactivators 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.

项目总结/文摘