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

• 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
• 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' 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

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.

项目概要/摘要 对数以万计的人类基因组进行测序揭示了大量的序列 差异或变体。大多数变体似乎没有或几乎没有功能后果；然而，一个小 这些变异的一部分可以改变基因组调控以及特定疾病的易感性和预后 疾病。我们的目标是开发和使用通用且有效的方法来识别和表征 存在于强大转录调控元件中的阿尔茨海默病相关变异体 (ADaV) （TRE）称为增强子，分布在基因组的广阔非编码区域，可以映射 与它们所调节的基因有相当大的距离。此处调用包含 ADaV 的 TRE ADaV-TRE。我们独特的方法依赖于我们最近的演示，即增强子的不同转录 RNA（eRNA；通过我们的 PRO-cap 检测最灵敏地检测到）是精确定义活性的最佳标记 全基因组增强子（通常为 300 bp 或更少）。我们专注于识别与以下相关的 ADaV-TRE： AD 是痴呆症最常见的原因，利用精确控制的分化 诱导多能干细胞系 WTC11，产生高度同质的兴奋性神经元和小胶质细胞， AD 中最相关的两种细胞类型。在目标 1 中，我们使用 PRO-cap 分析来识别和界定所有 TRE 在这对 CNS 细胞类型中。这些 TRE 与 ADaV 重叠，要么是全基因组的罕见变体 来自全基因组关联研究的测序或常见变异，提供了一组高度丰富的 可能与基因组调控和特定疾病（即 AD）相关的变异。在目标 2 中，我们检查 通过高通量 eSTARR-seq 测定相对于参考 (WT) 检测每个 ADaV-TRE 的增强子活性 等位基因。此外，我们将分析这些 TRE 中针对并削弱特定 TF 基序的合成突变， 以及指导不同增强子 RNA (eRNA) 转录的核心启动子对的特征，使用 高通量诱变和 eSTARR-seq 分析。在目标 3 中，表征 那些 ADaV-TRE 和合成突变显示出增强子活性最强烈的改变，我们将使用 CRISPR 将天然位点的这些扰动引入 WTC11 细胞并诱导它们分化为 相关的中枢神经系统细胞类型。然后，我们将使用以下方法来表征这些扰动的影响：1）PRO-seq 以碱基对分辨率测量全基因组转录的变化； 2) 染色质构象捕获 (4C-seq) 检查 3D 增强子-启动子相互作用谱的变化； 3) ChIP-qPCR 测量 转录因子 (TF) 和辅激活因子结合的改变； 4) 通过表型分析来揭示疾病 表型。我们的系统性和分子精确性分析将识别出发生改变的 TRE 变体以及转录因子和共激活因子的调节活性和远程相互作用 与 TRE 的关联受到影响。这些信息将用于假设生成并对建模至关重要 基因组调控和剖析 AD 的分子机制。