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.

项目摘要/摘要 数以万计的人类基因组的测序揭示了很多序列 差异或变体。大多数变体似乎没有功能后果。但是，一个小 这些变体的一部分可以改变基因组调节以及对特定的敏感性和预后的敏感性 疾病。我们的目标是开发和使用一种一般有效的方法来识别和表征 阿尔茨海默氏症疾病相关变体（ADAV），该变体位于强大的转录调节元件中 （TRE）称为增强剂，它们分布在基因组的巨大非编码区域上，可以映射 与它们调节的基因相当远。包含ADAV的TRE在这里调用 adav-tres。我们独特的方法取决于我们最近的演示，即增强剂的不同转录 RNA（ERNAS；由我们的Pro-CAP测定最敏感的最灵敏的检测）是精确定义活动的最佳标记 增强剂全基因组（通常为300 bp或更少）。我们专注于确定与 AD是痴呆症的最常见原因，使用了一个精确控制的分化 诱导的纯状干细胞系WTC11，以产生高度均匀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AIM 1中，我们使用支持CAP分析来识别和界定所有TRE 在这对CNS细胞类型中。这些与ADAV重叠的TRE，要么来自整个基因组的罕见变体 基因组广泛关联研究的测序或常见变体，提供了一组高度丰富的一组 可能与基因组调节和特定疾病有关的变体，即AD。在AIM 2中，我们检查 通过高通量ESTARR-SEQ分析相对于参考（WT），每个ADAV-tre的增强剂活性 等位基因。此外，我们将在这些靶向和削弱特定TF基序的TRE中测定合成突变， 和核心启动子对的特征，该对直接使用发散增强子RNA（ERNA）转录，使用 高通量诱变和estarr-seq分析。在AIM 3中，以表征全基因组的影响 那些ADAV-TRES和合成突变显示了增强剂活性中最强大的变化，我们将使用 CRISPR将本地基因座的这些扰动引入WTC11细胞中，并诱导它们区分 相关的CNS细胞类型。然后，我们将使用以下方式表征这些扰动的效果：1）Pro-Seq至 测量基本对分辨率下全基因组转录的变化； 2）染色质构象捕获 （4C-seq）检查3D增强器启动器相互作用曲线的变化； 3）芯片QPCR测量 转录因子（TF）和共激活因子结合的改变； 4）表型测定以揭示疾病 表型。我们的系统和分子专门的分析将识别出在其中发生变化的TRE 变体以及TFS和共激活因子的调节活动和远程相互作用 与TRE的关联受到影响。这些信息将是假设生成的，对于建模至关重要 基因组调节和解剖AD的分子机制。