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

项目摘要/摘要 对数以万计的人类基因组的测序揭示了过多的序列 差异或变种。大多数变体似乎没有或几乎没有功能后果；然而，一个小的 这些变异中的一部分可以改变基因组调节以及对特定疾病的易感性和预后 疾病。我们的目标是开发和使用一种通用和有效的方法来识别和表征 存在于强大的转录调控元件中的阿尔茨海默病相关变体(ADAV) (TRE)称为增强子，它们分布在基因组的大量非编码区，可以映射 与它们所调控的基因有相当大的距离。含有ADAV的树在这里被称为 ADAV-TRES。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 RNAs(eRNAs；我们的PRO-CAP检测最灵敏)是准确定义活性的最佳标志 全基因组的增强子(一般为300个碱基或更少)。我们专注于识别与以下相关的ADaV-TRES AD，痴呆症最常见的原因，使用了一种微妙控制的区分 诱导多能干细胞系WTC11产生高度均一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，我们使用我们的PRO-CAP分析来识别和界定所有的TRE 在这对中枢神经系统细胞类型中。这些与ADAV重叠的TRE要么是全基因组的稀有变体 测序或来自基因组广谱关联研究的常见变体，提供了一组高度丰富的 可能与基因组调控和一种特定疾病相关的变体，即AD。在目标2中，我们检查 高通量eSTARR-seq法检测每个ADAV-TrE相对于参照物(WT)的增强子活性 等位基因。此外，我们将分析这些TRE中的合成突变，这些突变针对并削弱特定的TF基序， 以及指导发散型增强子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，为了表征病毒的全基因组效应 这些ADaV-TRES和合成突变显示了增强子活性最强劲的变化，我们将使用 CRISPR将这些天然基因座的扰动引入WTC11细胞，并诱导它们分化为 相关中枢神经系统细胞类型。然后，我们将使用：1)PRO-SEQ TO来表征这些扰动的影响 在碱基对分辨率下测量全基因组转录的变化；2)染色质构象捕获 (4C-seq)检测3D增强子-启动子相互作用谱的变化；3)芯片-qPCR检测 转录因子(TF)和共激活子结合的改变；以及4)用于揭示疾病的表型分析 表型。我们的系统和分子精确的分析将识别在它们的 调控活性和变异体的远程相互作用，以及其转录因子和共激活因子 与TRES的关联受到影响。这些信息将产生假设，并对建模至关重要 基因组调控和阿尔茨海默病分子机制研究。

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.