High-throughput functional characterization of human enhancers

人类增强子的高通量功能表征

• 批准号: 10166068
• 负责人: JOHN T LIS
• 金额: $ 32.95万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-25 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10166068
• 关键词: Architecture; Base Pairing; Binding; Biological Assay; CRISPR/Cas technology; Cell Line; Characteristics; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; DNA Sequence; Data; Deoxyribonuclease I; Development; Disease; Distal; Elements; Enhancers; Environment; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genomics; HSF1; Heat-Shock Response; Histones; Human; Hypersensitivity; K-562; K562 Cells; Kinetics; Maintenance; Measures; Methods; Molecular; Mutate; Mutation; Oncogenes; Pattern; Personal Satisfaction; Production; Regulation; Regulator Genes; Resolution; Role; Site; Structure; Testing; To specify; Variant; base; chromosome conformation capture; design; embryo cell; experimental study; genome-wide; histone modification; in vivo; mutant; novel therapeutics; nutrition; promoter; transcription factor

PROJECT SUMMARY/ABSTRACT Specific enhancers interact with promoters to specify the cellular pattern, timing, and levels of gene expression. Enhancers can reside up to megabases away from their target gene promoters and strongly activate transcription. Aim 1 will characterize active enhancer elements and their relationship to promoter elements in vivo in human K562 (a tier 1 ENCODE cell line) by testing a broad array of Transcription Regulatory Elements (TREs) for their enhancer activity using eSTARR-seq, our modified element-clone-compatible STARR-seq assay. This collection of TREs will be selected based on a variety of criteria established by ENCODE and others. Large numbers of selected TREs can be handled using our new Clone- seq method, and then tested for enhancer activity by eSTARR-seq. For the TREs that have significant enhancer activity, ~10,000 synthetic mutations will be generated that are designed to destroy distinct TF binding motifs found within each enhancer. We will generate mutant clones using our en masse Clone-seq2 method and examine their impact on enhancer activity using eSTARR-seq. These data will be used to understand the underlying molecular architecture and function of enhancers and promoters. Aim 2 generates K562 cell lines using CRISPR/Cas9 that contain critical synthetic enhancer mutations identified in Aim 1. PRO- seq assays can then be used to measure with high sensitivity and resolution the transcription at the variant enhancers as well as all TREs and transcription units genome-wide. This will reveal the role of DNA sequence motifs within native enhancer loci in the regulatory crosstalk with distal gene promoters and enhancers. Circularized Chromosome Conformation Capture (4C) experiments with particular enhancers as the anchor site will provide an unbiased analysis of distal interactions, while targeted ChIP-qPCR experiments will test effects of these mutant enhancers on transcription factor binding and local histone marks at these genomic points of enhancer interaction. Thus, Aim 2 rigorously characterizes mutated enhancers from Aim 1 in their native chromatin environment. Aim 3 characterizes the de novo activation of enhancers, which are known to be triggered by the heat shock activation of HSF1, a master regulator. Because the sequence motif, HSE, to which HSF1 binds is well defined, targeted HSE mutations that cripple the enhancer activity will be made immediately using CRISPR at native loci and the effects on transcription genome-wide can be analyzed directly by PRO-seq. Additional critical motifs in these inducible enhancers will be identified in a less biased way by the more laborious, but high-throughput, eSTARR-seq approach described in Aim 1. Finally, tracking the kinetics with which the structural characteristics of these enhancers form in the minutes following heat shock relative to the induced transcriptional activity as measured by PRO-seq allows assessment of which characteristics (DNase I hypersensitivity, histone modifications, binding of HSF1 and other TFs, and eRNA production) correlate with functional transcription effects on distal promoters and other enhancers.

项目摘要/摘要 特定增强子与启动子相互作用，以指定基因表达的细胞模式，时机和水平。 增强剂可以居住到远离目标基因启动子的巨型蛋白酶，并强烈激活 转录。 AIM 1将表征主动增强子元素及其与启动子元素的关系 人体K562中的体内（一个层1编码单元线）通过测试一系列转录调节元件 （TRE）用于使用Estarr-Seq的增强剂活动，我们的修改元素 - Clone兼容starr-seq 测定。此收集的TRE将根据编码和其他人建立的各种标准选择。 可以使用我们的新clone-seq方法来处理大量选定的TRE，然后对增强剂进行测试 Estarr-Seq的活动。对于具有显着增强剂活性的TRE，约有10,000个合成突变将 生成旨在破坏每个增强子中发现的不同TF结合基序的生成。我们将 使用我们的Ense Clone-Seq2方法生成突变克隆，并检查其对增强剂活动的影响 使用estarr-seq。这些数据将用于了解基本的分子结构和功能 增强剂和促进者。 AIM 2使用包含关键合成的CRISPR/CAS9生成K562细胞系 在AIM 1中鉴定出的增强子突变。然后可以使用高灵敏度测量和 分辨出变体增强子以及所有TRE和转录单元的转录。 这将揭示DNA序列基序在天然增强子基因座中的作用 基因启动子和增强子。循环染色体构象捕获（4C）实验 特定的增强剂，因为锚点将提供对远端相互作用的无偏分析，而目标 CHIP-QPCR实验将测试这些突变体增强子对转录因子结合和局部的影响 组蛋白在增强子相互作用的这些基因组点处。因此，AIM 2严格地表征突变 AIM 1的增强剂在其本地染色质环境中。 AIM 3表征从头激活 增强剂，已知是由主调节器HSF1的热激活激活触发的。因为 HSF1结合的序列基序HSE定义得很好，有针对性的HSE突变使增强子削弱 活动将立即使用天然基因座的CRISPR进行，并且对整个转录基因组的影响可以 直接通过Pro-Seq分析。这些诱导型增强器中的其他关键基序将在较少的 以目标1中描述的更艰辛但高通量的Estarr-seq方法的偏见。最后， 跟踪这些增强剂在接下来的几分钟内形成这些增强剂的结构特征的动力学 相对于通过Pro-Seq测量的诱导转录活性的热休克允许评估哪个 特征（DNase I超敏反应，组蛋白修饰，HSF1和其他TF的结合以及ERNA 生产）与远端启动子和其他增强子的功能转录效应相关。