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.

项目概要/摘要 特定的增强子与启动子相互作用以指定基因表达的细胞模式、时间和水平。 增强子可以位于距离其目标基因启动子长达兆碱基的地方并强烈激活 转录。目标 1 将表征活性增强子元件及其与启动子元件的关系 通过测试广泛的转录调控元件，在人类 K562（一级 ENCODE 细胞系）中进行体内实验 (TRE) 使用 eSTARR-seq（我们修改后的元件克隆兼容 STARR-seq）来检测其增强子活性 化验。该 TRE 集合将根据 ENCODE 和其他机构制定的各种标准进行选择。 可以使用我们新的 Cloneseq 方法处理大量选定的 TRE，然后测试增强子 eSTARR-seq 的活性。对于具有显着增强子活性的 TRE，大约 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。我们将 使用我们的 en Masse Clone-seq2 方法生成突变克隆并检查它们对增强子活性的影响 使用 eSTARR-seq。这些数据将用于了解底层的分子结构和功能 的增强子和启动子。目标 2 使用 CRISPR/Cas9 生成 K562 细胞系，其中包含关键的合成材料 目标 1 中鉴定出的增强子突变。然后可以使用 PRO-seq 检测进行高灵敏度测量和 解析变体增强子处的转录以及全基因组范围内的所有 TRE 和转录单元。 这将揭示天然增强子基因座内 DNA 序列基序在与远端增强子的调控串扰中的作用。 基因启动子和增强子。环状染色体构象捕获 (4C) 实验 特定的增强子作为锚定位点将提供远端相互作用的公正分析，同时有针对性 ChIP-qPCR 实验将测试这些突变增强子对转录因子结合和局部的影响 组蛋白标记在这些增强子相互作用的基因组点上。因此，目标 2 严格表征突变 来自 Aim 1 的增强子在其天然染色质环境中。目标 3 的特征是从头激活 增强子，已知是由主调节因子 HSF1 的热休克激活触发的。因为 HSF1 结合的序列基序 HSE 明确，靶向 HSE 突变会削弱增强子 使用 CRISPR 在天然位点立即产生活性，并且对全基因组转录的影响可以 直接通过 PRO-seq 进行分析。这些诱导增强子中的其他关键基序将在更少的时间内被鉴定出来。 目标 1 中描述的更费力但高通量的 eSTARR-seq 方法有偏差。最后， 跟踪这些增强剂的结构特征在几分钟内形成的动力学 与通过 PRO-seq 测量的诱导转录活性相关的热休克可以评估哪些 特征（DNase I 超敏性、组蛋白修饰、HSF1 和其他 TF 的结合以及 eRNA 产生）与远端启动子和其他增强子的功能转录效应相关。