High-throughput functional characterization of human enhancers

人类增强子的高通量功能表征

• 批准号: 9904754
• 负责人: JOHN T LIS
• 金额: $ 75.05万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904754
• 关键词: Affect; Architecture; Base Pairing; Binding; Binding Sites; Biological Assay; CRISPR/Cas technology; Cell Line; Characteristics; Chromatin; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Computing Methodologies; DNA Sequence; Data; Deoxyribonuclease I; Development; Disease; Distal; Elements; Enhancers; Environment; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Genomic DNA; Genomics; Goals; HSF1; Heat-Shock Response; Histones; Human; Hypersensitivity; K-562; K562 Cells; Kinetics; Knock-in; Lead; Maintenance; Measures; Methods; Molecular; Mutate; Mutation; Oncogenes; Pattern; Personal Satisfaction; Phenotype; Process; Production; RNA; RNA Sequences; Regulation; Regulator Genes; Resolution; Role; Site; Structure; Testing; To specify; Transactivation; Transcriptional Regulation; Transgenic Organisms; Variant; base; biological adaptation to stress; chromatin immunoprecipitation; chromosome conformation capture; design; embryo cell; experimental study; follow-up; genome-wide; high throughput screening; 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. !

项目总结/摘要 特异性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。 表情增强子可以驻留在远离其靶基因启动子的地方， 激活转录。目的1将表征活性增强子元件及其与启动子的关系 通过测试广泛的转录水平， 使用eSTARR-seq，我们的修饰的元件克隆， 相容的STARR-seq测定。将根据各种标准选择此TREs集合 由ENCODE和其他人建立。使用我们的新克隆可以处理大量选定的TREs- seq方法，然后通过eSTARR-seq测试增强子活性。对于具有显著 增强子活性，将产生约10，000个合成突变，这些突变旨在破坏不同的TF 在每个增强子内发现的结合基序。我们将使用我们的基因克隆-seq 2产生突变克隆 方法并使用eSTARR-seq检查它们对增强子活性的影响。这些数据将用于 了解增强子和启动子的基本分子结构和功能。Aim 2生成 使用CRISPR/Cas9的K562细胞系，其含有Aim 1中鉴定的关键合成增强子突变。PRO- 然后可以使用SEQ测定以高灵敏度和分辨率测量在变异体上的转录 增强子以及全基因组的所有TREs和转录单位。这将揭示DNA序列的作用 在与远端基因启动子和增强子的调控串扰中天然增强子基因座内的基序。 用特定增强子作为锚的环化染色体构象捕获（4C）实验 研究中心将提供远端相互作用的无偏倚分析，而靶向ChIP-qPCR实验将检测 这些突变增强子对这些基因组中转录因子结合和局部组蛋白标记的影响 增强子相互作用点。因此，Aim 2严格地表征了来自Aim 1的突变增强子在其功能上的特性。 天然染色质环境。Aim 3描述了增强子的从头激活， 由热休克激活的HSF 1，主调节器触发。因为序列基序HSE， 如果HSF 1结合的基因是明确的，那么将产生削弱增强子活性的靶向HSE突变， 立即在天然基因座使用CRISPR，并且可以分析对全基因组转录的影响。 直接通过PRO-Seq。这些诱导型增强子中的其他关键基序将在偏倚较小的 通过Aim 1中描述的更费力但高通量的eSTARR-seq方法。最后，跟踪 这些增强剂的结构特征在加热后几分钟内形成的动力学 通过PRO-seq测量的相对于诱导的转录活性的休克允许评估 特征（DNA酶I超敏反应、组蛋白修饰、HSF 1和其他TF的结合以及eRNA 产生）与对远端启动子和其它增强子的功能性转录作用相关。 !