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。 增强子可以驻留在远离其靶基因启动子的地方， 转录。目的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， 使用eSTARR-seq，我们的修饰的元件克隆相容性STARR-seq， 比色法将根据ENCODE和其他机构制定的各种标准选择该TREs集合。 我们的新克隆测序方法可以处理大量选定的TREs，然后测试增强子 eSTARR-seq的活动。对于具有显著增强子活性的TREs，约10，000个合成突变将产生显著的增强子活性。 被设计为破坏在每个增强子内发现的不同TF结合基序。我们将 使用我们的enclone-seq 2方法产生突变克隆，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潜在的分子结构和功能 增强子和启动子。目的2使用CRISPR/Cas9产生K562细胞系，其含有关键的合成酶。 目的1中鉴定的增强子突变。然后可以使用PRO-seq测定以高灵敏度测量， 在变体增强子以及所有TREs和全基因组转录单位处解析转录。 这将揭示天然增强子基因座内的DNA序列基序在与远端转录因子的调控串扰中的作用。 基因启动子和增强子。环化染色体构象捕获（4C）实验， 作为锚位点的特定增强子将提供远端相互作用的无偏分析，而靶向的增强子将提供远端相互作用的无偏分析。 ChIP-qPCR实验将测试这些突变增强子对转录因子结合和局部表达的影响。 组蛋白标记在这些增强子相互作用的基因组位点上。因此，目标2严格表征突变的 来自Aim 1的增强子在其天然染色质环境中的表达。Aim 3描述了 增强剂，已知其由主调节剂HSF 1的热休克激活触发。因为 与HSF 1结合的序列基序HSE是明确定义的，靶向HSE突变使增强子失效 在天然基因座上使用CRISPR将立即产生活性，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较少的研究中鉴定。 通过目标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随后的几分钟内形成的动力学 相对于通过PRO-seq测量的诱导的转录活性的热休克允许评估 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。