High-throughput functional characterization of human enhancers

项目摘要/摘要 特定的增强子与启动子相互作用，以指定细胞模式、时机和基因表达水平。 增强子可以驻留在远离其目标基因启动子的几兆碱基的地方，并强烈激活 抄写。目标1将表征活性增强子元件及其与启动子元件的关系 通过检测广泛的转录调控元件在人K562(一种一级编码细胞系)中的活体表达 (TRES)使用eSTARR-seq的增强活性，我们的修改后的元件克隆兼容STARR-seq 化验。这个TRE集合将根据ENCODE和其他机构建立的各种标准进行选择。 可以使用我们新的Clone-Seq方法处理大量选定的TRE，然后进行增强剂测试 活动按eSTARR-seq.对于具有显著增强活性的TrE，大约10,000个合成突变将 被设计用来破坏在每个增强子中发现的不同的TF结合基序。我们会 使用我们的整体克隆-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.这些数据将被用来理解潜在的分子结构和功能 促进剂和促进剂。AIM 2使用CRISPR/Cas9建立K562细胞系，该细胞含有关键的合成 在AIM 1中发现的增强子突变。PRO-SEQ分析然后可以用于以高灵敏度和 在全基因组范围内解析变异增强子以及所有tres和转录单位的转录。 这将揭示天然增强子基因座中的DNA序列基序在调控与远端串扰中的作用 基因启动子和增强子。循环染色体构象捕获(4C)实验 作为锚点的特定增强剂将提供对远端相互作用的公正分析，同时具有针对性 芯片定量聚合酶链式反应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。 组蛋白标记在这些基因组点的增强子相互作用。因此，目标2严格地刻画了突变的特征 来自Aim 1的增强剂在其天然染色质环境中。目标3描述了从头开始的激活 增强剂，已知由主调节因子HSF1的热休克激活触发。因为 HSF1结合的序列基序HSE是明确定义的、具有靶向性的HSE突变，它会削弱增强子 将立即使用CRISPR在本地位置进行活性，并对全基因组转录产生影响 由PRO-SEQ直接分析。在这些可诱导的增强子中的其他关键基序将在较少的 目标1中描述的更费力但高吞吐量的eSTARR-SEQ方法的偏向方式。最后， 跟踪这些增强剂的结构特征在接下来的几分钟内形成的动力学 通过PRO-SEQ测量的与诱导转录活性相关的热休克可以评估 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。

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.