Mapping regulatory elements from transcription initation data

Determining enhancer and promoter activities is important for modeling gene regulation and interpreting the impact of genetic variation on disease. Studying regulatory element activity genome-wide has been challenging, partly due to a lack of benchmarks. Here, we systematically evaluate CAGE for detecting active regulatory elements and present PRIME, a framework for analyzing regulatory elements from transcription initiation data. We assess nuclear RNA CAGE (nucCAGE) versus total RNA CAGE using fine-mapped eQTLs, likely pathogenic variants, and CRISPRi loci. Transcribed loci identified by PRIME on CAGE data show superior performance in detecting active regulatory elements compared to transcription run-on assays, and nucCAGE improves recall. Importantly, nucCAGE maintains the ability of CAGE to study promoter activities at base-pair resolution. These assays and computational tools will aid in understanding gene regulatory circuits and interpreting pathogenic variants. Overall design: Transcription initiation was profiled in 5 human cell lines (i.e., A549, HCT116, GM12878, HepG2, K562) using CAGE. Selected samples were subjected to 1) nuclear extraction by applying osmotic pressure and sheer forces or 2) transcriptional perturbation by applying triptolide. Libraries were prepared from defined numbers of input cells to allow comparability of results and sequenced to acquire strand-specific transcription start site signals to summarize promoter and enhancer activities.

精准鉴定增强子（enhancer）与启动子（promoter）的活性，对于构建基因调控模型、解析遗传变异对疾病的影响具有重要意义。全基因组范围内研究调控元件活性的工作长期面临挑战，其中部分原因在于缺乏标准化基准数据集。本研究系统性评估了CAGE（Cap Analysis of Gene Expression，基因表达帽分析）在检测活性调控元件中的应用，并推出了PRIME——一种基于转录起始数据开展调控元件分析的分析框架。本研究利用精细定位的表达数量性状位点（expression quantitative trait locus, eQTL）、潜在致病变异以及CRISPR干扰（CRISPRi）靶点，对核RNA CAGE（nucCAGE）与总RNA CAGE进行了对比评估。基于CAGE数据通过PRIME鉴定出的转录位点，在检测活性调控元件方面相较于转录延续测定（transcription run-on assay）表现更优，且nucCAGE可提升召回率。尤为关键的是，nucCAGE保留了CAGE在碱基分辨率下解析启动子活性的能力。本研究开发的实验技术与计算工具，将有助于解析基因调控环路、阐释致病变异的致病机制。实验整体设计：本研究采用CAGE技术，对5种人类细胞系（即A549、HCT116、GM12878、HepG2及K562）的转录起始情况进行了谱型分析。选取的样本分别接受两种处理：1）通过渗透压与剪切力进行细胞核提取；2）利用雷公藤内酯醇诱导转录扰动。研究人员通过设定输入细胞的固定数量制备测序文库，以保障实验结果的可比性与可重复性；随后进行高通量测序，获取链特异性的转录起始位点信号，以此总结启动子与增强子的活性。