Sequence determinants of human gene regulatory elements, ATAC-seq experiments

DNA determines where and when genes are expressed, but the full set of sequence determinants that control gene expression is not known. Here, we measured transcriptional activity of DNA sequences that represent ~100 times larger sequence space than the human genome using massively parallel reporter assays. Machine learning models revealed that transcription factors (TFs) act generally in an additive manner with weak grammar, and that enhancers increase expression from a promoter by a mechanism that does not involve specific TF-TF interactions. The enhancers themselves can be classified into three distinct types: classical, closed chromatin and chromatin-dependent enhancers. We also show that few TFs are strongly active in a cell, with most activities similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening, enhancing, promoting and TSS determining activity – consistent with the view that the TF binding motif is the only atomic unit of gene expression. Chromatin accessibility was analyzed by ATAC-seq in human colon cancer cells line, GP5d and hepatocellular carcinoma cell line, HepG2 in control untreated and transfected with STARR-seq (SS) library or treated with 5-fluorouracil (5-FU).

DNA（Deoxyribonucleic Acid）决定了基因表达的时空位置，但目前学界尚未完全明确控制基因表达的全套序列决定因子。本研究借助大规模并行报告基因检测技术（Massively Parallel Reporter Assays），对覆盖规模达人类基因组约100倍的DNA序列空间的转录活性进行了定量测定。机器学习模型分析结果显示，转录因子（Transcription Factors，TFs）普遍以叠加模式发挥调控功能，且其作用规则缺乏严格的序列语法约束；增强子通过不依赖特异性转录因子-转录因子相互作用的机制，提升启动子介导的基因表达水平。增强子自身可被划分为三类截然不同的亚型：经典型增强子、封闭染色质型增强子以及染色质依赖型增强子。本研究同时证实，细胞内仅少数转录因子具有显著调控活性，且多数转录因子的调控活性在不同细胞类型间保持高度相似。单个转录因子可兼具多种基因调控活性，包括染色质开放活性、增强活性、启动活性以及转录起始位点（Transcription Start Site，TSS）决定活性——这与“转录因子结合基序是基因表达的唯一基本功能单位”的学术观点相一致。本研究通过ATAC-seq（转座酶可及性测序，Assay for Transposase-Accessible Chromatin using sequencing）技术，对人结肠癌细胞系GP5d与肝细胞癌细胞系HepG2的染色质可及性展开分析，实验处理分组包括未施加任何干预的空白对照组、转染STARR-seq（Self-transcribing Active Regulatory Region Sequencing，简称SS）文库组，以及经5-氟尿嘧啶（5-Fluorouracil，简称5-FU）处理组。