Sequence determinants of human gene regulatory elements, ATAC-seq experiments. 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. Overall design: 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决定基因的表达位置与时机，但目前尚未明确调控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（massively parallel reporter assays），对序列空间较人类基因组大约100倍的DNA序列的转录活性进行了测定。机器学习模型揭示：转录因子（transcription factors, TFs）通常以加性模式发挥作用，且其调控遵循的规则较弱；增强子通过不涉及特定转录因子-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。增强子自身可分为三类截然不同的类型：经典型增强子、封闭染色质型增强子及染色质依赖型增强子。研究同时发现，仅少数转录因子在细胞中具备强活性，且多数转录因子的活性在不同细胞类型间高度相似。单个转录因子可具备多种基因调控活性，包括染色质开放活性、增强活性、促进活性及转录起始位点（transcription start site, TSS）确定活性——这与"转录因子结合基序是基因表达的唯一基本原子单位"的观点相一致。整体实验设计：针对人类结肠癌细胞系GP5d与肝细胞癌细胞系HepG2，采用转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）分析其染色质开放性；实验设置未处理对照组、转染STARR-seq（SS）文库组以及5-氟尿嘧啶（5-fluorouracil, 5-FU）处理组。