Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/enhancer-binding protein beta binding sites

Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of transcription factor bound sites are actively involved in transcriptional regulation, nor are the key genomic features known that discriminate between active and inactive TF binding events. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a potential marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize systematically the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional assay, we performed cis-regulatory element sequencing (CRE-seq) reporter assays. We tested more than 1,000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB) bound sites in two human cell types, HepG2 and K562. We found that CEBPB bound sites that co-occur with RNAP2 were more likely to exhibit enhancer activity in our reporter assay and these active sequences display significantly stronger RNAP2 read enrichment and local enhancer RNA (eRNA) activity compared to inactive sites. CEBPB-bound sites further maintained substantial cell type specificity, indicating that DNA sequence information at such sites can accurately convey cell type-specific regulatory information, supporting a strong role for local DNA sequence in distinguishing active from inactive TF-bound loci. By comparing our CRE-seq results to a comprehensive set of over 1,000 genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity as well as cell type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can discriminate active versus inactive transcription factor bound sites. CRE-seq reporter assay of replicate barcodes from plasmid DNA, transfected plasmid DNA, expressed RNA (cDNA). CEBPB binding sites as well as scrambled control sequences were tested.

转录因子（Transcription factors, TFs）可结合哺乳动物基因组内数千个DNA序列，但此类结合事件大多对基因表达无直接影响。目前学界尚未明确，为何仅少数转录因子结合位点会主动参与转录调控，也未确定区分活跃与非活跃转录因子结合事件的关键基因组特征。近期研究发现，RNA聚合酶II（RNA polymerase II, RNAP2）可在增强子元件处形成启动子远端结合，提示此类相互作用或可作为活跃调控序列的潜在标志物。尽管已有此类相关性分析，但针对这些基因组共占据事件的全面功能验证仍存在空白。为通过功能实验系统表征两类DNA序列的基因调控活性：一类是与RNA聚合酶II共占据的启动子远端转录因子结合事件，另一类是缺乏RNA聚合酶II占据的转录因子结合位点，我们开展了顺式调控元件测序（cis-regulatory element sequencing, CRE-seq）报告基因实验。我们在两种人类细胞系HepG2与K562中，测试了超过1000个启动子远端CCAAT/增强子结合蛋白β（CCAAT/enhancer-binding protein beta, CEBPB）结合位点。实验结果显示，与RNA聚合酶II共占据的CEBPB结合位点在报告基因实验中更易表现出增强子活性；相较于非活跃位点，此类活跃序列的RNA聚合酶II读段富集程度与局部增强子RNA（enhancer RNA, eRNA）活性均显著更高。CEBPB结合位点还表现出显著的细胞类型特异性，表明此类位点的DNA序列信息可精准传递细胞类型特异性调控信息，佐证了局部DNA序列在区分活跃与非活跃转录因子结合位点中的核心作用。通过将CRE-seq实验结果与逾1000个基因组注释的综合数据集进行比对，我们鉴定出多种可有效预测调控元件活性及细胞类型特异性活性的基因组特征。综合来看，本研究的功能实验结果表明，RNA聚合酶II占据可作为区分活跃与非活跃转录因子结合位点的关键基因组标志物。本研究的CRE-seq报告基因实验涵盖了来自质粒DNA、转染后质粒DNA及表达RNA（互补DNA, cDNA）的重复条形码序列，并测试了CEBPB结合位点与随机对照序列。