Coordination of widespread transcription at enhancers and target genes through BRD4 (His-NET-Seq)

Gene transcription by RNA polymerase II (Pol II) is under the control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II which correlates with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcriptional enhancers in human cells. An integrated multi-omics analysis shows that transcription at most enhancers depends on the BET protein BRD4. We provide evidence for a direct BRD4-mediated coupling of Pol II transcription at enhancers and target genes. Specifically, BRD4 links elongation activation at enhancers and genes by maintaining their proximity. These studies reveal that transcription at enhancers and target genes is coordinated at the level of elongation. Overall design: Pol II occupancy profiling using standard NET-seq or HiS-NET-seq data for K562 WT cells and upon BRD4-specific degradation in K562 dTAG-BRD4 cells.

RNA聚合酶II（RNA polymerase II, Pol II）所介导的基因转录，受到启动子及被称为增强子（enhancers）的远端调控元件的调控。增强子自身亦可被Pol II转录，且该转录过程与其调控活性密切相关。目前，增强子转录的调控机制及其与靶基因转录的协同方式仍未明确。本研究开发了一种高灵敏度原生延伸转录本测序法，命名为HiS-NET-seq（High-sensitive Native Elongating Transcript Sequencing），以实现转录研究的高分辨率全景解析，尤其适用于增强子等低转录丰度区域。借助HiS-NET-seq，本研究在人类细胞中发现了全新的转录增强子。整合多组学分析结果表明，绝大多数增强子的转录依赖于BET家族蛋白BRD4。本研究提供了直接证据，证明BRD4可介导增强子与靶基因处Pol II转录的耦合过程。具体而言，BRD4通过维持增强子与靶基因的空间邻近性，将两者的转录延伸激活过程关联起来。本研究揭示，增强子与靶基因的转录可在延伸层面实现协同调控。
实验整体设计：针对K562野生型（WT）细胞，以及经BRD4特异性降解处理的K562 dTAG-BRD4细胞，分别采用标准NET-seq或HiS-NET-seq技术进行RNA聚合酶II占据谱分析。