BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation [RNA-seq]

Understanding the precise functions and relationship of BRD2 with other bromodomain and extraterminal motif (BET) proteins is central for the application of BET-specific and pan inhibitors. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at low levels of H3K4me3-modified promoters and Pol II elongation by suppressing R-loops. Single and double depletion revealed that BRD2 and BRD3, but not BRD4, redundantly and independently function in Pol II transcription at different promoters and cooperatively occupy enhancers. Interestingly, we found that depletion of BRD2 affects the expression of different genes during differentiation processes, priming with promoter regulation in ES cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state. Overall design: We have finished the ChIA-PET, ChIP-seq, RNA-seq and 4C-seq to investigate the roles of BRD2 in transcription initiation. The V6.5 mouse ES (mES) cell line was used to perform all the high-throughput analysis. BRD2-degron mES cells were pre-treated with 1 µg/ml doxycycline for 18 hours and treated with or without 500 µM indole-3-acetica cid for 24 hours.

准确解析BRD2与其他溴结构域和末端外基序（bromodomain and extraterminal motif, BET）家族蛋白的功能及相互关系，是BET亚型选择性抑制剂与泛BET抑制剂应用的核心前提。
本研究采用急性蛋白降解技术结合定量基因组学与蛋白质组学方法，探究BRD2在转录过程中的核心功能。
本研究发现，BRD2是TAF3介导的RNA聚合酶II（Pol II）在低水平H3K4me3修饰启动子处起始转录，以及通过抑制R环（R-loops）以促进Pol II延伸过程所必需的蛋白。
单基因与双基因敲降实验表明，BRD2与BRD3而非BRD4，可在不同启动子区域冗余且独立地参与Pol II转录过程，并协同结合增强子区域。
有趣的是，本研究观察到，在细胞分化过程中敲降BRD2会影响不同基因的表达，且在胚胎干细胞（embryonic stem cell, ES）中该影响主要通过调控启动子活性实现。
综上，本研究结果表明，BRD2与BRD3在启动子区域存在复杂的相互调控关系，可通过精细调控Pol II的起始与延伸过程，进而控制细胞状态。
实验整体设计：为探究BRD2在转录起始过程中的功能，本研究完成了染色质相互作用分析配对末端标签测序（ChIA-PET）、染色质免疫共沉淀测序（ChIP-seq）、RNA测序（RNA-seq）及染色体构象捕获测序（4C-seq）相关实验。所有高通量测序分析均使用V6.5小鼠胚胎干细胞（mouse ES, mES）系完成。实验中，BRD2-degron mES细胞先用1 μg/ml多西环素预处理18小时，随后分别用或不用500 μM吲哚-3-乙酸处理24小时。