BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation [4C-seq]. BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation [4C-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-acetic acid for 24 hours.

明确BRD2与其他溴结构域和额外末端结构域（bromodomain and extraterminal motif, BET）蛋白的具体功能及相互关系，是开发BET亚型特异性抑制剂与广谱抑制剂的核心前提。
本研究采用急性蛋白质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。
本研究揭示，BRD2对于TAF3介导的RNA聚合酶II（Pol II）在低水平H3K4me3修饰启动子处的转录起始，以及通过抑制R环（R-loops）实现的Pol II延伸过程均不可或缺。
单基因与双基因敲除实验显示，BRD2与BRD3（而非BRD4）可在不同启动子处冗余且独立地发挥Pol II转录调控功能，并协同结合增强子区域。
值得注意的是，本研究观察到BRD2敲除会改变细胞分化过程中不同基因的表达谱，并在胚胎干细胞（ES细胞）中通过启动子调控实现转录预编程。
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作用，通过精准调控Pol II的转录起始与延伸过程，进而控制细胞状态。
实验整体设计：为探究BRD2在转录起始中的调控作用，本研究完成了ChIA-PET、ChIP-seq、RNA-seq及4C-seq实验。
本研究选用V6.5小鼠胚胎干细胞（mES细胞系）开展所有高通量测序分析。
BRD2降解标签小鼠胚胎干细胞（BRD2-degron mES细胞）先用1 μg/ml多西环素（doxycycline）预处理18小时，随后分别用或不用500 μM吲哚-3-乙酸（indole-3-acetic acid）处理24小时。