LNCaP treated with iBET

BRD4 belongs to the bromodomain and extraterminal (BET) family of epigenetic reader proteins that bind acetylated histones and regulate gene expression. Pharmacological inhibition of BRD4 by BET inhibitors (BETi) has indicated antitumor activity against multiple cancer types. We define a new function for BRD4 that is distinct from its established role in transcriptional gene regulation. We show that BRD4 is essential for the repair of DNA double-strand breaks (DSBs), and mediates the formation of oncogenic gene rearrangements by engaging the non-homologous end joining (NHEJ) pathway. Mechanistically, genome-wide DNA breaks are associated with enhanced acetylation of histone H4, leading to BRD4 recruitment, and stable establishment of the DNA repair complex. Loss of BRD4 function blocks the recruitment of multiple DNA repair proteins to the chromatin upon DNA damage, and thereby results in defective DNA repair. In support of this, we also show that in clinical tumor samples, BRD4 protein levels are negatively associated with outcome after prostate cancer (PCa) radiation therapy. Thus, in addition to regulating gene-expression, BRD4 is also a central player in the repair of DNA breaks, with significant implications for cancer therapy. Overall design: LNCaP cellline treated iBET at two different doses with duplicates 6 samples including 2 (replicates) LNCaP cell line treated with DMSO, 2 (replicates) LNCaP treated with iBET at 0.5uM, and 2 (replicates) LNCaP treated with iBET at 2uM for 8 hoursBRD4属于溴结构域和额外末端（bromodomain and extraterminal, BET）家族的表观遗传阅读器蛋白，这类蛋白可结合乙酰化组蛋白并调控基因表达。BET抑制剂（BET inhibitor, BETi）对BRD4的药理学抑制已在多种癌症类型中展现出抗肿瘤活性。本研究定义了BRD4区别于其经典转录基因调控功能的全新生物学功能。我们证实，BRD4对DNA双链断裂（DNA double-strand break, DSB）的修复至关重要，并通过激活非同源末端连接（non-homologous end joining, NHEJ）通路介导致癌性基因重排的形成。从机制层面来看，全基因组DNA断裂会伴随组蛋白H4乙酰化水平升高，进而招募BRD4，并稳定DNA修复复合物的组装。BRD4功能缺失会阻断DNA损伤时多种DNA修复蛋白向染色质的招募，最终导致DNA修复缺陷。临床验证结果显示，在前列腺癌（prostate cancer, PCa）患者的临床肿瘤样本中，BRD4蛋白表达水平与放射治疗后的预后呈负相关。综上，除调控基因表达外，BRD4还是DNA断裂修复的核心调控因子，该发现对癌症治疗具有重要的指导价值。整体实验设计：将LNCaP细胞系以两种不同剂量的iBET进行处理并设置生物学重复，共包含6个样本：2个经二甲基亚砜（dimethyl sulfoxide, DMSO）处理的LNCaP细胞重复样本、2个经0.5μM iBET处理的LNCaP细胞重复样本，以及2个经2μM iBET处理的LNCaP细胞重复样本，所有样本的处理时长均为8小时。