The Bromodomain and Extra-Terminal Domain (BET) Family: Functional Anatomy of BET Paralogous Proteins
溴结构域和末端外域 (BET) 家族：BET 对映体蛋白的功能剖析

4.2. BRD4 Regulates Gene Transcription by Interacting with Acetylated Histones through Its Bromodomains
4.2.BRD4 通过其溴结构域与乙酰化组蛋白相互作用来调控基因转录

Immunoaffinity purification of nuclear extracts from HeLa cells expressing FLAG-tagged and HA-tagged BRD4 with an anti-FLAG or anti-HA antibody results in the precipitation of complexes composed of BRD4, Cdk9, and cyclinT1 [21,22]. This finding demonstrates that BRD4 interacts with Cdk9 and cyclinT1, which constitute the core positive transcription elongation factor b (P-TEFb). Active P-TEFb, which functions as a protein kinase, phosphorylates the RNA Pol II CTM, and results in releasing the Pol II from a pause in transcription elongation in the promoter-proximal region and promotes the efficient progression of Pol II along the gene [68,69,70]. About half of the cellular P-TEFb in HeLa cells also exists in an inactive complex with the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein [71,72]. Immunoblotting of extracts from BRD4 overexpressing HeLa cells with an antibody specific to Ser2 or Ser5 CTM phosphorylation results in a specific increase in Ser2 CTM phosphorylation. Conversely, in HeLa cells that express BRD4-siRNA, Ser2 phosphorylation is substantially reduced. Moreover, BRD4 promotes the activity of a reporter gene driven by the HIV-1 long terminal repeat (LTR) promoter and cellular promoters such as c-Myc and c-Jun in a dose-dependent manner. These results indicate that BRD4 has a positive effect on Ser2 phosphorylation of the RNA Pol II CTM by enhancing the recruitment of P-TEFb [21]. Immunoprecipitation experiments using 293T cells transfected with FLAG-tagged BRD4 deletion mutants reveal that the C-terminal 34 amino acids of BRD4 are crucial for interaction with P-TEFb [40]. BRD4 has also been reported to be an atypical kinase that directly phosphorylates a Ser2 residue in the RNA Pol II CTM [73]. In vitro kinase activity assays have been performed using the following four peptides: recombinant P-TEFb, the CTM of RNA Pol II, a C-terminal segment of 54 amino acid residues in BRD4 that is termed as the P-TEFb interacting domain (PID), and HEXIM1, sequestering P-TEFb into an inactive complex. The result showed that an antagonistic effect of HEXIM1 on P-TEFb-mediated CTM phosphorylation is canceled in the presence of PID, indicating that BRD4 relieves the inhibition of P-TEFb by HEXIM1 rather than phosphorylating P-TEFb [42].
用抗FLAG或抗HA抗体对表达FLAG标记和HA标记BRD4的HeLa细胞的核提取物进行免疫亲和纯化，结果沉淀出由BRD4、Cdk9和cyclinT1组成的复合物[ 21, 22]。这一发现表明，BRD4 与 Cdk9 和 cyclinT1 相互作用，它们构成了核心的正转录延伸因子 b（P-TEFb）。活跃的 P-TEFb 作为一种蛋白激酶，可使 RNA Pol II CTM 磷酸化，从而使 Pol II 从启动子近端区域的转录延伸暂停中释放出来，并促进 Pol II 沿着基因有效地向前推进[68, 69, 70]。在 HeLa 细胞中，大约一半的细胞 P-TEFb 也与 7SK 小核 RNA（snRNA）和 HEXIM1 蛋白以非活性复合物的形式存在 [ 71, 72]。用 Ser2 或 Ser5 CTM 磷酸化特异性抗体对过量表达 BRD4 的 HeLa 细胞提取物进行免疫印迹，结果发现 Ser2 CTM 磷酸化特异性增加。相反，在表达 BRD4-siRNA 的 HeLa 细胞中，Ser2 磷酸化显著降低。此外，BRD4 还能以剂量依赖的方式促进由 HIV-1 长末端重复（LTR）启动子和细胞启动子（如 c-Myc 和 c-Jun）驱动的报告基因的活性。这些结果表明，BRD4 通过增强 P-TEFb 的招募，对 RNA Pol II CTM 的 Ser2 磷酸化有积极作用[ 21]。使用转染了FLAG标记的BRD4缺失突变体的293T细胞进行的免疫沉淀实验表明，BRD4的C端34个氨基酸是与P-TEFb相互作用的关键[40]。另据报道，BRD4 是一种非典型激酶，可直接磷酸化 RNA Pol II CTM 中的 Ser2 残基[73]。体外激酶活性测定使用了以下四种肽：重组 P-TEFb、RNA Pol II 的 CTM、BRD4 中一个由 54 个氨基酸残基组成的 C 端片段（称为 P-TEFb 交互作用域（PID））以及 HEXIM1（将 P-TEFb 封闭在一个非活性复合物中）。结果表明，在PID存在的情况下，HEXIM1对P-TEFb介导的CTM磷酸化的拮抗作用被取消，这表明BRD4缓解了HEXIM1对P-TEFb的抑制作用，而不是使P-TEFb磷酸化[42]。

Mass spectrometry analysis of affinity-purified proteins from cell lysates of 293T cells stably expressing BRD4 Flag-HA-tagged fragments identified NSD3 and JMJD6 as interactors of the BRD4 ET domain [39]. NSD3 belongs to a subfamily of H3K36 methyltransferases [74], and JMJD6 is known to be a histone arginine demethylase [75]. Knockdown of NSD3 or JMJD6 using siRNA in C33A cells decreases the expression of BRD4 target genes by 2- to 3-fold, suggesting that NSD3 and JMJD6 are recruited to the regulated genes in a BRD4-dependent manner [39]. A pull-down assay of nuclear extracts from HEK293T cells with an anti-JMJD6 antibody results in the precipitation of BRD4 complexed with P-TEFb, suggesting that JMJD6 associates with the active form of the P-TEFb complex [24]. Whole genome ChIP-seq with an anti-Pol II antibody shows that knockdown of JMJD6 or BRD4 by siRNA in HEK293T cells significantly increases the Pol II occupancy rate at promoter-proximal regions and dramatically decreases its rate along the gene body, suggesting that association of JMJD6 and BRD4 promotes the Pol II promoter-proximal pause release [24]. Further, genome-wide ChIP-seq analysis with antibodies against several histone markers, JMJD6, and BRD4 identifies JMJD6- and BRD4-associated distal enhancers characterized by H3K4me1 [76], H3Ac, H4Ac, and H3K27Ac markers [24]. In concert, these findings raise the possibility that the next-nearest-neighbor interaction of the P-TEFb complex at the promoter-proximal region with BRD4 and JMJD6 co-bound to the distal enhancer permits the pause release for transcriptional elongation (Figure 3B).
对稳定表达 BRD4 Flag-HA 标记片段的 293T 细胞裂解液中亲和层析蛋白质的质谱分析发现，NSD3 和 JMJD6 与 BRD4 ET 结构域相互作用[39]。NSD3 属于 H3K36 甲基转移酶亚家族[74]，而 JMJD6 是已知的组蛋白精氨酸去甲基化酶[75]。在 C33A 细胞中使用 siRNA 敲除 NSD3 或 JMJD6 会使 BRD4 靶基因的表达量减少 2-3 倍，这表明 NSD3 和 JMJD6 是以依赖 BRD4 的方式被招募到受调控的基因上的[39]。用抗 JMJD6 抗体对 HEK293T 细胞的核提取物进行下拉实验，结果发现与 P-TEFb 复合物结合的 BRD4 会沉淀，这表明 JMJD6 与 P-TEFb 复合物的活性形式结合[24]。用抗Pol II抗体进行的全基因组ChIP-seq分析表明，在HEK293T细胞中用siRNA敲除JMJD6或BRD4会显著提高Pol II在启动子近端区域的占据率，并显著降低其在基因体上的占据率，这表明JMJD6和BRD4的结合促进了Pol II启动子近端暂停释放[24]。此外，利用针对几种组蛋白标记、JMJD6 和 BRD4 的抗体进行的全基因组 ChIP-seq 分析发现，JMJD6 和 BRD4 相关的远端增强子以 H3K4me1[76]、H3Ac、H4Ac 和 H3K27Ac 标记为特征[24]。这些发现共同提出了这样一种可能性，即启动子近端区域的 P-TEFb 复合物与共同结合在远端增强子上的 BRD4 和 JMJD6 的近邻相互作用允许暂停释放以促进转录延伸（图 3B）。

In addition to the role of recruiting P-TEFb to release RNA Pol II from pausing in the promoter-proximal region, BRD4 has been reported to play a role in supporting the progression of Pol II through hyperacetylated nucleosomes by interacting with acetylated histones via its bromodomains [25,77]. Assays with NIH3T3 cells stimulated with interferon (IFN)-β show that IFN-induced gene transcription is markedly suppressed by JQ1, which inhibits BRD4-acetyl histone binding, or by a transfected short hairpin RNA (shRNA) plasmid causing BRD4 knockdown [77]. This result indicates that inducible gene transcription is dependent on interactions between BRD4 and the acetylated lysine residues in histones. Metagene analysis of chromatin-bound nascent RNA-seq reads of serum-responsive genes, activated by serum re-stimulation after starvation in 3T3 cells shows that JQ1 antagonizes the downstream process of transcriptional elongation rather than the typical pause-releasing event occurring in the proximal region of transcription start sites [25]. c-Myc and Klf-4 are BRD4-dependent genes whose transcript levels are reduced upon BRD4 knockdown or JQ1 treatment. In BRD4-knockdown cells, wild type YFP-tagged BRD4 effectively rescues c-Myc and Klf-4 expression, whereas YFP-BRD4 whose bromodomains are mutated does not rescue their expression. Analysis of nascent chromatin-bound RNA-seq reads across the c-Myc and Klf-4 gene loci in NIH3T3 cells reveals that transcript elongation on these genes is clearly inhibited by JQ1. An in vitro transcription assay shows that BRD4 allows Pol II to transcribe through nucleosomes in a histone hyperacetylation dependent manner, and that JQ1 specifically inhibits the activity of BRD4 but not that of the histone chaperone FACT (facilitates chromatin transcription). In BRD4-knockdown cells, the short form of BRD4 lacking the C-terminal P-TEFb interacting domain (BRD4 short) rescues the expression of c-Myc and Klf-4, while the BRD4 short form whose bromodomains are mutated (BRD4 short mBD mutant) does not rescue the expression of these genes. ChIP and quantitative PCR (ChIP-qPCR) assays with an antibody specific for Ser2 CTM phosphorylation in RNA Pol II (Ser2P Pol II) reveal that Ser2P Pol II distribution on the Klf-4 locus is shifted towards and beyond the transcription end site in BRD4-knockdown cells reconstituted with the short BRD4 compared with those reconstituted with the BRD4 short mBD mutant. These observations collectively support the idea that BRD4 facilitates Pol II progression along the gene body independently of P-TEFb as it traverses hyperacetylated nucleosomes by interacting with acetylated histones through its bromodomains [25]. This assistance of BRD4 for Pol II elongation is due to its histone-chaperone activity. Figure 3B illustrates the manner by which BRD4 contributes to gene transcription together with other components.
除了招募 P-TEFb 使 RNA Pol II 从启动子近端区域的停顿中释放出来的作用外，据报道，BRD4 还通过其溴结构域与乙酰化组蛋白相互作用，在支持 Pol II 穿过超乙酰化核小体的过程中发挥作用[ 25, 77]。用干扰素（IFN）-β刺激 NIH3T3 细胞进行的试验表明，抑制 BRD4-乙酰组蛋白结合的 JQ1 或转染短发夹 RNA（shRNA）质粒导致 BRD4 基因敲除，会明显抑制 IFN 诱导的基因转录[77]。这一结果表明，诱导基因转录依赖于 BRD4 与组蛋白中乙酰化赖氨酸残基之间的相互作用。对 3T3 细胞饥饿后血清再刺激激活的血清反应基因的染色质结合新生 RNA-seq 读数进行的元基因分析表明，JQ1 可拮抗转录伸长的下游过程，而不是发生在转录起始位点近端区域的典型暂停释放事件[25]。在BRD4敲除的细胞中，野生型YFP标记的BRD4能有效地挽救c-Myc和Klf-4的表达，而溴域突变的YFP-BRD4不能挽救它们的表达。对 NIH3T3 细胞中 c-Myc 和 Klf-4 基因位点的新生染色质结合 RNA-seq 读数分析表明，这些基因的转录本延伸明显受到 JQ1 的抑制。体外转录试验表明，BRD4允许Pol II以组蛋白超乙酰化依赖的方式通过核小体进行转录，而JQ1能特异性地抑制BRD4的活性，但不能抑制组蛋白伴侣FACT（促进染色质转录）的活性。在BRD4敲除的细胞中，缺少C端P-TEFb相互作用结构域的短型BRD4（BRD4 short）能挽救c-Myc和Klf-4的表达，而溴结构域发生突变的短型BRD4（BRD4 short mBD突变体）不能挽救这些基因的表达。使用 RNA Pol II（Ser2P Pol II）中 Ser2 CTM 磷酸化特异性抗体进行的 ChIP 和定量 PCR（ChIP-qPCR）检测发现，与使用 BRD4 短 mBD 突变体重组的细胞相比，在使用短 BRD4 重组的 BRD4 敲除细胞中，Ser2P Pol II 在 Klf-4 基因座上的分布向转录末端位点和转录末端位点以外移动。这些观察结果共同支持了这样一种观点，即BRD4通过其溴结构域与乙酰化组蛋白相互作用，穿越超乙酰化核小体，从而在P-TEFb之外促进Pol II沿着基因体前进[ 25]。BRD4 协助 Pol II 延长是由于它具有组蛋白伴侣活性。 图 3B 说明了 BRD4 与其他成分共同促进基因转录的方式。

Rapid activation of immediate early genes (IEGs) in response to external signals is critical for the consolidation of synaptic modifications underlying synaptic plasticity and memory formation [78,79]. In cultured neurons isolated from mice, brain-derived neurotrophic factor (BDNF) causes a rapid increase in IEG transcripts. However, both JQ1-treatment and BRD4 knockdown by siRNA block the BDNF-induced increase in IEG expression [34]. These data support the idea that BRD4 recruits P-TEFb to promote Pol II phosphorylation and rapidly allows Pol II to elongate the IEG transcripts. Among the experimental models assessing cognitive functions in mice, the novel object recognition test can be evaluated by differences in the exploration time of novel and familiar objects [80]. If mice remember the previous objects, they will subsequently spend more time with a novel object. JQ1-injected mice showed no preference for novel objects compared with that of control mice, suggesting that JQ1 affects long-term memory [34]. Taken together, these findings indicate that BRD4 is critical for neuronal function and mediates the transcriptional regulation underlying learning and memory in mice. The heat shock response is a set of regulated responses to stress in the cell. The features of the heat shock response are the production of heat shock proteins and a partial inhibition of pre-mRNA splicing [81]. BRD4 has been identified as a regulator of the IFN-stimulated and oxidative stress-induced responses [77,82]. Alternative splicing analysis from RNA splicing data of heat shock-treated cells and heat shock-treated cells with BRD4 knockdown shows a significant increase in splicing inhibition, in particular intron retentions, in BRD4-depleted cells after heat shock, indicating that BRD4 prevents cells from heat stress-induced splicing inhibition [83]. Overall, BRD4 plays a critical role in controlling gene expression in response to external signals.
即时早期基因（IEG）对外界信号的快速激活对于突触可塑性和记忆形成所依赖的突触修饰的巩固至关重要[78, 79]。在从小鼠分离的培养神经元中，脑源性神经营养因子（BDNF）会导致 IEG 转录物的快速增加。然而，JQ1 处理和通过 siRNA 敲除 BRD4 都会阻断 BDNF 诱导的 IEG 表达的增加 [ 34]。这些数据支持这样一种观点，即 BRD4 招募 P-TEFb 以促进 Pol II 磷酸化，并迅速使 Pol II 延长 IEG 转录本。在评估小鼠认知功能的实验模型中，新物体识别测试可通过新物体和熟悉物体探索时间的差异进行评估[80]。如果小鼠记住了以前的物体，它们随后就会在新物体上花费更多时间。与对照组小鼠相比，注射了JQ1的小鼠没有表现出对新奇物体的偏好，这表明JQ1会影响长期记忆[34]。综上所述，这些研究结果表明，BRD4 对神经元功能至关重要，并介导了小鼠学习和记忆的转录调控。热休克反应是对细胞内压力的一系列调节反应。热休克反应的特征是产生热休克蛋白和部分抑制前核糖核酸的剪接[81]。BRD4 已被确定为 IFN 刺激和氧化应激诱导反应的调节因子[77, 82]。根据热休克处理细胞和敲除 BRD4 的热休克处理细胞的 RNA 剪接数据进行的替代剪接分析表明，热休克后去除了 BRD4 的细胞的剪接抑制显著增加，尤其是内含子的保留，这表明 BRD4 可防止细胞受到热应激诱导的剪接抑制[83]。总之，BRD4 在控制基因表达以响应外部信号方面发挥着关键作用。

5.2. BRDT Interacts with Various Proteins and Functions as a Transcriptional Regulator during Spermatogenesis
5.2.BRDT 与多种蛋白质相互作用，在精子发生过程中发挥转录调控作用

Immunoprecipitation of extracts from adult mouse testes with anti-Cdk 9 and anti-cyclin T1 antibodies showed BRDT as a factor binding to both Cdk 9 and cyclin T1, suggesting that BRDT as well as BRD4 is required for the recruitment of P-TEFb, which is known to bind the C-terminal region of BRD4 [27,40]. This finding is also supported by the fact that the C-terminal sequence of BRDT is strikingly similar to that of BRD4 (Figure 1). These data confirmed that BRDT is a true functional tissue-specific paralogue of BRD4. To determine the interacting partners of BRDT, immunoprecipitation of rat testicular nuclear extracts using an anti-BRDT antibody and mass spectrometry analysis of proteins derived from precipitates identified Smarce1 as an interacting partner of BRDT [95]. Smarce1 is a member of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family and a component of the multimeric ATP-dependent chromatin remodeling complexes that regulate subunit interactions and transcriptional activation. A pull-down assay of a FLAG-tagged BRDT deletion mutant and recombinant Smarce1 mixture using an anti-Smarce1 antibody shows that N-terminal deletion interferes with co-immunoprecipitation of BRDT with Smarce1, suggesting that the N-terminal region is necessary for BRDT to associate with Smarce1 [95]. Comparative microarray analysis of RNA from wild-type and Brdt^{∆BDI/∆BDI} mutant round spermatids reveals that, among the genes that are upregulated by BDI deletion, RNA splicing genes are enriched and that over 60% of these splicing genes have transcripts that lack the truncation of their 3′-untranslated region, that is typical of round spermatids [96]. Immunoprecipitation of mouse testicular lysates using an anti-BRDT antibody reveals the co-immunoprecipitation of Srsf2, Ddx5, Hnrnpk, and Tardbp that participate in RNA splicing, suggesting that BRDT complexes with these splicing proteins and functions as a part of the splicing machinery in the testis [96]. Further, BRDT has been reported to repress the expression of the testis-specific histone H1t during spermatogenesis by interacting with the histone deacetylase, HDAC1, the arginine-specific histone methyltransferase 5, PRMT5, and the Tripartite motif-containing 28 protein, TRIM28 [97]. Collectively, these results indicate that BRDT associates with a number of regulatory proteins such as P-TEFb and exerts its functions as a transcriptional activator or repressor during spermatogenesis.
用抗 Cdk 9 和抗细胞周期蛋白 T1 抗体对成年小鼠睾丸提取物进行免疫沉淀显示，BRDT 既是与 Cdk 9 结合的因子，也是与细胞周期蛋白 T1 结合的因子。BRDT 的 C 端序列与 BRD4 的 C 端序列极为相似（图 1），这也支持了上述发现。这些数据证实，BRDT 是 BRD4 的一个真正的功能性组织特异性同源物。为了确定BRDT的相互作用伙伴，使用抗BRDT抗体对大鼠睾丸核提取物进行免疫沉淀，并对沉淀物中的蛋白质进行质谱分析，发现Smarce1是BRDT的相互作用伙伴[95]。Smarce1 是 SWI/SNF（SWItch/蔗糖不发酵）家族的成员，也是多聚 ATP 依赖性染色质重塑复合物的组成成分，该复合物可调节亚基相互作用和转录激活。使用抗Smarce1抗体对FLAG标记的BRDT缺失突变体和重组Smarce1混合物进行的牵引试验表明，N端缺失会干扰BRDT与Smarce1的共免疫沉淀，这表明N端区域是BRDT与Smarce1结合所必需的[95]。对来自野生型和Brdt ^{∆ BDI/∆BDI} 突变圆精子的RNA进行芯片比较分析 ^BDI/∆BDI突变体圆型精子的 RNA 的比较微阵列分析表明，在因 BDI 缺失而上调的基因中，RNA 剪接基因富集，而且这些剪接基因中超过 60% 的转录本缺乏圆型精子典型的 3′-非翻译区截断[96]。使用抗 BRDT 抗体对小鼠睾丸裂解液进行免疫沉淀后，发现参与 RNA 剪接的 Srsf2、Ddx5、Hnrnpk 和 Tardbp 发生了共免疫沉淀，这表明 BRDT 与这些剪接蛋白复合，并作为睾丸中剪接机制的一部分发挥作用[96]。此外，有报道称，在精子发生过程中，BRDT通过与组蛋白去乙酰化酶HDAC1、精氨酸特异性组蛋白甲基转移酶5（PRMT5）和含三方基序的28蛋白TRIM28相互作用，抑制睾丸特异性组蛋白H1t的表达[97]。这些结果综合表明，BRDT与P-TEFb等多种调控蛋白结合，在精子发生过程中发挥转录激活剂或抑制剂的功能。

6.2. BRD2 and BRD4 Interact with Viral Proteins and Contribute to Oncogenesis in Host Cells Infected with Viruses
6.2.BRD2 和 BRD4 与病毒蛋白相互作用，促进受病毒感染的宿主细胞的肿瘤发生

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma (KS), a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma, and some types of multicentric Castleman’s disease. Its latent nuclear antigen (LANA), which is expressed in the nuclei of latently infected cells, mediates episomal replication and persistence of viral genomes [111]. Yeast two-hybrid screening using a carboxy-terminal fragment of LANA fused to the DNA binding domain of GAL4 (bait) and a human leukocyte cDNA library expressing proteins fused to the GAL4 activation domain (prey) identified the BRD2 protein as an activator of reporter gene expression, suggesting that LANA interacts with BRD2 [112]. Further, a pull-down assay of lysates from BCP-1 cells, a KSHV-infected B-cell lymphoma cell line, with an anti-BRD2 antibody results in the immunoprecipitation of both BRD2 and LANA, indicating interaction between BRD2 and LANA in vivo [112]. An immunoprecipitation assay using lysates from HEK 293T cells co-transfected with a series of LANA deletion constructs and a GFP-tagged BRD2 construct shows that deletions within the carboxy terminus prevent LANA from co-immunoprecipitating with an anti-GFP antibody, suggesting that the carboxy-terminal domain of LANA interacts with BRD2 [113]. Pull-down assays of lysates from SF9 insect cells expressing the recombinant LANA protein using glutathione beads coated with a series of GST-tagged BRD2 deletion proteins show that deletions within the ET domain interfere with the binding of BRD2 to LANA, suggesting that the ET domain of BRD2 interacts with LANA [112]. These results indicate that the carboxy-terminal domain of LANA interacts with a region in BRD2 that contains the ET domain. Like the interaction of LANA with BRD2, BRD4 short has been reported to interact directly with an element in the LANA carboxy-terminal domain through its carboxy-terminal region that contains the highly conserved ET domain [114,115]. Immunohistochemical staining of KSHV-infected BCLM cells shows clear colocalization of BRD4 with LANA in punctate speckles, wherein KSHV episomes are colocalized [111] on both mitotic chromosomes and interphase nuclei [114].
卡波西肉瘤相关疱疹病毒（KSHV）会导致卡波西肉瘤（KS）--一种常见于艾滋病患者的癌症，以及原发性渗出淋巴瘤和某些类型的多中心卡斯特曼病。它的潜伏核抗原（LANA）在潜伏感染细胞的细胞核中表达，介导外显子复制和病毒基因组的持续存在[ 111]。使用与 GAL4 DNA 结合域（诱饵）融合的 LANA 羧基末端片段和表达与 GAL4 激活域（猎物）融合的蛋白质的人类白细胞 cDNA 文库进行的酵母双杂交筛选发现，BRD2 蛋白是报告基因表达的激活剂，这表明 LANA 与 BRD2 相互作用[112]。此外，用抗 BRD2 抗体对受 KSHV 感染的 B 细胞淋巴瘤细胞系 BCP-1 细胞的裂解物进行下拉检测，结果发现 BRD2 和 LANA 都被免疫沉淀，表明 BRD2 和 LANA 在体内相互作用[112]。使用与一系列 LANA 缺失构建物和 GFP 标记的 BRD2 构建物共转染的 HEK 293T 细胞裂解物进行的免疫沉淀分析表明，羧基末端的缺失阻止 LANA 与抗 GFP 抗体共免疫沉淀，表明 LANA 的羧基末端结构域与 BRD2 相互作用[113]。使用涂有一系列GST标记的BRD2缺失蛋白的谷胱甘肽珠对表达重组LANA蛋白的SF9昆虫细胞裂解物进行的牵引检测表明，ET结构域内的缺失会干扰BRD2与LANA的结合，这表明BRD2的ET结构域与LANA相互作用[112]。这些结果表明，LANA 的羧基末端结构域与 BRD2 中包含 ET 结构域的区域相互作用。与 LANA 与 BRD2 的相互作用一样，据报道，短 BRD4 也通过其包含高度保守的 ET 结构域的羧基末端区域与 LANA 羧基末端结构域中的一个元件直接相互作用[114, 115]。对受 KSHV 感染的 BCLM 细胞进行免疫组化染色显示，BRD4 与 LANA 在点状斑点中明显共定位，而 KSHV 表位体则在有丝分裂染色体和间期细胞核上共定位[111][114]。

Papillomaviruses constitute a group of small DNA viruses that cause benign tumors in a variety of higher vertebrates. During the latent infection period, viral genomes persist as episomes in the nuclei of proliferating host cells. The papillomavirus genome includes an early region (E) encoding six (E1, E2, E4, E5, E6, and E7) open reading frames (ORF) that are expressed immediately after initial infection of the host cell. The E2 protein, which binds to the viral replication origin cooperatively with the E1 protein, plays a crucial role in long-term episomal maintenance and DNA replication of the viral genome [116,117]. The E6 protein mediates p53 degradation resulting in reduced ability to respond to DNA damage in host cells [118]. Because the E2 regulatory protein suppresses the enhancer and promoter activity that causes E6 and E7 transcription, integration of the papillomavirus DNA into cellular chromosomes in a manner that disrupts the E2 ORF, promotes oncogenesis in host cells [119,120]. The viral proteins need to interact with a variety of cellular proteins to efficiently execute their function.

Cellular E2-interacting proteins have been explored using the bovine papillomavirus (BPV) E2 protein. A proteomic analysis of nuclear lysates from human cervical carcinoma C33 cells transduced with a recombinant retrovirus expressing FLAG-HA-tagged BPV E2 shows that BRD4 is co-immunopurified with anti-FLAG and anti-HA antibodies, suggesting that BRD4 is a member of the cellular interacting partners of BPV E2 [121]. The overlap of E2 and BRD4 immunofluorescence images in E2-transfected C33 cells demonstrates that these two proteins colocalize on mitotic chromosomes [121]. In vitro binding assays of fragments from BRD4 covering different regions of the protein with the GST-E2 fusion protein immobilized on glutathione resin, show that deletion of the C-terminal region prevents BRD4 from binding to GST-E2, indicating that E2 binds to the BRD4 C-terminal domain [121]. Further, an immunohistochemical study of E2-transfected C33 cells stably expressing the BRD4 C-terminal domain shows disappearance of overlapping E2 and BRD4 staining, suggesting that expression of the BRD4 C-terminal domain blocks E2 interaction with BRD4 in a dominant-negative manner [121]. Collectively, these findings indicate that interaction of the BPV E2 protein with BRD4 is required to tether the viral genome to the host mitotic chromosomes.

The E2 protein encoded by human papillomaviruses (HPVs) has also been reported to be associated with BRD4. Using nuclear extracts of human embryonic kidney-derived 293 cells that express FLAG-tagged HPV E2, anti-FLAG immunoaffinity purification followed by mass spectrometry identified BRD4 as a cellular protein that is naturally associated with HPV E2 [122]. In vitro reconstituted chromatin transcription experiments illustrate that BRD4 confers the ability to silence HPV chromatin transcription on E2. BRD4 knockdown in C33 cells using an shRNA expressing retroviral vector causes a reduction in E2-mediated repression of E6 promoter activity, suggesting that BRD4 is a cellular co-repressor essential for E2-mediated repression of HPV E6 promoter activity [122]. Chromatin samples isolated from E2-expressing HeLa cells that harbor endogenous HPV genomes, using anti-TAF1 and Pol II antibodies, do not include DNA from the E6 promoter region, suggesting that E2 complexed with BRD4 blocks the recruitment of TAF1 (a subunit of TFIID) and RNA Pol II to the HPV E6 promoter region [122]. Further, a genome-wide siRNA screen using BPV E2-expressing C33 cells transfected with an E2-repressible reporter gene identified BRD4 as a cooperator contributing to E2-mediated transcriptional repression [123]. In contrast, both competitive inhibition of E2 binding to BRD4 by the C-terminal fragment of BRD4 and BRD4 knockdown by siRNA compromise transcriptional activation from an E2-responsive promoter, suggesting that BRD4 is required to mediate E2 transcriptional activation function [124,125]. Further, transfection of E2 fused with Cdk9, a subunit of P-TEFb, in C33 cells more efficiently enhances the activity of E2-responsive reporter genes, indicating that the recruitment of P-TEFb by BRD4 is important for E2-dependent transactivation [126]. Notably, BRD4 has dual effects on the transcriptional regulation of E2 in papillomaviruses.

6.3. BET Proteins Are Potential Therapeutic Targets in a Wide Range of Cancers

To identify and optimize therapeutic lead compounds for translation of small-molecule modulators of epigenetic targets as cancer therapeutics, high-throughput screening has been conducted. JQ1, a thienotriazolodiazepine, is identified as a potent and selective small-molecule inhibitor of BET proteins that competitively binds to the acetyl-lysine recognition pocket of their bromodomains [30]. FRAP assays with human osteosarcoma cells transfected with GFP-BRD4 and GFP-BRD4-NUT reveal accelerated fluorescence recovery in the presence of JQ1, indicating that JQ1 displaces BRD4 from nuclear chromatin and increases the levels of freely diffusing BRD4 [30]. Treatment of the NMC patient-derived cell line with JQ1 eliminates discrete nuclear foci composed of the BRD4-NUT oncoprotein, arrests proliferation at the G1 cell-cycle stage, and prompts terminal squamous differentiation. Analysis of NMC xenografts in mice by positron-emission tomography (PET) imaging of ¹⁸F-fluorodeoxyglucose (FDG) uptake shows that JQ1-treated mice exhibit marked reduction of FDG uptake, whereas vehicle-treated mice develop progressive cancer. In fact, remarkable tumor regression and prolonged survival are observed in mice treated with JQ1 compared to that in the vehicle control [30]. These observations highlight the immediate therapeutic potential of direct-acting inhibitors of BET proteins.

c-MYC is a transcription factor that regulates cell proliferation. Aberrant c-MYC expression leads to cancer by the coordinated activation of transcriptional pathways involved in cell division, metabolic adaptation, and survival. c-MYC is thus viewed as a promising target for anti-cancer drugs. To investigate whether BET inhibition specifically abrogates MYC-dependent transcription, global transcriptional profiling and unbiased gene set enrichment analysis (GSEA) [127] was performed with human multiple myeloma (MM) cell lines that highly express BET proteins. The result showed that downregulation by JQ1 is strongly correlated with canonical transcriptional signatures of MYC-dependent genes, indicating that BET bromodomain inhibition by JQ1 gives rise to selective repression of the transcriptional network induced by c-MYC [31]. JQ1 treatment in tumor-bearing SCID mice orthotopically xenografted with an intravenous injection of MM cells significantly decreases the disease burden and results in marked prolongation of overall survival compared to that in vehicle-treated animals [31]. Likewise, small-molecule BET inhibitors (JQ1 and I-BET) show anticancer effects in vitro and in vivo on acute myeloid leukemia (AML) [32,33], mixed lineage leukemia (MLL) [128], Burkitt’s lymphoma [33], medulloblastoma, which is the most common brain tumor in childhood [129], MYCN-amplified neuroblastoma [130], castration-resistant prostate cancer [131], Ewing’s sarcoma [132], and colorectal cancer [133]. These results demonstrate the pre-clinical efficacy of BET inhibitors in a wide range of malignancies, which reinforces the therapeutic potential of these drugs. Currently, clinical trials are under way by means of small-molecule bromodomain inhibitors to treat some of the most common cancers, including lymphomas, leukemias, multiple myelomas and solid tumours (such as cancers of the pancreas and prostate) [134].

To optimize the clinical efficacy of BET inhibition, mechanisms of drug resistance have been evaluated. Comparison between I-BET-resistant MLL cells and vehicle MLL cells by GSEA reveals the enrichment of leukemia stem cell signatures and significant upregulation of Wnt/β-catenin signaling in the resistant clones. Negative regulation of the Wnt/β-catenin pathway results in restoration of sensitivity to I-BET both in vitro and in vivo [135]. A chromatin-focused RNAi screen in mouse AML cells shows that suppression of PRC2 (Polycomb Repressive Complex 2) histone methyltransferase to transcriptionally silence chromatin, promotes JQ1 resistance in AML. GSEA with the transcriptomes of JQ1-resistant AML cells generated by PRC2 shRNA and JQ1-sensitive AML cells reveals that loss of PRC2 can facilitate transcriptional activation of Wnt signaling, which can drive MYC transcription [136]. These results identify and validate Wnt signaling as a driver and candidate biomarker of BET resistance in leukemia. Genome-wide shRNA “dropout screens” for functional genomic analyses on 77 breast cancer cell lines show that BRD4 is preferentially essential for cell viability in luminal/HER2 lines. Although many luminal/HER2 lines that are sensitive to BRD4 knockdown are JQ1-resistant, most basal lines sensitive to BRD4 knockdown are JQ1-sensitive. Further, integrative analysis reveals a strong correlation between JQ1 resistance and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation, which is supported by the fact that PIK3CA overexpression confers JQ1 resistance to JQ1-sensitive cells whereas a PIK3CA-specific inhibitor increases JQ1 sensitivity in resistant cells [137]. These findings highlight the identity of BRD4 as a potential therapeutic target and the significance of PIK3CA mutation leading to BET-inhibitor resistance in luminal breast cancer.

Financial support from Tokai University is gratefully acknowledged.