BRD4蛋白在磷酸化激活条件下的结构和机制研究

BRD4 belongs to the bromodomain and extra-terminal (BET) family of transcriptional co-activators, which use tandem bromodomain modules (BD1 and BD2) to recognize acetylated-lysine residues on histones and transcription-associated proteins in chromatin. BRD4 is important in regulation of gene transcription in chromatin through its bromodomain-mediated interactions with lysine-acetylated histones, and with lysine-acetylated transcription factors to enhancer and promoter regions of target genes, but BRD4 also plays essential roles in human diseases, including viral infections and cancer. The extraterminal (ET) domain in the BRD4 has been reported to interact with transcription-associated proteins such as NSD3 that is reportedly to be overexpressed in human cancer, its specific molecular function has remained elusive. Our ongoing study discovered that the BRD4 is capable of directly binding DNA; and serine/threonine phosphorylation of BRD4 between the BD1 and the ET domain can switch off its DNA binding activity. Our findings provide an unprecedented opportunity to resolve some of long puzzling questions regarding the mechanism of BRD4 functions in gene transcriptional regulation. ..BRD4 is known to bind to acetylated histones via BD1 with modest affinity, yet the association with chromatin is effective and dynamic. Our newly discovered DNA binding activity likely contributes to the sophisticated regulatory capability of BRD4 in directing ordered gene transcription in chromatin. Specifically, BRD4 gene-specific targeting may be jointly conferred by its own DNA-binding function, and the cooperative binding, triggered by three modest-affinity interactions such as BRD4-acetylated histone tail, BRD4-DNA and BRD4-trascription factor, leads to a ‘‘coded-commitment’’ stage for gene-specific regulation. In our ongoing research, we will investigate the testable hypothesis, in which the coordinated histone and DNA binding activities enable BRD4 to effectively recruit to target gene loci and coordinate long-range interactions between the enhancer and promoter sites, while the process of BRD4 phosphorylation serves as an important regulating mechanism that controls BRD4 functions through fine-tuning the efficiency of its DNA binding and possibly modulating the BD1/2 and ET domain activities. In this study, we will use NMR and X-ray crystallography technologies to conduct a detailed structure-function relationship analysis to determine how BRD4/DNA binding is coordinated with BRD4 interactions with histones and transcription factors through its BD1/2 and ET domain, and how these interactions are modulated by serine/threonine phosphorylation in BRD4. We expect that the outcome of our study will greatly enhance our mechanistic understanding of complex functions and regulatory capability of BRD4 in gene transcription in biology and disease. Such new knowledge will provide a clear guidance in developing rational therapeutic agents that target the disease-causing properties of BRD4, which recently has gained major attention as a promising drug target for new treatments of cancer and inflammatory disorders.

转录调控因子BRD4蛋白调控染色质相关的基因转录活动，与炎症和癌症等疾病紧密相关，是新药研发的重要靶标。本项目组发现：BRD4能同时结合染色质上DNA和乙酰基化组蛋白，招募转录因子形成“染色质-BRD4-转录因子”复合结构，阻碍转录因子特异性结合DNA，并导致这种结构不能激活基因转录。然而，当BRD4被CK2磷酸化后，其内部结构域之间发生重组转换，使招募的转录因子能够在染色质位点上结合DNA，并激活基因转录启动。因此，本课题希望利用核磁共振和X-光谱深度解析BRD4与DNA结合的结构原理，以及磷酸化BRD4内部结构域之间的重组转换机制，探究磷酸化后BRD4招募的转录因子在基因转录调控中激活启动的作用功能。本课题成果能有助于明确BRD4在调控基因转录中的分子结构原理和机制，阐明BRD4抑制剂产生抗药耐药性的原因，指导BRD4抑制剂的临床应用和优化筛选，为开发BRD4抑制剂类创新药物奠定基础。

BRD4蛋白是BET蛋白家族的重要成员之一，能通过其串联的双溴域（BD1和BD2）识别染色质上的组蛋白乙酰化赖氨酸侧链，结合到基因组的增强子和启动子区域，并招募转录机器形成转录复合体，调控基因转录活性。然而，BRD4的表达失调与多种癌症的发生发展相关，因此它是重要的药物靶标蛋白。BRD4蛋白有两个亚型，BRD4L长亚型和BRD4S短亚型蛋白。前期报道，BRD4L通过羧基C末端的无序结构（IDR），结合介导蛋白MED1在超级增强子区域形成液态凝集，并聚集转录机器蛋白组件，调控基因转录。BRD4S缺失BRD4L长亚型的C末端序列，其调控功能尚不十分清楚。.我们发现，BRD4S短亚型在不同细胞系细胞核内形成液态性质的核斑点（nuclear puncta），并与BRD4L、MED1、乙酰化组蛋白H3K27ac核斑点共定位。不同细胞系中，BRD4核斑点体积与BRD4S在细胞核内的表达水平呈现正相关性，但与BRD4L表达水平未见明显相关。在C33A细胞内通过shRNA敲低BRD4S（而不是敲低BRD4L），BRD4蛋白核斑点荧光就会减弱或消失，靶标癌基因的转录水平也会相应地下降。在C33A细胞外源表达BRD4S，BRD4的核斑点得到加强或增多，而靶基因的转录水平也对应提高。这说明BRD4S短亚型对BRD4蛋白在细胞内凝聚核斑点以及调控基因转录有重要作用。.我们在体外重组并荧光标记Fluor-BRD4S蛋白质。Fluor-BRD4S在低盐浓度条件下能够形成液相分离（LLPS）的液滴。BRD4S可以通过其有序结构和IDRs与DNA结合，并促进BRD4S液滴的数量增加和体积增大，说明BRD4S与DNA相互作用增强BRD4S的液相分离。而BRD4S蛋白质的磷酸化明显地降低BRD4S结合DNA的能力，减弱BRD4S液滴形成。BRD4S在HeLa核提取液中也能形成相分离的液滴，并招募H3K27ac、BRD4L、MED1、RNA Pol II和CDK9等转录相关蛋白于凝聚体中，增加模版DNA的转录水平。.本研究显示BRD4S能够在细胞内形成液态凝聚体，并通过无序结构和有序结构溴域双共价作用DNA和乙酰化组蛋白聚集于染色质，招募转录相关蛋白，调控染色质上的基因转录，揭示了BRD4S短亚型蛋白在调控基因转录中的功能，显示了BRD4S在招募转录相关蛋白因子，调控基因转录中的重要性。