E2-Cellular Complexes in HPV Chromatin Transcription

描述（由申请人提供）：人乳头瘤病毒（HPV）是许多人类疾病的病原体，包括生殖器疣、皮肤疣、宫颈癌和某些类型的头颈癌和皮肤癌。虽然目前有两种预防HPV感染的疫苗，并推荐用于年轻女孩/男孩的免疫接种，但大多数HPV感染人群最需要的治疗性疫苗仍然缺乏。由于HPV编码的早期蛋白2（E2）对于病毒DNA复制、HPV基因组分离、控制病毒基因表达以及诱导细胞凋亡和衰老至关重要，因此其已成为开发抗病毒疗法的有希望的靶点。 在最后一个资助期，我们已经鉴定了含溴结构域蛋白4（Brd4）作为介导HPV编码的E2蛋白的多种功能的中心细胞因子，特别是通过形成能够阻断激活蛋白-1（AP-1）触发的来自不表现出转录活性的潜伏HPV染色质的基因激活的E2-Brd4沉默复合物。在这个更新申请中，我们提出了三个目的来剖析HPV染色质依赖性转录调控的机制：1。明确AP-1依赖的HPV染色质转录机制2.明确E2-Brd4在HPV染色质转录中的抑制机制3.为了定义HPV特异性与AP-1，Brd4和E2调控的全局细胞基因转录，我们将首先应用我们开发的创新HPV染色质转录系统来定义细胞染色质修饰酶（包括p300和CBP组蛋白乙酰转移酶（HAT））在HPV染色质转录的AP-1依赖性激活中的作用。然后，我们将剖析E2-Brd4沉默复合物在抑制HPV染色质转录中的步骤，并进一步阐明两个新鉴定的Brd4相互作用蛋白的功能作用，即，G9a组蛋白甲基转移酶（HMT）和拓扑异构酶II结合蛋白1（TopBP 1），在E2调节的HPV转录中。 为了区分HPV特异性和全球宿主细胞基因转录，我们还将与加州大学圣地亚哥分校的Bing Ren博士合作，应用全基因组方法来分析这些HPV转录调节因子和染色质修饰剂的转录谱和结合模式，并在佛罗里达斯克里普斯的托马斯科达德克博士的帮助下，我们将利用合成化学来开发阻断HPV转录中Brd4作用的小化合物。我们的中心假设是Brd4的构象变化为Brd4募集不同的细胞因子以调节HPV特异性和细胞基因转录提供了分子开关。 机制研究和全基因组分析的结合将显着推进我们对调节HPV转录的病毒和细胞因子的理解，并促进阻断HPV诱导的人类疾病的治疗剂的开发。

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are etiological agents for many human diseases, including genital warts, skin warts, cervical cancer, and certain types of head-and-neck and skin cancers. Although two prophylactic vaccines against HPV infection are presently available and recommended for immunization of young girls/boys, therapeutic vaccines that are most needed for the majority of HPV-infected populations are still lacking. Since early protein 2 (E2) encoded by HPVs is crucial for viral DNA replication, HPV genome segregation, control of viral gene expression, and induction of cellular apoptosis and senescence, it has become a promising target for the development of anti-viral therapeutics. In the last funding period, we have identified bromodomain- containing protein 4 (Brd4) as a central cellular factor mediating diverse functions of HPV-encoded E2 protein, particularly via formation of an E2-Brd4 silencing complex able to block activator protein-1 (AP-1)-triggered gene activation from latent HPV chromatin that exhibits no transcriptional activity. In this renewal application, we propose three aims to dissect the mechanisms of HPV chromatin-dependent transcriptional regulation: 1. To define the mechanism of AP-1-dependent HPV chromatin transcription 2. To define the repression mechanism of E2-Brd4 in HPV chromatin transcription 3. To define HPV-specific versus global cellular gene transcription regulated by AP-1, Brd4, and E2 We will first apply an innovative HPV chromatin transcription system that we have developed to define the role of cellular chromatin-modifying enzymes, including p300 and CBP histone acetyltransferases (HATs), in AP-1-dependent activation of HPV chromatin transcription. We will then dissect the steps of E2-Brd4 silencing complex in suppressing HPV chromatin transcription and further elucidate the functional role of two newly identified Brd4-interacting proteins, i.e., G9a histone methyltransferase (HMT) and topoisomerase II¿-binding protein 1 (TopBP1), in E2-regulated HPV transcription. To distinguish HPV-specific and global host cell gene transcription, we will also apply genome-wide approaches, in collaboration with Dr. Bing Ren at UC-San Diego, to analyze the transcriptional profiles and binding patterns of these HPV transcriptional regulators and chromatin modifiers, and with the help from Dr. Thomas Kodadek at Florida Scripps, we will utilize synthetic chemistry to develop small compounds blocking Brd4 action in HPV transcription. Our central hypothesis here is that conformational changes in Brd4 provide a molecular switch for Brd4 recruitment of distinct cellular factors to regulate HPV-specific and cellular gene transcription. The combination of mechanistic studies and genome-wide analyses will significantly advance our understanding of viral and cellular factors modulating HPV transcription and facilitate the development of therapeutic agents blocking HPV-induced human diseases.