E2-Cellular Complexes in HPV Chromatin Transcription

HPV 染色质转录中的 E2 细胞复合物

基本信息

批准号：
7895006
负责人：
CHENG-MING CHIANG
金额：
$ 28.29万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7895006
关键词：
Accounting Address Anogenital venereal warts Binding Biochemical Biological Biological Assay Bovine Papillomavirus Bromodomain CCAAT-Enhancer-Binding Proteins Cells Cervix carcinoma Chromatin Chromatin Remodeling Factor Complex Family Fractionation Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Transcription HPV-High Risk Human Human Cell Line Human Papilloma Virus Vaccine Human Papillomavirus Human papilloma virus infection Human papillomavirus 11 Human papillomavirus 16 Human papillomavirus 18 In Vitro Individual Ion Exchange Laboratories Lead Length Low risk HPV Malignant neoplasm of cervix uteri Malignant neoplasm of penis Mass Spectrum Analysis Mediating Methylation Modification Molecular Names Pathogenesis Phosphorylation Play Proteins Regulation Repression Research Research Personnel Resolution Risk Role Skin System Testing Transcription Repressor/Corepressor Transcriptional Regulation Viral Virus base cell type drug development genome-wide high risk histone acetyltransferase human disease inhibitor/antagonist malignant mouth neoplasm pathogen polypeptide promoter prophylactic public health relevance reconstitution therapeutic vaccine transcription factor virus host interaction

项目摘要

Human papillomaviruses (HPVs) induce genital warts, cervical cancer, and many other human diseases. Regulation of HPV gene expression is mainly controlled by virus-encoded E2 proteins and cellular transcription factors. Using biochemical approaches, we isolated two E2 complexes from human 293 cells conditionally expressing the HPV type 11 (HPV-11) E2 protein. These two complexes, eluting at the 0.5 M and 0.85 M KCI fraction of a P11 ion-exchange column, were named E2-P.5 and E2-P.85, respectively. E2-P.5 is an abundant complex containing two predominant proteins: E2 and cellular bromodomain-containing protein 4 (Brd4). We demonstrated that E2-Brd4 functions as a silencing complex inhibiting HPV chromatin transcription. In contrast, E2-P.85 is a less abundant but much larger (>7 MDa) complex containing E2 in association with core promoter-binding factor TFIID, histone acetyltransferase GCN5 and chromatin remodeling factor SWI/SNF. In this renewal application, we will continue the characterization and mechanistic studies of these two E2 complexes. The Specific Aims are: 1) To Define the repression mechanism of E2-Brd4 in HPV Chromatin Transcription. Our recent identification of the chromatin adaptor Brd4 as a transcriptional corepressor for HPV E2 indicates that Brd4 plays an important role for E2 targeting to HPV chromatin. We will define the repression mechanism by which E2-Brd4 inhibits HPV chromatin transcription using in vitro-reconstituted HPV chromatin and cell-based assays performed with HPV-harboring human cell lines. 2) To Define the role of E2-P.85 in HPV Chromatin Transcription. Both candidate and unbiased mass spectrometry approaches will be employed to identify the protein composition of the E2-P.85 complex. The coexistence of TFIID, GCN5 and SWI/SNF in the same E2 complex indicates that E2-P.85 is likely the long-sought E2-activating complex for HPV chromatin transcription. We will explore this possibility using our in vitro-reconstituted HPV chromatin transcription systems and further define the role of Brd4 in E2-mediated activation of HPV chromatin transcription. Collectively, these studies will facilitate the identification of cellular factors involved in E2- regulated transcription and further unravel the mechanisms of HPV transcriptional regulation.

人乳头瘤病毒（HPV）诱导生殖器疣，宫颈癌和许多其他人类疾病。 HPV基因表达的调节主要由病毒编码的E2蛋白和细胞转录控制因素。使用生化方法，我们从有条件的293个细胞中分离了两个E2复合物表达HPV型11（HPV-11）E2蛋白。这两个配合物在0.5 m和0.85 m kci处洗脱 P11离子交换列的一部分分别命名为E2-P.5和E2-P.85。 E2-P.5是丰富的含有两种主要蛋白质的复合物：E2和含细胞溴结构域的蛋白4（BRD4）。我们证明E2-BRD4充当抑制HPV染色质转录的沉默复合物。在对比度，E2-P.85是一个不那么丰富，但包含E2的较大（> 7 MDA）复合物与Core相关启动子结合因子TFIID，组蛋白乙酰转移酶GCN5和染色质重塑因子SWI/SNF。在这种续订应用，我们将继续对这两个E2的表征和机械研究复合物。具体目的是：1）定义HPV染色质中E2-BRD4的抑制机制转录。我们最近将染色质衔接子BRD4识别为转录核心的鉴定 HPV E2表明BRD4对于E2靶向HPV染色质起着重要作用。我们将定义 E2-BRD4使用体外重新定位的HPV抑制HPV染色质转录的抑制机制用HPV捕获的人类细胞系进行的染色质和基于细胞的测定。 2）定义角色 HPV染色质转录中的E2-P.85。候选人和公正的质谱方法都将被用来识别E2-P.85复合物的蛋白质组成。 TFIID，GCN5和同一E2复合物中的SWI/SNF表明E2-P.85可能是长期追求的E2激活复合物 HPV染色质转录。我们将使用我们的体外重新确定的HPV染色质探索这种可能性转录系统并进一步定义了BRD4在E2介导的HPV染色质激活中的作用转录。总的来说，这些研究将促进鉴定E2-涉及的细胞因素调节转录并进一步揭示了HPV转录调控的机制。