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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）可诱发生殖器疣、宫颈癌和许多其他人类疾病。 HPV基因表达调控主要受病毒编码的E2蛋白和细胞转录调控因素利用生物化学方法，我们从人293细胞中条件性分离出两种E2复合物，表达HPV 11型（HPV-11）E2蛋白。这两种复合物，在0.5 M和0.85 M KCl洗脱 P11离子交换柱的洗脱级分分别命名为E2-P.5和E2-P.85。E2-P.5是一种丰富的该复合物含有两种主要蛋白：E2和细胞含溴结构域蛋白4（Brd 4）。我们证明E2-Brd 4作为抑制HPV染色质转录的沉默复合物起作用。在相比之下，E2-P.85是一种丰度较低但大得多（>7 MDa）的复合物，含有与核心结合的E2 启动子结合因子TFIID、组蛋白乙酰转移酶GCN 5和染色质重塑因子SWI/SNF。在本次更新申请，我们将继续这两个E2的表征和机制研究配合物具体目的：1）明确E2-Brd 4在HPV染色质中的阻遏机制转录。我们最近鉴定了染色质接头Brd 4作为转录辅阻遏物， HPV E2表明Brd 4在E2靶向HPV染色质中起重要作用。我们将定义 E2-Brd 4抑制HPV染色质转录的抑制机制用携带HPV的人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。候选质谱法和无偏质谱法都将用于鉴定E2-P.85复合物的蛋白组成。TFIID、GCN 5和 SWI/SNF在同一E2复合物中的表达表明E2-P.85可能是长期寻找的E2激活复合物， HPV染色质转录。我们将使用我们的体外重组HPV染色质来探索这种可能性进一步确定Brd 4在E2介导的HPV染色质活化中的作用转录。总的来说，这些研究将有助于识别参与E2-E3的细胞因子。调节转录，并进一步阐明HPV转录调节的机制。