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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.

人类乳头瘤病毒(HPV)会导致生殖器疣、宫颈癌和许多其他人类疾病。 HPV基因表达的调控主要受病毒编码的E2蛋白和细胞转录调控各种因素。利用生化方法，我们有条件地从人293细胞中分离出两个E2复合体表达人乳头瘤病毒11型(HPV-11)E2蛋白。这两种络合物，在0.5M和0.85M KCI处洗脱分别命名为E_2-P_5和E_2-P_(85)。E2-P.5是一种丰富的包含两种主要蛋白质的复合体：E2和细胞内含溴结构域蛋白4(Brd4)。我们证明了E2-Brd4作为沉默复合体抑制HPV染色质转录。在……里面相比之下，E2-p.85是一个含量较少但要大得多的(&gt；7 MDA)复合体，包含与核心相关的E2 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……里面本次续展申请，我们将继续对这两个E2的表征和机理进行研究复合体。具体目的是：1)明确E2-Brd4在HPV染色质中的抑制机制抄写。我们最近发现染色质接头Brd4是一种转录抑制因子 HPV E2提示Brd4在E2靶向HPV染色质中起重要作用。我们将定义体外重组HPV中E2-Brd4抑制HPV染色质转录的机制染色质和基于细胞的分析是对携带HPV的人类细胞株进行的。2)确定…的角色 HPV染色质转录中的E2-p.85。候选和无偏的质谱学方法都将用来鉴定E2-P.85复合体的蛋白质组成。TFIID、GCN5和在同一个E2复合体中的Swi/Snf表明，E2-p.85很可能是长期寻找的E2激活复合体 HPV染色质转录。我们将使用我们的体外重组的HPV染色质来探索这种可能性转录系统，并进一步确定Brd4在E2介导的HPV染色质激活中的作用抄写。总而言之，这些研究将有助于确定参与E2的细胞因子- 调控转录，进一步揭示HPV转录调控的机制。