E2-Cellular Complexes in HPV Chromatin Transcription

描述(申请人提供)：人乳头瘤病毒(HPV)可引起多种人类疾病，包括生殖器疣和宫颈癌。HPV基因表达的调控主要受病毒编码的E2蛋白和细胞转录因子的调控。虽然已有许多研究利用病毒基因组对HPV转录进行了研究，但对导致HPV染色质转录启动的分子事件知之甚少。染色质是HPV基因组的浓缩形式，与细胞核心组蛋白形成复合体。染色质的形成阻止了细胞转录机制的进入，从而抑制了HPV基因的表达。为了确定促进HPV染色质转录的蛋白质因子，并进一步确定转录事件的分子机制，我们提出了以下两个目标。1)鉴定与HPVE2蛋白协同作用的细胞蛋白。自然组装的E2细胞复合体已经从人293来源的细胞系中部分纯化出来，这些细胞系有条件地表达不同形式的标记HPV-11E2蛋白。我们的发现表明，在2-MDA-E2细胞复合体中发现了人SWI/SNF染色质重塑复合体、GCN5组蛋白乙酰转移酶和通用转录因子TFIID，而另一组具有辅阻遏子功能的细胞蛋白与另一个E2细胞复合体相关。这一发现表明，E2的N-末端结构域可能在功能上招募SWI/SNF、GCN5和TFIID来启动HPV染色质转录，也可能在转录过程中切换其相互作用的伙伴以进一步调节HPV基因的表达。这些假说将通过进行染色质重塑和组蛋白乙酰转移酶/去乙酰酶分析，以及通过进行重组的无细胞转录和蛋白质-蛋白质-DNA相互作用研究来验证。2)剖析E2细胞复合体在HPV染色质转录中的功能作用。我们将通过使用单独纯化的染色质组装因子进行重组的HPV染色质转录分析，以及使用有条件地表达不同形式的HPVE2和E1蛋白的人HeLa来源的细胞株进行体内染色质免疫沉淀研究，来确定E2细胞复合体的作用。总之，这些研究将揭示人类SWI/SNF、GCN5、TFIID和其他细胞因子在E2介导的HPV染色质转录中的作用，这种转录模仿在体内发现的自然发生的HPV基因组，并揭示导致染色质转录的分子事件。

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) induce a variety of human diseases including genital warts and cervical carcinomas. Regulation of HPV gene expression is mainly controlled by virus-encoded E2 proteins and cellular transcription factors. Although many studies have been conducted on HPV transcription using naked viral DNA genomes, very little is known about the molecular events leading to initiation of transcription from HPV chromatin, which represents a condensed form of the HPV genome in complex with cellular core histone proteins. Formation of chromatin prevents access of the cellular transcription machinery and thereby inhibits HPV gene expression. To identify protein factors that facilitate transcription from HPV chromatin and to further define the molecular mechanism underlying transcriptional events, we propose the following two aims. 1) To identify cellular proteins working in conjunction with HPV E2 protein. Naturally assembled E2- cellular complexes have been partially purified from human 293-derived cell lines that conditionally express different forms of FLAG-tagged HPV-11 E2 proteins. Our findings that human SWI/SNF chromatin remodeling complex, GCN5 histone acetyltransferase and general transcription factor TFIID are found in a 2-MDa E2-cellular complex and a different set of cellular proteins with putative corepressor function is associated with another E2-cellular complex suggest that the N-terminal domain of E2 may functionally recruit SWI/SNF, GCN5 and TFIID to initiate HPV chromatin transcription and also E2 may switch its interacting partners during the transcriptional process to further modulate HPV gene expression. These hypotheses will be tested by performing chromatin remodeling and histone acetyltransferase/deacetylase assays, as well as by conducting reconstituted cell-free transcription and protein-protein and protein-DNA interaction studies. 2) To dissect the functional role of E2-cellular complexes in HPV chromatin transcription. We will define the role of E2-cellular complexes by reconstituted HPV chromatin transcription assays using individually purified chromatin assembly factors as well as by in vivo chromatin immunoprecipitation studies using human HeLa-derived cell lines conditionally expressing different forms of HPV E2 and E1 proteins. Collectively, these studies will uncover the role of human SWI/SNF, GCN5, TFIID and other cellular factors in E2-mediated transcription of HPV chromatin, which mimics naturally occurring HPV genomes found in vivo, and also shed light on the molecular events leading to a productive initiation of chromatin transcription.