E2-Cellular Complexes in HPV Chromatin Transcription

描述（由申请人提供）：人乳头瘤病毒（hpv）诱导多种人类疾病，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E2蛋白和细胞转录因子的调控。尽管使用裸病毒DNA基因组对HPV转录进行了许多研究，但对于导致HPV染色质开始转录的分子事件知之甚少，染色质代表HPV基因组与细胞核心组蛋白复合物的浓缩形式。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染色质转录的蛋白因子，并进一步确定转录事件的分子机制，我们提出以下两个目标。1)鉴定与HPV E2蛋白联合作用的细胞蛋白。自然组装的E2-细胞复合物已经从人293衍生的细胞系中部分纯化，这些细胞系有条件地表达不同形式的flag标记的HPV-11 E2蛋白。我们发现，人类SWI/SNF染色质重塑复合体、GCN5组蛋白乙酰转移酶和一般转录因子TFIID存在于2-MDa E2细胞复合体中，另外一组具有协同抑制功能的细胞蛋白与另一E2细胞复合体相关，这表明E2的n端结构域可能在功能上招募SWI/SNF。GCN5和TFIID启动HPV染色质转录，E2可能在转录过程中改变其相互作用的伙伴，进一步调节HPV基因表达。这些假设将通过进行染色质重塑和组蛋白乙酰转移酶/去乙酰化酶测定，以及进行重组无细胞转录和蛋白质-蛋白质和蛋白质- dna相互作用研究来验证。2)分析e2细胞复合物在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，以及使用人类hela来源的细胞系有条件地表达不同形式的HPV E2和E1蛋白，通过体内染色质免疫沉淀研究来确定E2细胞复合物的作用。总的来说，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，该转录模拟了体内自然发生的HPV基因组，并揭示了导致染色质转录起始的分子事件。

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.