E2-Cellular Complexes in HPV Chromatin Transcription

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 在这次更新申请中，我们将继续对这两个E2进行表征和机制研究 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。

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.