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.
Human papillomaviruses (HPVs) induces cervical cancer, penile cancer, oral cancer, genital warts, skin warts, and many other human diseases. Although prophylactic HPV vaccines developed by different investigators have recently become available, therapeutic vaccines that are most needed for HPV-infected individuals are still lacking. The research described in this proposal will unravel the role of viral and cellular proteins involved in HPV transcriptional regulation and gene expression, and further facilitate the development of drug inhibitors able to block HPV propagation.
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