E2-Cellular Complexes in HPV Chromatin Transcription

Ut Southwestern Medical Center, Dallas TX

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Abstract

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.