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.
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