title: Regulation of RNA polymerase I-dependent promoters by the hepatitis B virus X protein via activated Ras and TATA-binding protein.
abstract: The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with an X gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838-6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.
authors.affiliation: Departments of Molecular Pharmacology and Biochemistry, Schools of Pharmacy and Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: H D
authors.initials: HD
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Trivedi
authors.forename: D L
authors.initials: DL
authors.lastname: Johnson
mesh_terms: D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D004330:Drosophila / Q000201:enzymology*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012318:RNA Polymerase I / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins; D018631:ras Proteins / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D012335:RNA, Ribosomal; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D012318:RNA Polymerase I; D018631:ras Proteins