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. 1996 Dec;70(12):8813–8820. doi: 10.1128/jvi.70.12.8813-8820.1996

A functional hepatocyte nuclear factor 3 binding site is a critical component of the duck hepatitis B virus major surface antigen promoter.

T Welsheimer 1, J E Newbold 1
PMCID: PMC190978  PMID: 8971010

Abstract

The gene coding for the S protein, the smaller of the two envelope antigens of the duck hepatitis B virus (DHBV), is transcribed from a TATA-less promoter. In this study, we localized the promoter to a 245-bp segment of the genome that was capable of efficiently driving expression of a linked reporter gene upon transient transfection into the differentiated hepatoma cell lines LMH and HepG2. However, no measurable activity from this construct could be detected in similar assays with the dedifferentiated cell line HepG2.1 or the nonhepatic cell line HeLa. Located at position -25 relative to the transcriptional start site was a sequence conforming to the consensus binding site for hepatocyte nuclear factor 3 (HNF3). Deletion of this region reduced activity of the reporter gene to barely detectable levels in LMH cells. The results of electrophoretic mobility shift analysis (EMSA) demonstrated that a double-stranded oligonucleotide containing this sequence formed a specific complex with DNA-binding proteins from LMH and HepG2 cells but not with nuclear extracts obtained from HepG2.1 or HeLa cells. Cotransfection of HepG2.1 cells with DHBV S promoter constructs and a rat HNF3beta expression plasmid resulted in transactivation of only those constructs in which the candidate HNF3 site was present. Furthermore, EMSA using HepG2.1 nuclear extracts containing exogenously expressed HNF3 formed complexes with the same migration and competition properties as those in which the proteins were derived from the differentiated hepatoma cells. Thus, several lines of evidence suggest a critical role for HNF3 in activity from the DHBV S promoter.

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Selected References

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  1. Beg A. A., Finco T. S., Nantermet P. V., Baldwin A. S., Jr Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993 Jun;13(6):3301–3310. doi: 10.1128/mcb.13.6.3301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Büscher M., Reiser W., Will H., Schaller H. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription. Cell. 1985 Mar;40(3):717–724. doi: 10.1016/0092-8674(85)90220-x. [DOI] [PubMed] [Google Scholar]
  3. Chang H. K., Ting L. P. The surface gene promoter of the human hepatitis B virus displays a preference for differentiated hepatocytes. Virology. 1989 May;170(1):176–183. doi: 10.1016/0042-6822(89)90364-4. [DOI] [PubMed] [Google Scholar]
  4. Chang H. K., Wang B. Y., Yuh C. H., Wei C. L., Ting L. P. A liver-specific nuclear factor interacts with the promoter region of the large surface protein gene of human hepatitis B virus. Mol Cell Biol. 1989 Nov;9(11):5189–5197. doi: 10.1128/mcb.9.11.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Condreay L. D., Aldrich C. E., Coates L., Mason W. S., Wu T. T. Efficient duck hepatitis B virus production by an avian liver tumor cell line. J Virol. 1990 Jul;64(7):3249–3258. doi: 10.1128/jvi.64.7.3249-3258.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Costa R. H., Grayson D. R., Darnell J. E., Jr Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes. Mol Cell Biol. 1989 Apr;9(4):1415–1425. doi: 10.1128/mcb.9.4.1415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crescenzo-Chaigne B., Pillot J., Lilienbaum A. Interplay between a new HNF3 and the HNF1 transcriptional factors in the duck hepatitis B virus enhancer. Virology. 1995 Oct 20;213(1):231–240. doi: 10.1006/viro.1995.1563. [DOI] [PubMed] [Google Scholar]
  8. Crescenzo-Chaigne B., Pillot J., Lilienbaum A., Levrero M., Elfassi E. Identification of a strong enhancer element upstream from the pregenomic RNA start site of the duck hepatitis B virus genome. J Virol. 1991 Jul;65(7):3882–3886. doi: 10.1128/jvi.65.7.3882-3886.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cullen B. R., Lomedico P. T., Ju G. Transcriptional interference in avian retroviruses--implications for the promoter insertion model of leukaemogenesis. Nature. 1984 Jan 19;307(5948):241–245. doi: 10.1038/307241a0. [DOI] [PubMed] [Google Scholar]
  10. Faktor O., De-Medina T., Shaul Y. Regulation of hepatitis B virus S gene promoter in transfected cell lines. Virology. 1988 Feb;162(2):362–368. doi: 10.1016/0042-6822(88)90476-x. [DOI] [PubMed] [Google Scholar]
  11. Huang M., Summers J. pet, a small sequence distal to the pregenome cap site, is required for expression of the duck hepatitis B virus pregenome. J Virol. 1994 Mar;68(3):1564–1572. doi: 10.1128/jvi.68.3.1564-1572.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson D. A., Rowader K. E., Stevens K., Jiang C., Milos P., Zaret K. S. Modulation of liver-specific transcription by interactions between hepatocyte nuclear factor 3 and nuclear factor 1 binding DNA in close apposition. Mol Cell Biol. 1993 Apr;13(4):2401–2410. doi: 10.1128/mcb.13.4.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Johnson J. L., Raney A. K., McLachlan A. Characterization of a functional hepatocyte nuclear factor 3 binding site in the hepatitis B virus nucleocapsid promoter. Virology. 1995 Apr 1;208(1):147–158. doi: 10.1006/viro.1995.1138. [DOI] [PubMed] [Google Scholar]
  14. Kawaguchi T., Nomura K., Hirayama Y., Kitagawa T. Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res. 1987 Aug 15;47(16):4460–4464. [PubMed] [Google Scholar]
  15. Lilienbaum A., Crescenzo-Chaigne B., Sall A. A., Pillot J., Elfassi E. Binding of nuclear factors to functional domains of the duck hepatitis B virus enhancer. J Virol. 1993 Oct;67(10):6192–6200. doi: 10.1128/jvi.67.10.6192-6200.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu C., Condreay L. D., Burch J. B., Mason W. Characterization of the core promoter and enhancer of duck hepatitis B virus. Virology. 1991 Sep;184(1):242–252. doi: 10.1016/0042-6822(91)90841-x. [DOI] [PubMed] [Google Scholar]
  17. Liu C., Mason W. S., Burch J. B. Identification of factor-binding sites in the duck hepatitis B virus enhancer and in vivo effects of enhancer mutations. J Virol. 1994 Apr;68(4):2286–2296. doi: 10.1128/jvi.68.4.2286-2296.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mandart E., Kay A., Galibert F. Nucleotide sequence of a cloned duck hepatitis B virus genome: comparison with woodchuck and human hepatitis B virus sequences. J Virol. 1984 Mar;49(3):782–792. doi: 10.1128/jvi.49.3.782-792.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Overdier D. G., Porcella A., Costa R. H. The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix. Mol Cell Biol. 1994 Apr;14(4):2755–2766. doi: 10.1128/mcb.14.4.2755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Raney A. K., Le H. B., McLachlan A. Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor. J Virol. 1992 Dec;66(12):6912–6921. doi: 10.1128/jvi.66.12.6912-6921.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raney A. K., Milich D. R., Easton A. J., McLachlan A. Differentiation-specific transcriptional regulation of the hepatitis B virus large surface antigen gene in human hepatoma cell lines. J Virol. 1990 May;64(5):2360–2368. doi: 10.1128/jvi.64.5.2360-2368.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Raney A. K., Zhang P., McLachlan A. Regulation of transcription from the hepatitis B virus large surface antigen promoter by hepatocyte nuclear factor 3. J Virol. 1995 Jun;69(6):3265–3272. doi: 10.1128/jvi.69.6.3265-3272.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schneider R., Will H. Regulatory sequences of duck hepatitis B virus C gene transcription. J Virol. 1991 Nov;65(11):5693–5701. doi: 10.1128/jvi.65.11.5693-5701.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Standring D. N., Rutter W. J., Varmus H. E., Ganem D. Transcription of the hepatitis B surface antigen gene in cultured murine cells initiates within the presurface region. J Virol. 1984 May;50(2):563–571. doi: 10.1128/jvi.50.2.563-571.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Xanthopoulos K. G., Mirkovitch J. Gene regulation in rodent hepatocytes during development, differentiation and disease. Eur J Biochem. 1993 Sep 1;216(2):353–360. doi: 10.1111/j.1432-1033.1993.tb18152.x. [DOI] [PubMed] [Google Scholar]

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