Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1989 Mar 15;258(3):769–776. doi: 10.1042/bj2580769

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

S X Cao 1, H Mishoe 1, J Elion 1, P E Berg 1, A N Schechter 1
PMCID: PMC1138431  PMID: 2525024

Abstract

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.

Full text

PDF
769

Images in this article

771Fig. 2.
on p.771
772Fig. 3.
on p.772
772Fig. 4.
on p.772
773Fig. 5.
on p.773
774Fig. 7.
on p.774

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allan M., Lanyon W. G., Paul J. Multiple origins of transcription in the 4.5 Kb upstream of the epsilon-globin gene. Cell. 1983 Nov;35(1):187–197. doi: 10.1016/0092-8674(83)90221-0. [DOI] [PubMed] [Google Scholar]
  2. Alwine J. C., Reed S. I., Stark G. R. Characterization of the autoregulation of simian virus 40 gene A. J Virol. 1977 Oct;24(1):22–27. doi: 10.1128/jvi.24.1.22-27.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alwine J. C. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol. 1985 May;5(5):1034–1042. doi: 10.1128/mcb.5.5.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  5. Baron M. H., Maniatis T. Rapid reprogramming of globin gene expression in transient heterokaryons. Cell. 1986 Aug 15;46(4):591–602. doi: 10.1016/0092-8674(86)90885-8. [DOI] [PubMed] [Google Scholar]
  6. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
  7. Borrelli E., Hen R., Chambon P. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature. 1984 Dec 13;312(5995):608–612. doi: 10.1038/312608a0. [DOI] [PubMed] [Google Scholar]
  8. Brady J., Bolen J. B., Radonovich M., Salzman N., Khoury G. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2040–2044. doi: 10.1073/pnas.81.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brady J., Khoury G. trans Activation of the simian virus 40 late transcription unit by T-antigen. Mol Cell Biol. 1985 Jun;5(6):1391–1399. doi: 10.1128/mcb.5.6.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brash D. E., Reddel R. R., Quanrud M., Yang K., Farrell M. P., Harris C. C. Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells. Mol Cell Biol. 1987 May;7(5):2031–2034. doi: 10.1128/mcb.7.5.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fordis C. M., Nelson N., McCormick M., Padmanabhan R., Howard B., Schechter A. N. The 5'-flanking sequences of human globin genes contribute to tissue specific expression. Biochem Biophys Res Commun. 1986 Jan 14;134(1):128–133. doi: 10.1016/0006-291x(86)90536-x. [DOI] [PubMed] [Google Scholar]
  12. Gaynor R. B., Hillman D., Berk A. J. Adenovirus early region 1A protein activates transcription of a nonviral gene introduced into mammalian cells by infection or transfection. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1193–1197. doi: 10.1073/pnas.81.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Green M. R., Treisman R., Maniatis T. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell. 1983 Nov;35(1):137–148. doi: 10.1016/0092-8674(83)90216-7. [DOI] [PubMed] [Google Scholar]
  15. Grinnell B. W., Berg D. T., Walls J. Activation of the adenovirus and BK virus late promoters: effects of the BK virus enhancer and trans-acting viral early proteins. Mol Cell Biol. 1986 Nov;6(11):3596–3605. doi: 10.1128/mcb.6.11.3596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  17. Hartzell S. W., Byrne B. J., Subramanian K. N. The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6335–6339. doi: 10.1073/pnas.81.20.6335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hen R., Borrelli E., Chambon P. Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products. Science. 1985 Dec 20;230(4732):1391–1394. doi: 10.1126/science.2999984. [DOI] [PubMed] [Google Scholar]
  19. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  20. Humphries R. K., Ley T., Turner P., Moulton A. D., Nienhuis A. W. Differences in human alpha-, beta- and delta-globin gene expression in monkey kidney cells. Cell. 1982 Aug;30(1):173–183. doi: 10.1016/0092-8674(82)90023-x. [DOI] [PubMed] [Google Scholar]
  21. Imperiale M. J., Feldman L. T., Nevins J. R. Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element. Cell. 1983 Nov;35(1):127–136. doi: 10.1016/0092-8674(83)90215-5. [DOI] [PubMed] [Google Scholar]
  22. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  24. Khoury G., May E. Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of a functional T-antigen. J Virol. 1977 Jul;23(1):167–176. doi: 10.1128/jvi.23.1.167-176.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Langner K. D., Weyer U., Doerfler W. Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1598–1602. doi: 10.1073/pnas.83.6.1598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Loeken M. R., Khoury G., Brady J. Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms. Mol Cell Biol. 1986 Jun;6(6):2020–2026. doi: 10.1128/mcb.6.6.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  28. Mitchell P. J., Wang C., Tjian R. Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen. Cell. 1987 Sep 11;50(6):847–861. doi: 10.1016/0092-8674(87)90512-5. [DOI] [PubMed] [Google Scholar]
  29. Myers R. M., Tilly K., Maniatis T. Fine structure genetic analysis of a beta-globin promoter. Science. 1986 May 2;232(4750):613–618. doi: 10.1126/science.3457470. [DOI] [PubMed] [Google Scholar]
  30. Nevins J. R. Mechanism of activation of early viral transcription by the adenovirus E1A gene product. Cell. 1981 Oct;26(2 Pt 2):213–220. doi: 10.1016/0092-8674(81)90304-4. [DOI] [PubMed] [Google Scholar]
  31. Pozzatti R., Muschel R., Williams J., Padmanabhan R., Howard B., Liotta L., Khoury G. Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials. Science. 1986 Apr 11;232(4747):223–227. doi: 10.1126/science.3456644. [DOI] [PubMed] [Google Scholar]
  32. Reed S. I., Stark G. R., Alwine J. C. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. doi: 10.1073/pnas.73.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rio D., Robbins A., Myers R., Tjian R. Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5706–5710. doi: 10.1073/pnas.77.10.5706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Robbins P. D., Rio D. C., Botchan M. R. trans Activation of the simian virus 40 enhancer. Mol Cell Biol. 1986 Apr;6(4):1283–1295. doi: 10.1128/mcb.6.4.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sassone-Corsi P., Wildeman A., Chambon P. A trans-acting factor is responsible for the simian virus 40 enhancer activity in vitro. Nature. 1985 Feb 7;313(6002):458–463. doi: 10.1038/313458a0. [DOI] [PubMed] [Google Scholar]
  36. Schöler H. R., Gruss P. Specific interaction between enhancer-containing molecules and cellular components. Cell. 1984 Feb;36(2):403–411. doi: 10.1016/0092-8674(84)90233-2. [DOI] [PubMed] [Google Scholar]
  37. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tegtmeyer P., Schwartz M., Collins J. K., Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. doi: 10.1128/jvi.16.1.168-178.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Velcich A., Ziff E. Adenovirus E1a proteins repress transcription from the SV40 early promoter. Cell. 1985 Mar;40(3):705–716. doi: 10.1016/0092-8674(85)90219-3. [DOI] [PubMed] [Google Scholar]
  41. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES