Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Nov;8(11):4799–4807. doi: 10.1128/mcb.8.11.4799

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

L J Brunet 1, A J Berk 1
PMCID: PMC365573  PMID: 2974921

Abstract

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

Full text

PDF
4799

Images in this article

4802Image
on p.4802
4803Image
on p.4803
4804Image
on p.4804
4806Image
on p.4806

Selected References

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

  1. Babiss L. E., Ginsberg H. S. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis. J Virol. 1984 Apr;50(1):202–212. doi: 10.1128/jvi.50.1.202-212.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babiss L. E., Young C. S., Fisher P. B., Ginsberg H. S. Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells. J Virol. 1983 May;46(2):454–465. doi: 10.1128/jvi.46.2.454-465.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J. Adenovirus promoters and E1A transactivation. Annu Rev Genet. 1986;20:45–79. doi: 10.1146/annurev.ge.20.120186.000401. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Enoch T., Zinn K., Maniatis T. Activation of the human beta-interferon gene requires an interferon-inducible factor. Mol Cell Biol. 1986 Mar;6(3):801–810. doi: 10.1128/mcb.6.3.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferguson B., Krippl B., Andrisani O., Jones N., Westphal H., Rosenberg M. E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA. Mol Cell Biol. 1985 Oct;5(10):2653–2661. doi: 10.1128/mcb.5.10.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Garcia J., Wu F., Gaynor R. Upstream regulatory regions required to stabilize binding to the TATA sequence in an adenovirus early promoter. Nucleic Acids Res. 1987 Oct 26;15(20):8367–8385. doi: 10.1093/nar/15.20.8367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gaynor R. B., Berk A. J. Cis-acting induction of adenovirus transcription. Cell. 1983 Jul;33(3):683–693. doi: 10.1016/0092-8674(83)90011-9. [DOI] [PubMed] [Google Scholar]
  10. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  11. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  12. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  13. Hoeffler W. K., Roeder R. G. Enhancement of RNA polymerase III transcription by the E1A gene product of adenovirus. Cell. 1985 Jul;41(3):955–963. doi: 10.1016/s0092-8674(85)80076-3. [DOI] [PubMed] [Google Scholar]
  14. Jochemsen A. G., Peltenburg L. T., te Pas M. F., de Wit C. M., Bos J. L., van der Eb A. J. Activation of adenovirus 5 E1A transcription by region E1B in transformed primary rat cells. EMBO J. 1987 Nov;6(11):3399–3405. doi: 10.1002/j.1460-2075.1987.tb02663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
  16. Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
  17. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  18. Kingston R. E., Baldwin A. S., Sharp P. A. Transcription control by oncogenes. Cell. 1985 May;41(1):3–5. doi: 10.1016/0092-8674(85)90049-2. [DOI] [PubMed] [Google Scholar]
  19. Klessig D. F., Brough D. E., Cleghon V. Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell. Mol Cell Biol. 1984 Jul;4(7):1354–1362. doi: 10.1128/mcb.4.7.1354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klock G., Strähle U., Schütz G. Oestrogen and glucocorticoid responsive elements are closely related but distinct. Nature. 1987 Oct 22;329(6141):734–736. doi: 10.1038/329734a0. [DOI] [PubMed] [Google Scholar]
  21. Kovesdi I., Reichel R., Nevins J. R. Identification of a cellular transcription factor involved in E1A trans-activation. Cell. 1986 Apr 25;45(2):219–228. doi: 10.1016/0092-8674(86)90386-7. [DOI] [PubMed] [Google Scholar]
  22. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  23. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  24. Lee K. A., Bindereif A., Green M. R. A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech. 1988 Mar-Apr;5(2):22–31. doi: 10.1016/0735-0651(88)90023-4. [DOI] [PubMed] [Google Scholar]
  25. Leff T., Elkaim R., Goding C. R., Jalinot P., Sassone-Corsi P., Perricaudet M., Kédinger C., Chambon P. Individual products of the adenovirus 12S and 13S EIa mRNAs stimulate viral EIIa and EIII expression at the transcriptional level. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4381–4385. doi: 10.1073/pnas.81.14.4381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leong K., Brunet L., Berk A. J. Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor. Mol Cell Biol. 1988 Apr;8(4):1765–1774. doi: 10.1128/mcb.8.4.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Montell C., Courtois G., Eng C., Berk A. Complete transformation by adenovirus 2 requires both E1A proteins. Cell. 1984 Apr;36(4):951–961. doi: 10.1016/0092-8674(84)90045-x. [DOI] [PubMed] [Google Scholar]
  29. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. doi: 10.1038/295380a0. [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. Ngai J., Bond V. C., Wold B. J., Lazarides E. Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences. Mol Cell Biol. 1987 Nov;7(11):3955–3970. doi: 10.1128/mcb.7.11.3955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Osborne T. F., Arvidson D. N., Tyau E. S., Dunsworth-Browne M., Berk A. J. Transcription control region within the protein-coding portion of adenovirus E1A genes. Mol Cell Biol. 1984 Jul;4(7):1293–1305. doi: 10.1128/mcb.4.7.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Osborne T. F., Schell R. E., Burch-Jaffe E., Berget S. J., Berk A. J. Mapping a eukaryotic promoter: a DNA sequence required for in vivo expression of adenovirus pre-early functions. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1381–1385. doi: 10.1073/pnas.78.3.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paek I., Axel R. Glucocorticoids enhance stability of human growth hormone mRNA. Mol Cell Biol. 1987 Apr;7(4):1496–1507. doi: 10.1128/mcb.7.4.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pilder S., Moore M., Logan J., Shenk T. The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Mol Cell Biol. 1986 Feb;6(2):470–476. doi: 10.1128/mcb.6.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Raychaudhuri P., Rooney R., Nevins J. R. Identification of an E1A-inducible cellular factor that interacts with regulatory sequences within the adenovirus E4 promoter. EMBO J. 1987 Dec 20;6(13):4073–4081. doi: 10.1002/j.1460-2075.1987.tb02753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shiroki K., Saito I., Maruyama K., Fukui Y., Imatani Y., Oda K. I., Shimojo H. Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene. J Virol. 1983 Mar;45(3):1074–1082. doi: 10.1128/jvi.45.3.1074-1082.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simon M. C., Kitchener K., Kao H. T., Hickey E., Weber L., Voellmy R., Heintz N., Nevins J. R. Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product. Mol Cell Biol. 1987 Aug;7(8):2884–2890. doi: 10.1128/mcb.7.8.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  40. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Staufenbiel M., Epple P., Deppert W. Progressive reorganization of the host cell cytoskeleton during adenovirus infection. J Virol. 1986 Dec;60(3):1186–1191. doi: 10.1128/jvi.60.3.1186-1191.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weinberg D. H., Ketner G. A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5383–5386. doi: 10.1073/pnas.80.17.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  44. Winberg G., Shenk T. Dissection of overlapping functions within the adenovirus type 5 E1A gene. EMBO J. 1984 Aug;3(8):1907–1912. doi: 10.1002/j.1460-2075.1984.tb02066.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wittek R., Hänggi M., Hiller G. Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J Virol. 1984 Feb;49(2):371–378. doi: 10.1128/jvi.49.2.371-378.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yoshida K., Venkatesh L., Kuppuswamy M., Chinnadurai G. Adenovirus transforming 19-kD T antigen has an enhancer-dependent trans-activation function and relieves enhancer repression mediated by viral and cellular genes. Genes Dev. 1987 Sep;1(7):645–658. doi: 10.1101/gad.1.7.645. [DOI] [PubMed] [Google Scholar]
  47. Yoshinaga S., Dean N., Han M., Berk A. J. Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J. 1986 Feb;5(2):343–354. doi: 10.1002/j.1460-2075.1986.tb04218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhai Z. H., Nickerson J. A., Krochmalnic G., Penman S. Alterations in nuclear matrix structure after adenovirus infection. J Virol. 1987 Apr;61(4):1007–1018. doi: 10.1128/jvi.61.4.1007-1018.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES