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. 1994 Feb;14(2):1333–1346. doi: 10.1128/mcb.14.2.1333

The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

S Obert 1, R J O'Connor 1, S Schmid 1, P Hearing 1
PMCID: PMC358488  PMID: 8289811

Abstract

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

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Selected References

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

  1. Bagchi S., Raychaudhuri P., Nevins J. R. Adenovirus E1A proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A trans-activation. Cell. 1990 Aug 24;62(4):659–669. doi: 10.1016/0092-8674(90)90112-r. [DOI] [PubMed] [Google Scholar]
  2. Bagchi S., Weinmann R., Raychaudhuri P. The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell. 1991 Jun 14;65(6):1063–1072. doi: 10.1016/0092-8674(91)90558-g. [DOI] [PubMed] [Google Scholar]
  3. Bandara L. R., Adamczewski J. P., Hunt T., La Thangue N. B. Cyclin A and the retinoblastoma gene product complex with a common transcription factor. Nature. 1991 Jul 18;352(6332):249–251. doi: 10.1038/352249a0. [DOI] [PubMed] [Google Scholar]
  4. Bandara L. R., Buck V. M., Zamanian M., Johnston L. H., La Thangue N. B. Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F. EMBO J. 1993 Nov;12(11):4317–4324. doi: 10.1002/j.1460-2075.1993.tb06116.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bandara L. R., La Thangue N. B. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature. 1991 Jun 6;351(6326):494–497. doi: 10.1038/351494a0. [DOI] [PubMed] [Google Scholar]
  6. Blaber M., Zhang X. J., Matthews B. W. Structural basis of amino acid alpha helix propensity. Science. 1993 Jun 11;260(5114):1637–1640. doi: 10.1126/science.8503008. [DOI] [PubMed] [Google Scholar]
  7. Blake M. C., Azizkhan J. C. Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo. Mol Cell Biol. 1989 Nov;9(11):4994–5002. doi: 10.1128/mcb.9.11.4994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cao L., Faha B., Dembski M., Tsai L. H., Harlow E., Dyson N. Independent binding of the retinoblastoma protein and p107 to the transcription factor E2F. Nature. 1992 Jan 9;355(6356):176–179. doi: 10.1038/355176a0. [DOI] [PubMed] [Google Scholar]
  9. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  10. Chellappan S., Kraus V. B., Kroger B., Munger K., Howley P. M., Phelps W. C., Nevins J. R. Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4549–4553. doi: 10.1073/pnas.89.10.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chittenden T., Livingston D. M., Kaelin W. G., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. doi: 10.1016/0092-8674(91)90559-h. [DOI] [PubMed] [Google Scholar]
  12. Cutt J. R., Shenk T., Hearing P. Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells. J Virol. 1987 Feb;61(2):543–552. doi: 10.1128/jvi.61.2.543-552.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis R. L., Cheng P. F., Lassar A. B., Weintraub H. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell. 1990 Mar 9;60(5):733–746. doi: 10.1016/0092-8674(90)90088-v. [DOI] [PubMed] [Google Scholar]
  14. Devoto S. H., Mudryj M., Pines J., Hunter T., Nevins J. R. A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin A complex. Cell. 1992 Jan 10;68(1):167–176. doi: 10.1016/0092-8674(92)90215-x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Girling R., Partridge J. F., Bandara L. R., Burden N., Totty N. F., Hsuan J. J., La Thangue N. B. A new component of the transcription factor DRTF1/E2F. Nature. 1993 Mar 4;362(6415):83–87. doi: 10.1038/362083a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  19. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  20. Halbert D. N., Cutt J. R., Shenk T. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J Virol. 1985 Oct;56(1):250–257. doi: 10.1128/jvi.56.1.250-257.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hardy S., Shenk T. E2F from adenovirus-infected cells binds cooperatively to DNA containing two properly oriented and spaced recognition sites. Mol Cell Biol. 1989 Oct;9(10):4495–4506. doi: 10.1128/mcb.9.10.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Helin K., Lees J. A., Vidal M., Dyson N., Harlow E., Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. doi: 10.1016/0092-8674(92)90107-n. [DOI] [PubMed] [Google Scholar]
  23. Helin K., Wu C. L., Fattaey A. R., Lees J. A., Dynlacht B. D., Ngwu C., Harlow E. Heterodimerization of the transcription factors E2F-1 and DP-1 leads to cooperative trans-activation. Genes Dev. 1993 Oct;7(10):1850–1861. doi: 10.1101/gad.7.10.1850. [DOI] [PubMed] [Google Scholar]
  24. Hiebert S. W., Chellappan S. P., Horowitz J. M., Nevins J. R. The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev. 1992 Feb;6(2):177–185. doi: 10.1101/gad.6.2.177. [DOI] [PubMed] [Google Scholar]
  25. Hiebert S. W., Lipp M., Nevins J. R. E1A-dependent trans-activation of the human MYC promoter is mediated by the E2F factor. Proc Natl Acad Sci U S A. 1989 May;86(10):3594–3598. doi: 10.1073/pnas.86.10.3594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huang M. M., Hearing P. The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex. Genes Dev. 1989 Nov;3(11):1699–1710. doi: 10.1101/gad.3.11.1699. [DOI] [PubMed] [Google Scholar]
  27. Huber H. E., Edwards G., Goodhart P. J., Patrick D. R., Huang P. S., Ivey-Hoyle M., Barnett S. F., Oliff A., Heimbrook D. C. Transcription factor E2F binds DNA as a heterodimer. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3525–3529. doi: 10.1073/pnas.90.8.3525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  29. Kovesdi I., Reichel R., Nevins J. R. E1A transcription induction: enhanced binding of a factor to upstream promoter sequences. Science. 1986 Feb 14;231(4739):719–722. doi: 10.1126/science.2935935. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Kovesdi I., Reichel R., Nevins J. R. Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2180–2184. doi: 10.1073/pnas.84.8.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  34. Lees E., Faha B., Dulic V., Reed S. I., Harlow E. Cyclin E/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner. Genes Dev. 1992 Oct;6(10):1874–1885. doi: 10.1101/gad.6.10.1874. [DOI] [PubMed] [Google Scholar]
  35. Manohar C. F., Kratochvil J., Thimmapaya B. The adenovirus EII early promoter has multiple EIA-sensitive elements, two of which function cooperatively in basal and virus-induced transcription. J Virol. 1990 Jun;64(6):2457–2466. doi: 10.1128/jvi.64.6.2457-2466.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Marton M. J., Baim S. B., Ornelles D. A., Shenk T. The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA. J Virol. 1990 May;64(5):2345–2359. doi: 10.1128/jvi.64.5.2345-2359.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mudryj M., Devoto S. H., Hiebert S. W., Hunter T., Pines J., Nevins J. R. Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A. Cell. 1991 Jun 28;65(7):1243–1253. doi: 10.1016/0092-8674(91)90019-u. [DOI] [PubMed] [Google Scholar]
  38. Mudryj M., Hiebert S. W., Nevins J. R. A role for the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway. EMBO J. 1990 Jul;9(7):2179–2184. doi: 10.1002/j.1460-2075.1990.tb07387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Murthy S. C., Bhat G. P., Thimmappaya B. Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2230–2234. doi: 10.1073/pnas.82.8.2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Neill S. D., Nevins J. R. Genetic analysis of the adenovirus E4 6/7 trans activator: interaction with E2F and induction of a stable DNA-protein complex are critical for activity. J Virol. 1991 Oct;65(10):5364–5373. doi: 10.1128/jvi.65.10.5364-5373.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nevins J. R. Transcriptional regulation. A closer look at E2F. Nature. 1992 Jul 30;358(6385):375–376. doi: 10.1038/358375a0. [DOI] [PubMed] [Google Scholar]
  42. O'Connor R. J., Hearing P. The C-terminal 70 amino acids of the adenovirus E4-ORF6/7 protein are essential and sufficient for E2F complex formation. Nucleic Acids Res. 1991 Dec 11;19(23):6579–6586. doi: 10.1093/nar/19.23.6579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
  44. Pagano M., Draetta G., Jansen-Dürr P. Association of cdk2 kinase with the transcription factor E2F during S phase. Science. 1992 Feb 28;255(5048):1144–1147. doi: 10.1126/science.1312258. [DOI] [PubMed] [Google Scholar]
  45. Qin X. Q., Chittenden T., Livingston D. M., Kaelin W. G., Jr Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 1992 Jun;6(6):953–964. doi: 10.1101/gad.6.6.953. [DOI] [PubMed] [Google Scholar]
  46. Raychaudhuri P., Bagchi S., Neill S. D., Nevins J. R. Activation of the E2F transcription factor in adenovirus-infected cells involves E1A-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product. J Virol. 1990 Jun;64(6):2702–2710. doi: 10.1128/jvi.64.6.2702-2710.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  48. Shan B., Zhu X., Chen P. L., Durfee T., Yang Y., Sharp D., Lee W. H. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. Mol Cell Biol. 1992 Dec;12(12):5620–5631. doi: 10.1128/mcb.12.12.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shirodkar S., Ewen M., DeCaprio J. A., Morgan J., Livingston D. M., Chittenden T. The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner. Cell. 1992 Jan 10;68(1):157–166. doi: 10.1016/0092-8674(92)90214-w. [DOI] [PubMed] [Google Scholar]
  50. SivaRaman L., Thimmappaya B. Two promoter-specific host factors interact with adjacent sequences in an EIA-inducible adenovirus promoter. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6112–6116. doi: 10.1073/pnas.84.17.6112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Slansky J. E., Li Y., Kaelin W. G., Farnham P. J. A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. Mol Cell Biol. 1993 Mar;13(3):1610–1618. doi: 10.1128/mcb.13.3.1610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stinski M. F., Roehr T. J. Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components. J Virol. 1985 Aug;55(2):431–441. doi: 10.1128/jvi.55.2.431-441.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  54. Thalmeier K., Synovzik H., Mertz R., Winnacker E. L., Lipp M. Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter. Genes Dev. 1989 Apr;3(4):527–536. doi: 10.1101/gad.3.4.527. [DOI] [PubMed] [Google Scholar]
  55. Virtanen A., Gilardi P., Näslund A., LeMoullec J. M., Pettersson U., Perricaudet M. mRNAs from human adenovirus 2 early region 4. J Virol. 1984 Sep;51(3):822–831. doi: 10.1128/jvi.51.3.822-831.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Webster N. J., Green S., Tasset D., Ponglikitmongkol M., Chambon P. The transcriptional activation function located in the hormone-binding domain of the human oestrogen receptor is not encoded in a single exon. EMBO J. 1989 May;8(5):1441–1446. doi: 10.1002/j.1460-2075.1989.tb03526.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weintraub S. J., Prater C. A., Dean D. C. Retinoblastoma protein switches the E2F site from positive to negative element. Nature. 1992 Jul 16;358(6383):259–261. doi: 10.1038/358259a0. [DOI] [PubMed] [Google Scholar]
  58. Yee A. S., Raychaudhuri P., Jakoi L., Nevins J. R. The adenovirus-inducible factor E2F stimulates transcription after specific DNA binding. Mol Cell Biol. 1989 Feb;9(2):578–585. doi: 10.1128/mcb.9.2.578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yee A. S., Reichel R., Kovesdi I., Nevins J. R. Promoter interaction of the E1A-inducible factor E2F and its potential role in the formation of a multi-component complex. EMBO J. 1987 Jul;6(7):2061–2068. doi: 10.1002/j.1460-2075.1987.tb02471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zajchowski D. A., Boeuf H., Kédinger C. The adenovirus-2 early EIIa transcription unit possesses two overlapping promoters with different sequence requirements for EIa-dependent stimulation. EMBO J. 1985 May;4(5):1293–1300. doi: 10.1002/j.1460-2075.1985.tb03775.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zamanian M., La Thangue N. B. Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor. EMBO J. 1992 Jul;11(7):2603–2610. doi: 10.1002/j.1460-2075.1992.tb05325.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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