Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Oct;83(20):7688–7692. doi: 10.1073/pnas.83.20.7688

Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein.

J P Thompson, A Granoff, D B Willis
PMCID: PMC386786  PMID: 3463992

Abstract

The high degree of methylation of the frog virus 3 (FV3) genome suggests that FV3-infected cells are capable of transcribing highly methylated DNA. We tested this hypothesis by assaying the transcriptional activity of adenovirus promoters known to be inhibited by methylation. Plasmid constructs containing the E1a and E2aE promoters of adenovirus type 12 linked to the gene for chloramphenicol acetyltransferase [(CAT) EC 2.3.1.28], when methylated and introduced into eukaryotic cells, promoted CAT synthesis only when the cells were subsequently infected with FV3. Mapping of transcriptional initiation sites revealed that the same sites in the E1a promoter were used for the initiation of transcription in uninfected and infected cells. Moreover, Southern blots showed that transfected plasmid DNA from FV3-infected cells was not demethylated. The absence of CAT-specific RNA in transfected cells infected with FV3 in the presence of protein synthesis inhibitors demonstrated that a virus-induced protein was responsible for the trans-activation. Inhibition of transcription from the methylated template by alpha-amanitin indicated that a functional host RNA polymerase II is required for transcription of methylated DNA in FV3-infected cells. The virus-induced trans-acting protein presumably alters either host RNA polymerase II or the methylated DNA template to allow transcription from the methylated adenovirus promoters.

Full text

PDF
7688

Images in this article

7689Image
on p.7689
7690Image
on p.7690
7690Image
on p.7690
7691Image
on p.7691
7691Image
on p.7691
7691Image
on p.7691

Selected References

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

  1. 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]
  2. Campadelli-Fiume G., Costanzo F., Foa'-Tomasi L., La Placa M. Modifications of cellular RNA-polymerase II after infection with frog virus 3. J Gen Virol. 1975 Jun;27(3):391–394. doi: 10.1099/0022-1317-27-3-391. [DOI] [PubMed] [Google Scholar]
  3. Chan V. L., Whitmore G. F., Siminovitch L. Mammalian cells with altered forms of RNA polymerase II. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3119–3123. doi: 10.1073/pnas.69.11.3119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Doerfler W. DNA methylation and gene activity. Annu Rev Biochem. 1983;52:93–124. doi: 10.1146/annurev.bi.52.070183.000521. [DOI] [PubMed] [Google Scholar]
  6. Doerfler W. DNA methylation and its functional significance: studies on the adenovirus system. Curr Top Microbiol Immunol. 1984;108:79–98. doi: 10.1007/978-3-642-69370-0_6. [DOI] [PubMed] [Google Scholar]
  7. Doerfler W., Langner K. D., Knebel D., Weyer U., Dobrzanski P., Knust-Kron B. Site-specific promoter methylations and gene inactivation. Prog Clin Biol Res. 1985;198:133–155. [PubMed] [Google Scholar]
  8. Flavell R. A., Grosveld F., Busslinger M., de Boer E., Kioussis D., Mellor A. L., Golden L., Weiss E., Hurst J., Bud H. Structure and expression of the human globin genes and murine histocompatibility antigen genes. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1067–1078. doi: 10.1101/sqb.1983.047.01.119. [DOI] [PubMed] [Google Scholar]
  9. Goorha R. Frog virus 3 requires RNA polymerase II for its replication. J Virol. 1981 Jan;37(1):496–499. doi: 10.1128/jvi.37.1.496-499.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Grodzicker T., Klessig D. F. Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA. Cell. 1980 Sep;21(2):453–463. doi: 10.1016/0092-8674(80)90482-1. [DOI] [PubMed] [Google Scholar]
  13. Haigh L. S., Owens B. B., Hellewell O. S., Ingram V. M. DNA methylation in chicken alpha-globin gene expression. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5332–5336. doi: 10.1073/pnas.79.17.5332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Imperiale M. J., Hart R. P., Nevins J. R. An enhancer-like element in the adenovirus E2 promoter contains sequences essential for uninduced and E1A-induced transcription. Proc Natl Acad Sci U S A. 1985 Jan;82(2):381–385. doi: 10.1073/pnas.82.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keshet I., Yisraeli J., Cedar H. Effect of regional DNA methylation on gene expression. Proc Natl Acad Sci U S A. 1985 May;82(9):2560–2564. doi: 10.1073/pnas.82.9.2560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kruczek I., Doerfler W. Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters: effect of promoter methylation on gene expression. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7586–7590. doi: 10.1073/pnas.80.24.7586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kruczek I., Doerfler W. The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression. EMBO J. 1982;1(4):409–414. doi: 10.1002/j.1460-2075.1982.tb01183.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. La Volpe A., Taggart M., Macleod D., Bird A. Coupled demethylation of sites in a conserved sequence of Xenopus ribosomal DNA. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):585–592. doi: 10.1101/sqb.1983.047.01.069. [DOI] [PubMed] [Google Scholar]
  20. Langner K. D., Vardimon L., Renz D., Doerfler W. DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2. Proc Natl Acad Sci U S A. 1984 May;81(10):2950–2954. doi: 10.1073/pnas.81.10.2950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Naegele R. F., Granoff A. Viruses and renal carcinoma of Rana pipiens. XI. Isolation of frog virus 3 temperature-sensitive mutants; complementation and genetic recombination. Virology. 1971 May;44(2):286–295. doi: 10.1016/0042-6822(71)90260-1. [DOI] [PubMed] [Google Scholar]
  23. Nevins J. R. Definition and mapping of adenovirus 2 nuclear transcription. Methods Enzymol. 1980;65(1):768–785. doi: 10.1016/s0076-6879(80)65072-1. [DOI] [PubMed] [Google Scholar]
  24. Osborne T. F., Gaynor R. B., Berk A. J. The TATA homology and the mRNA 5' untranslated sequence are not required for expression of essential adenovirus E1A functions. Cell. 1982 May;29(1):139–148. doi: 10.1016/0092-8674(82)90098-8. [DOI] [PubMed] [Google Scholar]
  25. Ott M. O., Sperling L., Cassio D., Levilliers J., Sala-Trepat J., Weiss M. C. Undermethylation at the 5' end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture. Cell. 1982 Oct;30(3):825–833. doi: 10.1016/0092-8674(82)90287-2. [DOI] [PubMed] [Google Scholar]
  26. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Price R., Penman S. Transcription of the adenovirus genome by an -amanitine-sensitive ribonucleic acid polymerase in HeLa cells. J Virol. 1972 Apr;9(4):621–626. doi: 10.1128/jvi.9.4.621-626.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  29. Riggs A. D., Jones P. A. 5-methylcytosine, gene regulation, and cancer. Adv Cancer Res. 1983;40:1–30. doi: 10.1016/s0065-230x(08)60678-8. [DOI] [PubMed] [Google Scholar]
  30. Sambrook J., Greene R., Stringer J., Mitchison T., Hu S. L., Botchan M. Analysis of the sites of integration of viral DNA sequences in rat cells transformed by adenovirus 2 or SV40. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):569–584. doi: 10.1101/sqb.1980.044.01.059. [DOI] [PubMed] [Google Scholar]
  31. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  32. Stein R., Sciaky-Gallili N., Razin A., Cedar H. Pattern of methylation of two genes coding for housekeeping functions. Proc Natl Acad Sci U S A. 1983 May;80(9):2422–2426. doi: 10.1073/pnas.80.9.2422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sugisaki H., Sugimoto K., Takanami M., Shiroki K., Saito I., Shimojo H., Sawada Y., Uemizu Y., Uesugi S., Fujinaga K. Structure and gene organization in the transformed Hind III-G fragment of Ad12. Cell. 1980 Jul;20(3):777–786. doi: 10.1016/0092-8674(80)90324-4. [DOI] [PubMed] [Google Scholar]
  34. Waalwijk C., Flavell R. A. MspI, an isoschizomer of hpaII which cleaves both unmethylated and methylated hpaII sites. Nucleic Acids Res. 1978 Sep;5(9):3231–3236. doi: 10.1093/nar/5.9.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wallace R. D., Kates J. State of adenovirus 2 deoxyribonucleic acid in the nucleus and its mode of transcription: studies with isolated viral deoxyribonucleic acid-protein complexes and isolated nuclei. J Virol. 1972 Apr;9(4):627–635. doi: 10.1128/jvi.9.4.627-635.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilks A. F., Cozens P. J., Mattaj I. W., Jost J. P. Estrogen induces a demethylation at the 5' end region of the chicken vitellogenin gene. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4252–4255. doi: 10.1073/pnas.79.14.4252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Willis D. B., Goorha R., Granoff A. DNA methyltransferase induced by frog virus 3. J Virol. 1984 Jan;49(1):86–91. doi: 10.1128/jvi.49.1.86-91.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Willis D. B., Granoff A. Frog virus 3 DNA is heavily methylated at CpG sequences. Virology. 1980 Nov;107(1):250–257. doi: 10.1016/0042-6822(80)90290-1. [DOI] [PubMed] [Google Scholar]
  39. Willis D. B., Granoff A. Macromolecular synthesis in cells infected by frog virus 3. IX. Two temporal classes of early viral RNA. Virology. 1978 May 15;86(2):443–453. doi: 10.1016/0042-6822(78)90084-3. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES