Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Jul 11;20(13):3287–3295. doi: 10.1093/nar/20.13.3287

Analysis of proteins binding to the proximal promoter region of the human cytomegalovirus IE-1/2 enhancer/promoter reveals both consensus and aberrant recognition sequences for transcription factors Sp1 and CREB.

D Lang 1, H Fickenscher 1, T Stamminger 1
PMCID: PMC312479  PMID: 1385862

Abstract

Expression of the immediate early 1 and 2 gene (IE-1/2) of human cytomegalovirus, an important pathogen in immunosuppressed patients, is controlled by a strong enhancer/promoter. To define the promoter domain within this large cis-active region of about 550 nucleotides, DNA-protein interactions were studied. DNase I footprinting experiments using procaryotically expressed transcription factor Sp1 revealed an extensive interaction of this transcription factor with both consensus and aberrant recognition elements within the IE-1/2 promoter region. Protection of these Sp1 binding sites could also be observed when nuclear extracts prepared from HeLa cells and permissive human fibroblast cells were used. After in vitro mutagenesis of Sp1 targets and transient expression of mutagenized CAT-expression plasmids, however, no significant reduction in CAT activities was found. By analyzing a series of 5' deletion mutants of the IE-1/2 promoter region, a strong cis-acting element was localized between nucleotides -94 and -78, upstream of sites that interact with Sp1. Gel retardation experiments demonstrated binding of recombinant transcription factor CREB to this motif which reveals it as an aberrant CREB recognition sequence. Thus, this study identifies several previously unknown binding sites for transcription factors Sp1 and CREB within the proximal promoter region of the IE-1/2 gene, which differ markedly in their relevance for constitutive promoter function.

Full text

PDF
3295

Images in this article

3289Image
on p.3289
3290Image
on p.3290
3291Image
on p.3291
3292Image
on p.3292
3293Image
on p.3293
3293Image
on p.3293

Selected References

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

  1. Ammendola R., Gounari F., Piaggio G., De Simone V., Cortese R. Transcription of the promoter of the rat NF-1 gene depends on the integrity of an Sp1 recognition site. Mol Cell Biol. 1990 Jan;10(1):387–390. doi: 10.1128/mcb.10.1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson G. M., Freytag S. O. Synergistic activation of a human promoter in vivo by transcription factor Sp1. Mol Cell Biol. 1991 Apr;11(4):1935–1943. doi: 10.1128/mcb.11.4.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berkowitz L. A., Gilman M. Z. Two distinct forms of active transcription factor CREB (cAMP response element binding protein). Proc Natl Acad Sci U S A. 1990 Jul;87(14):5258–5262. doi: 10.1073/pnas.87.14.5258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohmann D., Keller W., Dale T., Schöler H. R., Tebb G., Mattaj I. W. A transcription factor which binds to the enhancers of SV40, immunoglobulin heavy chain and U2 snRNA genes. Nature. 1987 Jan 15;325(6101):268–272. doi: 10.1038/325268a0. [DOI] [PubMed] [Google Scholar]
  5. Boshart M., Weber F., Jahn G., Dorsch-Häsler K., Fleckenstein B., Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985 Jun;41(2):521–530. doi: 10.1016/s0092-8674(85)80025-8. [DOI] [PubMed] [Google Scholar]
  6. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  7. Cherrington J. M., Khoury E. L., Mocarski E. S. Human cytomegalovirus ie2 negatively regulates alpha gene expression via a short target sequence near the transcription start site. J Virol. 1991 Feb;65(2):887–896. doi: 10.1128/jvi.65.2.887-896.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dorsch-Häsler K., Keil G. M., Weber F., Jasin M., Schaffner W., Koszinowski U. H. A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8325–8329. doi: 10.1073/pnas.82.24.8325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drew W. L. Cytomegalovirus infection in patients with AIDS. J Infect Dis. 1988 Aug;158(2):449–456. doi: 10.1093/infdis/158.2.449. [DOI] [PubMed] [Google Scholar]
  10. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  11. Ellinger S., Mach M., Korn K., Jahn G. Cleavage and purification of prokaryotically expressed HIV gag and env fusion proteins for detection of HIV antibodies in the ELISA. Virology. 1991 Feb;180(2):811–813. doi: 10.1016/0042-6822(91)90097-u. [DOI] [PubMed] [Google Scholar]
  12. Fickenscher H., Stamminger T., Rüger R., Fleckenstein B. The role of a repetitive palindromic sequence element in the human cytomegalovirus major immediate early enhancer. J Gen Virol. 1989 Jan;70(Pt 1):107–123. doi: 10.1099/0022-1317-70-1-107. [DOI] [PubMed] [Google Scholar]
  13. Ghazal P., Lubon H., Fleckenstein B., Hennighausen L. Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus enhancer. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3658–3662. doi: 10.1073/pnas.84.11.3658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ghazal P., Lubon H., Hennighausen L. Specific interactions between transcription factors and the promoter-regulatory region of the human cytomegalovirus major immediate-early gene. J Virol. 1988 Mar;62(3):1076–1079. doi: 10.1128/jvi.62.3.1076-1079.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ghazal P., Lubon H., Reynolds-Kohler C., Hennighausen L., Nelson J. A. Interactions between cellular regulatory proteins and a unique sequence region in the human cytomegalovirus major immediate-early promoter. Virology. 1990 Jan;174(1):18–25. doi: 10.1016/0042-6822(90)90049-w. [DOI] [PubMed] [Google Scholar]
  16. Gidoni D., Kadonaga J. T., Barrera-Saldaña H., Takahashi K., Chambon P., Tjian R. Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. Science. 1985 Nov 1;230(4725):511–517. doi: 10.1126/science.2996137. [DOI] [PubMed] [Google Scholar]
  17. Harrich D., Garcia J., Wu F., Mitsuyasu R., Gonazalez J., Gaynor R. Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1989 Jun;63(6):2585–2591. doi: 10.1128/jvi.63.6.2585-2591.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hennighausen L., Fleckenstein B. Nuclear factor 1 interacts with five DNA elements in the promoter region of the human cytomegalovirus major immediate early gene. EMBO J. 1986 Jun;5(6):1367–1371. doi: 10.1002/j.1460-2075.1986.tb04368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jeang K. T., Rawlins D. R., Rosenfeld P. J., Shero J. H., Kelly T. J., Hayward G. S. Multiple tandemly repeated binding sites for cellular nuclear factor 1 that surround the major immediate-early promoters of simian and human cytomegalovirus. J Virol. 1987 May;61(5):1559–1570. doi: 10.1128/jvi.61.5.1559-1570.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jones K. A., Kadonaga J. T., Luciw P. A., Tjian R. Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. Science. 1986 May 9;232(4751):755–759. doi: 10.1126/science.3008338. [DOI] [PubMed] [Google Scholar]
  21. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  22. Kadonaga J. T., Courey A. J., Ladika J., Tjian R. Distinct regions of Sp1 modulate DNA binding and transcriptional activation. Science. 1988 Dec 16;242(4885):1566–1570. doi: 10.1126/science.3059495. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Lehner R., Stamminger T., Mach M. Comparative sequence analysis of human cytomegalovirus strains. J Clin Microbiol. 1991 Nov;29(11):2494–2502. doi: 10.1128/jcm.29.11.2494-2502.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Letovsky J., Dynan W. S. Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence. Nucleic Acids Res. 1989 Apr 11;17(7):2639–2653. doi: 10.1093/nar/17.7.2639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li R., Knight J. D., Jackson S. P., Tjian R., Botchan M. R. Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell. 1991 May 3;65(3):493–505. doi: 10.1016/0092-8674(91)90467-d. [DOI] [PubMed] [Google Scholar]
  27. Liu B., Hermiston T. W., Stinski M. F. A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2. J Virol. 1991 Feb;65(2):897–903. doi: 10.1128/jvi.65.2.897-903.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lubon H., Ghazal P., Hennighausen L., Reynolds-Kohler C., Lockshin C., Nelson J. Cell-specific activity of the modulator region in the human cytomegalovirus major immediate-early gene. Mol Cell Biol. 1989 Mar;9(3):1342–1345. doi: 10.1128/mcb.9.3.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Malone C. L., Vesole D. H., Stinski M. F. Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins. J Virol. 1990 Apr;64(4):1498–1506. doi: 10.1128/jvi.64.4.1498-1506.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Niller H. H., Hennighausen L. Formation of several specific nucleoprotein complexes on the human cytomegalovirus immediate early enhancer. Nucleic Acids Res. 1991 Jul 11;19(13):3715–3721. doi: 10.1093/nar/19.13.3715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pizzorno M. C., Hayward G. S. The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site. J Virol. 1990 Dec;64(12):6154–6165. doi: 10.1128/jvi.64.12.6154-6165.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Puchtler E., Stamminger T. An inducible promoter mediates abundant expression from the immediate-early 2 gene region of human cytomegalovirus at late times after infection. J Virol. 1991 Nov;65(11):6301–6306. doi: 10.1128/jvi.65.11.6301-6306.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sambucetti L. C., Cherrington J. M., Wilkinson G. W., Mocarski E. S. NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J. 1989 Dec 20;8(13):4251–4258. doi: 10.1002/j.1460-2075.1989.tb08610.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stamminger T., Fickenscher H., Fleckenstein B. Cell type-specific induction of the major immediate early enhancer of human cytomegalovirus by cyclic AMP. J Gen Virol. 1990 Jan;71(Pt 1):105–113. doi: 10.1099/0022-1317-71-1-105. [DOI] [PubMed] [Google Scholar]
  35. Stamminger T., Fleckenstein B. Immediate-early transcription regulation of human cytomegalovirus. Curr Top Microbiol Immunol. 1990;154:3–19. doi: 10.1007/978-3-642-74980-3_1. [DOI] [PubMed] [Google Scholar]
  36. Stamminger T., Puchtler E., Fleckenstein B. Discordant expression of the immediate-early 1 and 2 gene regions of human cytomegalovirus at early times after infection involves posttranscriptional processing events. J Virol. 1991 May;65(5):2273–2282. doi: 10.1128/jvi.65.5.2273-2282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stenberg R. M., Depto A. S., Fortney J., Nelson J. A. Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region. J Virol. 1989 Jun;63(6):2699–2708. doi: 10.1128/jvi.63.6.2699-2708.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Urban M., Britt W., Mach M. The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific. J Virol. 1992 Mar;66(3):1303–1311. doi: 10.1128/jvi.66.3.1303-1311.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES