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. 1995 Dec;69(12):7612–7621. doi: 10.1128/jvi.69.12.7612-7621.1995

Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter.

L Huang 1, M F Stinski 1
PMCID: PMC189701  PMID: 7494269

Abstract

We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect.

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Selected References

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  1. Arlt H., Lang D., Gebert S., Stamminger T. Identification of binding sites for the 86-kilodalton IE2 protein of human cytomegalovirus within an IE2-responsive viral early promoter. J Virol. 1994 Jul;68(7):4117–4125. doi: 10.1128/jvi.68.7.4117-4125.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berkhout B., Jeang K. T. Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1992 Jan;66(1):139–149. doi: 10.1128/jvi.66.1.139-149.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caswell R., Hagemeier C., Chiou C. J., Hayward G., Kouzarides T., Sinclair J. The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation. J Gen Virol. 1993 Dec;74(Pt 12):2691–2698. doi: 10.1099/0022-1317-74-12-2691. [DOI] [PubMed] [Google Scholar]
  4. Chang C. P., Malone C. L., Stinski M. F. A human cytomegalovirus early gene has three inducible promoters that are regulated differentially at various times after infection. J Virol. 1989 Jan;63(1):281–290. doi: 10.1128/jvi.63.1.281-290.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Chiou C. J., Zong J., Waheed I., Hayward G. S. Identification and mapping of dimerization and DNA-binding domains in the C terminus of the IE2 regulatory protein of human cytomegalovirus. J Virol. 1993 Oct;67(10):6201–6214. doi: 10.1128/jvi.67.10.6201-6214.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeMarchi J. M. Post-transcriptional control of human cytomegalovirus gene expression. Virology. 1983 Jan 30;124(2):390–402. doi: 10.1016/0042-6822(83)90355-0. [DOI] [PubMed] [Google Scholar]
  9. DeMarchi J. M., Schmidt C. A., Kaplan A. S. Patterns of transcription of human cytomegalovirus in permissively infected cells. J Virol. 1980 Aug;35(2):277–286. doi: 10.1128/jvi.35.2.277-286.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Depto A. S., Stenberg R. M. Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products. J Virol. 1989 Mar;63(3):1232–1238. doi: 10.1128/jvi.63.3.1232-1238.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Dong G., Broker T. R., Chow L. T. Human papillomavirus type 11 E2 proteins repress the homologous E6 promoter by interfering with the binding of host transcription factors to adjacent elements. J Virol. 1994 Feb;68(2):1115–1127. doi: 10.1128/jvi.68.2.1115-1127.1994. [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. Hagemeier C., Caswell R., Hayhurst G., Sinclair J., Kouzarides T. Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J. 1994 Jun 15;13(12):2897–2903. doi: 10.1002/j.1460-2075.1994.tb06584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hagemeier C., Walker S. M., Sissons P. J., Sinclair J. H. The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements. J Gen Virol. 1992 Sep;73(Pt 9):2385–2393. doi: 10.1099/0022-1317-73-9-2385. [DOI] [PubMed] [Google Scholar]
  16. Hagemeier C., Walker S., Caswell R., Kouzarides T., Sinclair J. The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID. J Virol. 1992 Jul;66(7):4452–4456. doi: 10.1128/jvi.66.7.4452-4456.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  18. Hermiston T. W., Malone C. L., Witte P. R., Stinski M. F. Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter. J Virol. 1987 Oct;61(10):3214–3221. doi: 10.1128/jvi.61.10.3214-3221.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horikoshi N., Maguire K., Kralli A., Maldonado E., Reinberg D., Weinmann R. Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5124–5128. doi: 10.1073/pnas.88.12.5124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hou X., Johnson A. C., Rosner M. R. Identification of an epidermal growth factor receptor transcriptional repressor. J Biol Chem. 1994 Feb 11;269(6):4307–4312. [PubMed] [Google Scholar]
  21. Huang L., Malone C. L., Stinski M. F. A human cytomegalovirus early promoter with upstream negative and positive cis-acting elements: IE2 negates the effect of the negative element, and NF-Y binds to the positive element. J Virol. 1994 Apr;68(4):2108–2117. doi: 10.1128/jvi.68.4.2108-2117.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Imbalzano A. N., DeLuca N. A. Substitution of a TATA box from a herpes simplex virus late gene in the viral thymidine kinase promoter alters ICP4 inducibility but not temporal expression. J Virol. 1992 Sep;66(9):5453–5463. doi: 10.1128/jvi.66.9.5453-5463.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ingles C. J., Shales M., Cress W. D., Triezenberg S. J., Greenblatt J. Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature. 1991 Jun 13;351(6327):588–590. doi: 10.1038/351588a0. [DOI] [PubMed] [Google Scholar]
  24. Jupp R., Hoffmann S., Stenberg R. M., Nelson J. A., Ghazal P. Human cytomegalovirus IE86 protein interacts with promoter-bound TATA-binding protein via a specific region distinct from the autorepression domain. J Virol. 1993 Dec;67(12):7539–7546. doi: 10.1128/jvi.67.12.7539-7546.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Klucher K. M., Rabert D. K., Spector D. H. Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene. J Virol. 1989 Dec;63(12):5334–5343. doi: 10.1128/jvi.63.12.5334-5343.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klucher K. M., Sommer M., Kadonaga J. T., Spector D. H. In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins. Mol Cell Biol. 1993 Feb;13(2):1238–1250. doi: 10.1128/mcb.13.2.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Klucher K. M., Spector D. H. The human cytomegalovirus 2.7-kilobase RNA promoter contains a functional binding site for the adenovirus major late transcription factor. J Virol. 1990 Sep;64(9):4189–4198. doi: 10.1128/jvi.64.9.4189-4198.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kuwabara M. D., Sigman D. S. Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry. 1987 Nov 17;26(23):7234–7238. doi: 10.1021/bi00397a006. [DOI] [PubMed] [Google Scholar]
  29. Lang D., Stamminger T. Minor groove contacts are essential for an interaction of the human cytomegalovirus IE2 protein with its DNA target. Nucleic Acids Res. 1994 Aug 25;22(16):3331–3338. doi: 10.1093/nar/22.16.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lieberman P. M., Berk A. J. The Zta trans-activator protein stabilizes TFIID association with promoter DNA by direct protein-protein interaction. Genes Dev. 1991 Dec;5(12B):2441–2454. doi: 10.1101/gad.5.12b.2441. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Liu B., Stinski M. F. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol. 1992 Jul;66(7):4434–4444. doi: 10.1128/jvi.66.7.4434-4444.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lukac D. M., Manuppello J. R., Alwine J. C. Transcriptional activation by the human cytomegalovirus immediate-early proteins: requirements for simple promoter structures and interactions with multiple components of the transcription complex. J Virol. 1994 Aug;68(8):5184–5193. doi: 10.1128/jvi.68.8.5184-5193.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Macias M. P., Stinski M. F. An in vitro system for human cytomegalovirus immediate early 2 protein (IE2)-mediated site-dependent repression of transcription and direct binding of IE2 to the major immediate early promoter. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):707–711. doi: 10.1073/pnas.90.2.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. McDonough S. H., Spector D. H. Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169. Virology. 1983 Feb;125(1):31–46. doi: 10.1016/0042-6822(83)90061-2. [DOI] [PubMed] [Google Scholar]
  37. Olsen H. S., Rosen C. A. Contribution of the TATA motif to Tat-mediated transcriptional activation of human immunodeficiency virus gene expression. J Virol. 1992 Sep;66(9):5594–5597. doi: 10.1128/jvi.66.9.5594-5597.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Pizzorno M. C., Mullen M. A., Chang Y. N., Hayward G. S. The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal. J Virol. 1991 Jul;65(7):3839–3852. doi: 10.1128/jvi.65.7.3839-3852.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reifel-Miller A. E., Berg D. T., Grinnell B. W. Repression of enhancer activity by the adenovirus E1A tumor protein(s) is mediated through a novel HeLa cell nuclear factor. J Biol Chem. 1991 Jul 25;266(21):13873–13882. [PubMed] [Google Scholar]
  41. Renkawitz R. Transcriptional repression in eukaryotes. Trends Genet. 1990 Jun;6(6):192–197. doi: 10.1016/0168-9525(90)90176-7. [DOI] [PubMed] [Google Scholar]
  42. Schwartz R., Sommer M. H., Scully A., Spector D. H. Site-specific binding of the human cytomegalovirus IE2 86-kilodalton protein to an early gene promoter. J Virol. 1994 Sep;68(9):5613–5622. doi: 10.1128/jvi.68.9.5613-5622.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Skalnik D. G., Strauss E. C., Orkin S. H. CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter. J Biol Chem. 1991 Sep 5;266(25):16736–16744. [PubMed] [Google Scholar]
  44. Smith C. A., Bates P., Rivera-Gonzalez R., Gu B., DeLuca N. A. ICP4, the major transcriptional regulatory protein of herpes simplex virus type 1, forms a tripartite complex with TATA-binding protein and TFIIB. J Virol. 1993 Aug;67(8):4676–4687. doi: 10.1128/jvi.67.8.4676-4687.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sommer M. H., Scully A. L., Spector D. H. Transactivation by the human cytomegalovirus IE2 86-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein. J Virol. 1994 Oct;68(10):6223–6231. doi: 10.1128/jvi.68.10.6223-6231.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Spector D. H., Klucher K. M., Rabert D. K., Wright D. A. Human cytomegalovirus early gene expression. Curr Top Microbiol Immunol. 1990;154:21–45. doi: 10.1007/978-3-642-74980-3_2. [DOI] [PubMed] [Google Scholar]
  47. Staprans S. I., Rabert D. K., Spector D. H. Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene. J Virol. 1988 Sep;62(9):3463–3473. doi: 10.1128/jvi.62.9.3463-3473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stenberg R. M., Fortney J., Barlow S. W., Magrane B. P., Nelson J. A., Ghazal P. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains. J Virol. 1990 Apr;64(4):1556–1565. doi: 10.1128/jvi.64.4.1556-1565.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stinski M. F. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides. J Virol. 1978 Jun;26(3):686–701. doi: 10.1128/jvi.26.3.686-701.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stringer K. F., Ingles C. J., Greenblatt J. Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature. 1990 Jun 28;345(6278):783–786. doi: 10.1038/345783a0. [DOI] [PubMed] [Google Scholar]
  51. Wade E. J., Klucher K. M., Spector D. H. An AP-1 binding site is the predominant cis-acting regulatory element in the 1.2-kilobase early RNA promoter of human cytomegalovirus. J Virol. 1992 Apr;66(4):2407–2417. doi: 10.1128/jvi.66.4.2407-2417.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wathen M. W., Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982 Feb;41(2):462–477. doi: 10.1128/jvi.41.2.462-477.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wathen M. W., Thomsen D. R., Stinski M. F. Temporal regulation of human cytomegalovirus transcription at immediate early and early times after infection. J Virol. 1981 May;38(2):446–459. doi: 10.1128/jvi.38.2.446-459.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. West M., Mikovits J., Princler G., Liu Y. L., Ruscetti F. W., Kung H. F., Raziuddin Characterization and purification of a novel transcriptional repressor from HeLa cell nuclear extracts recognizing the negative regulatory element region of human immunodeficiency virus-1 long terminal repeat. J Biol Chem. 1992 Dec 15;267(35):24948–24952. [PubMed] [Google Scholar]
  55. Williams M., Brys A., Weiner A. M., Maizels N. A rapid method for determining the molecular weight of a protein bound to nucleic acid in a mobility shift assay. Nucleic Acids Res. 1992 Sep 25;20(18):4935–4936. doi: 10.1093/nar/20.18.4935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yang T. H., Tsai W. H., Lee Y. M., Lei H. Y., Lai M. Y., Chen D. S., Yeh N. H., Lee S. C. Purification and characterization of nucleolin and its identification as a transcription repressor. Mol Cell Biol. 1994 Sep;14(9):6068–6074. doi: 10.1128/mcb.14.9.6068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yeung K. C., Stoltzfus C. M., Stinski M. F. Mutations of the human cytomegalovirus immediate-early 2 protein defines regions and amino acid motifs important in transactivation of transcription from the HIV-1 LTR promoter. Virology. 1993 Aug;195(2):786–792. doi: 10.1006/viro.1993.1431. [DOI] [PubMed] [Google Scholar]

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