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. 1994 Mar 15;13(6):1460–1466. doi: 10.1002/j.1460-2075.1994.tb06400.x

The E6/E7 promoter of extrachromosomal HPV16 DNA in cervical cancers escapes from cellular repression by mutation of target sequences for YY1.

M May 1, X P Dong 1, E Beyer-Finkler 1, F Stubenrauch 1, P G Fuchs 1, H Pfister 1
PMCID: PMC394965  PMID: 8137827

Abstract

Human papillomavirus type 16 (HPV16) induces squamous intraepithelial lesions of the cervical mucosa which may develop into invasive cancer. The expression of viral oncogenes in advanced neoplasias appears increased relative to the proliferating cell layers of low grade lesions raising questions about molecular mechanisms of deregulation of transcription. In a lymph node metastasis of a cervical cancer, we observed full-length HPV16 plasmids and molecules with a small deletion, which was mapped to the long control region (LCR). Both wild type and shortened LCR were amplified by PCR, cloned into the promoter test plasmid pBLCAT6 and sequenced to identify a 107 bp deletion from position 7794 to 7901 in the short LCR. CAT expression in cervical cancer-derived HT3, SiHa and CaSki cells appeared 5- to 6-fold increased under the control of the short LCR. This could be traced back to elevated levels of mRNA initiated at the viral oncogene promoter. A slight further increase in CAT expression was noted in the presence of the HPV16 E2 protein which is probably due to the deletion of one E2 binding site and consequent relief from E2 repression. Computer-assisted sequence analysis and band-shift experiments with purified YY1 protein and wild type or mutated oligonucleotides identified four binding sites for this cellular transcriptional repressor within the promoter-proximal segment of the HPV16 LCR, three of which were removed by the deletion. A LCR fragment comprising these YY1 binding sites was cloned in front of the heterologous thymidine kinase gene promoter and suppressed CAT expression 3- to 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

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  1. Baker C. C., Phelps W. C., Lindgren V., Braun M. J., Gonda M. A., Howley P. M. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J Virol. 1987 Apr;61(4):962–971. doi: 10.1128/jvi.61.4.962-971.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauknecht T., Angel P., Royer H. D., zur Hausen H. Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1. EMBO J. 1992 Dec;11(12):4607–4617. doi: 10.1002/j.1460-2075.1992.tb05563.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beyer-Finkler E., Stoler M. H., Girardi F., Pfister H. Cell differentiation-related gene expression of human papillomavirus 33. Med Microbiol Immunol. 1990;179(4):185–192. doi: 10.1007/BF00195249. [DOI] [PubMed] [Google Scholar]
  4. Boshart M., Klüppel M., Schmidt A., Schütz G., Luckow B. Reporter constructs with low background activity utilizing the cat gene. Gene. 1992 Jan 2;110(1):129–130. doi: 10.1016/0378-1119(92)90456-y. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Chan S. Y., Ho L., Ong C. K., Chow V., Drescher B., Dürst M., ter Meulen J., Villa L., Luande J., Mgaya H. N. Molecular variants of human papillomavirus type 16 from four continents suggest ancient pandemic spread of the virus and its coevolution with humankind. J Virol. 1992 Apr;66(4):2057–2066. doi: 10.1128/jvi.66.4.2057-2066.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Chong T., Apt D., Gloss B., Isa M., Bernard H. U. The enhancer of human papillomavirus type 16: binding sites for the ubiquitous transcription factors oct-1, NFA, TEF-2, NF1, and AP-1 participate in epithelial cell-specific transcription. J Virol. 1991 Nov;65(11):5933–5943. doi: 10.1128/jvi.65.11.5933-5943.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chong T., Chan W. K., Bernard H. U. Transcriptional activation of human papillomavirus 16 by nuclear factor I, AP1, steroid receptors and a possibly novel transcription factor, PVF: a model for the composition of genital papillomavirus enhancers. Nucleic Acids Res. 1990 Feb 11;18(3):465–470. doi: 10.1093/nar/18.3.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Choo K. B., Pan C. C., Liu M. S., Ng H. T., Chen C. P., Lee Y. N., Chao C. F., Meng C. L., Yeh M. Y., Han S. H. Presence of episomal and integrated human papillomavirus DNA sequences in cervical carcinoma. J Med Virol. 1987 Feb;21(2):101–107. doi: 10.1002/jmv.1890210202. [DOI] [PubMed] [Google Scholar]
  11. Cripe T. P., Alderborn A., Anderson R. D., Parkkinen S., Bergman P., Haugen T. H., Pettersson U., Turek L. P. Transcriptional activation of the human papillomavirus-16 P97 promoter by an 88-nucleotide enhancer containing distinct cell-dependent and AP-1-responsive modules. New Biol. 1990 May;2(5):450–463. [PubMed] [Google Scholar]
  12. Cullen A. P., Reid R., Campion M., Lörincz A. T. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J Virol. 1991 Feb;65(2):606–612. doi: 10.1128/jvi.65.2.606-612.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Dostatni N., Lambert P. F., Sousa R., Ham J., Howley P. M., Yaniv M. The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex. Genes Dev. 1991 Sep;5(9):1657–1671. doi: 10.1101/gad.5.9.1657. [DOI] [PubMed] [Google Scholar]
  15. Dürst M., Glitz D., Schneider A., zur Hausen H. Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization. Virology. 1992 Jul;189(1):132–140. doi: 10.1016/0042-6822(92)90688-l. [DOI] [PubMed] [Google Scholar]
  16. Fogh J., Wright W. C., Loveless J. D. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J Natl Cancer Inst. 1977 Feb;58(2):209–214. doi: 10.1093/jnci/58.2.209. [DOI] [PubMed] [Google Scholar]
  17. Friedl F., Kimura I., Osato T., Ito Y. Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology. Proc Soc Exp Biol Med. 1970 Nov;135(2):543–545. doi: 10.3181/00379727-135-35091a. [DOI] [PubMed] [Google Scholar]
  18. Fuchs P. G., Girardi F., Pfister H. Human papillomavirus 16 DNA in cervical cancers and in lymph nodes of cervical cancer patients: a diagnostic marker for early metastases? Int J Cancer. 1989 Jan 15;43(1):41–44. doi: 10.1002/ijc.2910430110. [DOI] [PubMed] [Google Scholar]
  19. Fuchs P. G., Girardi F., Pfister H. Human papillomavirus DNA in normal, metaplastic, preneoplastic and neoplastic epithelia of the cervix uteri. Int J Cancer. 1988 Jan 15;41(1):41–45. doi: 10.1002/ijc.2910410109. [DOI] [PubMed] [Google Scholar]
  20. Girardi F., Pickel H., Beyer-Finkler E., Pfister H. Specific rearrangements of human papillomavirus DNA provide molecular evidence for genetic heterogeneity of primary cervical cancers, recurrencies, and lymph node metastases in two patients. Gynecol Oncol. 1993 Nov;51(2):281–286. doi: 10.1006/gyno.1993.1288. [DOI] [PubMed] [Google Scholar]
  21. Gloss B., Bernard H. U. The E6/E7 promoter of human papillomavirus type 16 is activated in the absence of E2 proteins by a sequence-aberrant Sp1 distal element. J Virol. 1990 Nov;64(11):5577–5584. doi: 10.1128/jvi.64.11.5577-5584.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Higgins G. D., Uzelin D. M., Phillips G. E., McEvoy P., Marin R., Burrell C. J. Transcription patterns of human papillomavirus type 16 in genital intraepithelial neoplasia: evidence for promoter usage within the E7 open reading frame during epithelial differentiation. J Gen Virol. 1992 Aug;73(Pt 8):2047–2057. doi: 10.1099/0022-1317-73-8-2047. [DOI] [PubMed] [Google Scholar]
  23. Krajinović M., Savić A. Sequencing data on the long control region of human papillomavirus type 16. J Gen Virol. 1991 Oct;72(Pt 10):2573–2576. doi: 10.1099/0022-1317-72-10-2573. [DOI] [PubMed] [Google Scholar]
  24. Lee T. C., Shi Y., Schwartz R. J. Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal alpha-actin transcription in embryonic myoblasts. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9814–9818. doi: 10.1073/pnas.89.20.9814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsukura T., Kanda T., Furuno A., Yoshikawa H., Kawana T., Yoshiike K. Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma. J Virol. 1986 Jun;58(3):979–982. doi: 10.1128/jvi.58.3.979-982.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matsukura T., Koi S., Sugase M. Both episomal and integrated forms of human papillomavirus type 16 are involved in invasive cervical cancers. Virology. 1989 Sep;172(1):63–72. doi: 10.1016/0042-6822(89)90107-4. [DOI] [PubMed] [Google Scholar]
  28. May M., Helbl V., Pfister H., Fuchs P. G. Unique topography of DNA-protein interactions in the non-coding region of epidermodysplasia verruciformis-associated human papillomaviruses. J Gen Virol. 1991 Dec;72(Pt 12):2989–2997. doi: 10.1099/0022-1317-72-12-2989. [DOI] [PubMed] [Google Scholar]
  29. Montalvo E. A., Shi Y., Shenk T. E., Levine A. J. Negative regulation of the BZLF1 promoter of Epstein-Barr virus. J Virol. 1991 Jul;65(7):3647–3655. doi: 10.1128/jvi.65.7.3647-3655.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pattillo R. A., Hussa R. O., Story M. T., Ruckert A. C., Shalaby M. R., Mattingly R. F. Tumor antigen and human chorionic gonadotropin in CaSki cells: a new epidermoid cervical cancer cell line. Science. 1977 Jun 24;196(4297):1456–1458. doi: 10.1126/science.867042. [DOI] [PubMed] [Google Scholar]
  31. Phelps W. C., Howley P. M. Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product. J Virol. 1987 May;61(5):1630–1638. doi: 10.1128/jvi.61.5.1630-1638.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Romanczuk H., Thierry F., Howley P. M. Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters. J Virol. 1990 Jun;64(6):2849–2859. doi: 10.1128/jvi.64.6.2849-2859.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schwarz E., Freese U. K., Gissmann L., Mayer W., Roggenbuck B., Stremlau A., zur Hausen H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature. 1985 Mar 7;314(6006):111–114. doi: 10.1038/314111a0. [DOI] [PubMed] [Google Scholar]
  34. Seedorf K., Krämmer G., Dürst M., Suhai S., Röwekamp W. G. Human papillomavirus type 16 DNA sequence. Virology. 1985 Aug;145(1):181–185. doi: 10.1016/0042-6822(85)90214-4. [DOI] [PubMed] [Google Scholar]
  35. Shi Y., Seto E., Chang L. S., Shenk T. Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein. Cell. 1991 Oct 18;67(2):377–388. doi: 10.1016/0092-8674(91)90189-6. [DOI] [PubMed] [Google Scholar]
  36. Shirasawa H., Tomita Y., Kubota K., Kasai T., Sekiya S., Takamizawa H., Simizu B. Transcriptional differences of the human papillomavirus type 16 genome between precancerous lesions and invasive carcinomas. J Virol. 1988 Mar;62(3):1022–1027. doi: 10.1128/jvi.62.3.1022-1027.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smotkin D., Prokoph H., Wettstein F. O. Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. J Virol. 1989 Mar;63(3):1441–1447. doi: 10.1128/jvi.63.3.1441-1447.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stoler M. H., Rhodes C. R., Whitbeck A., Wolinsky S. M., Chow L. T., Broker T. R. Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Hum Pathol. 1992 Feb;23(2):117–128. doi: 10.1016/0046-8177(92)90232-r. [DOI] [PubMed] [Google Scholar]
  39. Stubenrauch F., Malejczyk J., Fuchs P. G., Pfister H. Late promoter of human papillomavirus type 8 and its regulation. J Virol. 1992 Jun;66(6):3485–3493. doi: 10.1128/jvi.66.6.3485-3493.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tan S. H., Gloss B., Bernard H. U. During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element. Nucleic Acids Res. 1992 Jan 25;20(2):251–256. doi: 10.1093/nar/20.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thierry F., Howley P. M. Functional analysis of E2-mediated repression of the HPV18 P105 promoter. New Biol. 1991 Jan;3(1):90–100. [PubMed] [Google Scholar]
  42. Yang Y. C., Okayama H., Howley P. M. Bovine papillomavirus contains multiple transforming genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1030–1034. doi: 10.1073/pnas.82.4.1030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yee C., Krishnan-Hewlett I., Baker C. C., Schlegel R., Howley P. M. Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am J Pathol. 1985 Jun;119(3):361–366. [PMC free article] [PubMed] [Google Scholar]
  44. Zock C., Iselt A., Doerfler W. A unique mitigator sequence determines the species specificity of the major late promoter in adenovirus type 12 DNA. J Virol. 1993 Feb;67(2):682–693. doi: 10.1128/jvi.67.2.682-693.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. von Knebel Doeberitz M., Rittmüller C., zur Hausen H., Dürst M. Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 anti-sense RNA. Int J Cancer. 1992 Jul 9;51(5):831–834. doi: 10.1002/ijc.2910510527. [DOI] [PubMed] [Google Scholar]
  47. zur Hausen H. Human papillomaviruses in the pathogenesis of anogenital cancer. Virology. 1991 Sep;184(1):9–13. doi: 10.1016/0042-6822(91)90816-t. [DOI] [PubMed] [Google Scholar]

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