Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Mar;11(3):1214–1221. doi: 10.1128/mcb.11.3.1214

A stem cell-specific silencer in the primer-binding site of a retrovirus.

R Petersen 1, G Kempler 1, E Barklis 1
PMCID: PMC369392  PMID: 1996087

Abstract

Retrovirus expression in embryonal carcinoma (EC) cells is blocked at a postintegration stage of the viral life cycle, in part because of the inadequate function of the viral long terminal repeat promoter in this cell type. However, selection for retrovirus expression in EC cells has identified mutations in Moloney murine leukemia virus (M-MuLV) located in the tRNA primer-binding site (PBS) region which relieve the EC cell-specific repression. We have found that exchanging the M-MuLV proline PBS for a glutamine one in a recombinant virus permits expression in EC cells. By using the recombinant virus as a backbone, the EC cell-specific repressor-binding site (RBS) element has been mapped to M-MuLV nucleotides 147 to 174. The RBS does not require precise positioning downstream of the M-MuLV promoter and can function in either orientation and in an intron, indicating that the regulatory effect is probably at the DNA, rather than RNA, level. We also show that the RBS element can repress heterologous promoters from an upstream position. Our results indicate that the RBS acts as a silencer that its inhibitory effect is mediated by a trans-acting factor, and that the mechanism of action is probably at the level of transcription. Through in vitro binding assays we have identified a binding factor which specifically recognizes the wild-type RBS sequence (binding factor A). The binding characteristics of factor A suggest that it is a stem cell repressor which acts at the M-MuLV RBS. Our DNA-binding assays also have identified a unique binding factor (binding factor Hp) which specifically recognizes a hemimethylated form of the wild-type RBS. This factor may play a role in methylation mediated control of retrovirus expression in EC cells.

Full text

PDF
1214

Images in this article

1217Image
on p.1217
1218Image
on p.1218
1218Image
on p.1218
1219Image
on p.1219

Selected References

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

  1. Antequera F., Macleod D., Bird A. P. Specific protection of methylated CpGs in mammalian nuclei. Cell. 1989 Aug 11;58(3):509–517. doi: 10.1016/0092-8674(89)90431-5. [DOI] [PubMed] [Google Scholar]
  2. Baeuerle P. A., Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. doi: 10.1016/0092-8674(88)90382-0. [DOI] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. doi: 10.1126/science.3140380. [DOI] [PubMed] [Google Scholar]
  4. Barklis E., Lodish H. F. Regulation of dictyostelium discoideum mRNAs specific for prespore or prestalk cells. Cell. 1983 Apr;32(4):1139–1148. doi: 10.1016/0092-8674(83)90297-0. [DOI] [PubMed] [Google Scholar]
  5. Barklis E., Mulligan R. C., Jaenisch R. Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells. Cell. 1986 Nov 7;47(3):391–399. doi: 10.1016/0092-8674(86)90596-9. [DOI] [PubMed] [Google Scholar]
  6. Berstine E. G., Hooper M. L., Grandchamp S., Ephrussi B. Alkaline phosphatase activity in mouse teratoma. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3899–3903. doi: 10.1073/pnas.70.12.3899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colicelli J., Goff S. P. Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. J Virol. 1986 Jan;57(1):37–45. doi: 10.1128/jvi.57.1.37-45.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cone R. D., Weber-Benarous A., Baorto D., Mulligan R. C. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol. 1987 Feb;7(2):887–897. doi: 10.1128/mcb.7.2.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Evans M. J., Kaufman M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981 Jul 9;292(5819):154–156. doi: 10.1038/292154a0. [DOI] [PubMed] [Google Scholar]
  11. Feuer G., Taketo M., Hanecak R. C., Fan H. Two blocks in Moloney murine leukemia virus expression in undifferentiated F9 embryonal carcinoma cells as determined by transient expression assays. J Virol. 1989 May;63(5):2317–2324. doi: 10.1128/jvi.63.5.2317-2324.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorman C. M., Rigby P. W., Lane D. P. Negative regulation of viral enhancers in undifferentiated embryonic stem cells. Cell. 1985 Sep;42(2):519–526. doi: 10.1016/0092-8674(85)90109-6. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Johnson T. R., Ilan J. Proteins associated with poly(A)+RNA of cockerel liver: effects of estradiol stimulation. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4088–4092. doi: 10.1073/pnas.79.13.4088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones N. C., Rigby P. W., Ziff E. B. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 1988 Mar;2(3):267–281. doi: 10.1101/gad.2.3.267. [DOI] [PubMed] [Google Scholar]
  16. Jones T. A., Blaug G., Hansen M., Barklis E. Assembly of gag-beta-galactosidase proteins into retrovirus particles. J Virol. 1990 May;64(5):2265–2279. doi: 10.1128/jvi.64.5.2265-2279.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Korman A. J., Frantz J. D., Strominger J. L., Mulligan R. C. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2150–2154. doi: 10.1073/pnas.84.8.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Levy D. E., Kessler D. S., Pine R., Darnell J. E., Jr Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro. Genes Dev. 1989 Sep;3(9):1362–1371. doi: 10.1101/gad.3.9.1362. [DOI] [PubMed] [Google Scholar]
  19. Linney E., Davis B., Overhauser J., Chao E., Fan H. Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells. 1984 Mar 29-Apr 4Nature. 308(5958):470–472. doi: 10.1038/308470a0. [DOI] [PubMed] [Google Scholar]
  20. Loh T. P., Sievert L. L., Scott R. W. Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells. Mol Cell Biol. 1990 Aug;10(8):4045–4057. doi: 10.1128/mcb.10.8.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Loh T. P., Sievert L. L., Scott R. W. Negative regulation of retrovirus expression in embryonal carcinoma cells mediated by an intragenic domain. J Virol. 1988 Nov;62(11):4086–4095. doi: 10.1128/jvi.62.11.4086-4095.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loh T. P., Sievert L. L., Scott R. W. Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells. Mol Cell Biol. 1987 Oct;7(10):3775–3784. doi: 10.1128/mcb.7.10.3775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  24. Meehan R. R., Lewis J. D., McKay S., Kleiner E. L., Bird A. P. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell. 1989 Aug 11;58(3):499–507. doi: 10.1016/0092-8674(89)90430-3. [DOI] [PubMed] [Google Scholar]
  25. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Niwa O., Yokota Y., Ishida H., Sugahara T. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells. Cell. 1983 Apr;32(4):1105–1113. doi: 10.1016/0092-8674(83)90294-5. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Peckham I., Sobel S., Comer J., Jaenisch R., Barklis E. Retrovirus activation in embryonal carcinoma cells by cellular promoters. Genes Dev. 1989 Dec;3(12B):2062–2071. doi: 10.1101/gad.3.12b.2062. [DOI] [PubMed] [Google Scholar]
  29. Saito H., Kranz D. M., Takagaki Y., Hayday A. C., Eisen H. N., Tonegawa S. A third rearranged and expressed gene in a clone of cytotoxic T lymphocytes. Nature. 1984 Nov 1;312(5989):36–40. doi: 10.1038/312036a0. [DOI] [PubMed] [Google Scholar]
  30. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  31. Speck N. A., Baltimore D. Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemia virus enhancer. Mol Cell Biol. 1987 Mar;7(3):1101–1110. doi: 10.1128/mcb.7.3.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stevens L. C. The biology of teratomas. Adv Morphog. 1967;6:1–31. doi: 10.1016/b978-1-4831-9953-5.50005-6. [DOI] [PubMed] [Google Scholar]
  33. Teich N. M., Weiss R. A., Martin G. R., Lowy D. R. Virus infection of murine teratocarcinoma stem cell lines. Cell. 1977 Dec;12(4):973–982. doi: 10.1016/0092-8674(77)90162-3. [DOI] [PubMed] [Google Scholar]
  34. Thornell A., Hallberg B., Grundström T. Differential protein binding in lymphocytes to a sequence in the enhancer of the mouse retrovirus SL3-3. Mol Cell Biol. 1988 Apr;8(4):1625–1637. doi: 10.1128/mcb.8.4.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Weiher H., Barklis E., Ostertag W., Jaenisch R. Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells. J Virol. 1987 Sep;61(9):2742–2746. doi: 10.1128/jvi.61.9.2742-2746.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Winoto A., Baltimore D. Alpha beta lineage-specific expression of the alpha T cell receptor gene by nearby silencers. Cell. 1989 Nov 17;59(4):649–655. doi: 10.1016/0092-8674(89)90010-x. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES