Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Feb;69(2):1142–1149. doi: 10.1128/jvi.69.2.1142-1149.1995

Stem cell factor binding to retrovirus primer binding site silencers.

M Yamauchi 1, B Freitag 1, C Khan 1, B Berwin 1, E Barklis 1
PMCID: PMC188687  PMID: 7529329

Abstract

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and overlaps the Moloney murine leukemia virus (M-MuLV) proline primer binding site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silencer. Factor A bound specifically to the wild-type silencer element at room temperature and 30 degrees C, but not at 4 degrees C, and bound 10-fold better to the full-length silencer than to a minimal silencer core element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiated cells in which the silencer is nonfunctional. Salt and ion requirements for factor A binding were investigated, and partial purification steps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silencer and PBS activities, sequences representing phenylalanine, isoleucine, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell-specific repression of M-MuLV expression and in vitro in DNA binding assays. Of these PBS elements, only the lysine-1,2 PBS DNA fragment showed consistently high levels of repression. Interestingly, the lysine-1,2 PBS DNA fragment also formed a complex with an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding studies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna virus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited in undifferentiated stem cells.

Full Text

The Full Text of this article is available as a PDF (441.4 KB).

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. 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]
  5. Berwin B., Barklis E. Retrovirus-mediated insertion of expressed and non-expressed genes at identical chromosomal locations. Nucleic Acids Res. 1993 May 25;21(10):2399–2407. doi: 10.1093/nar/21.10.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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: 10.1006/abio.1976.9999. [DOI] [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. 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]
  9. 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]
  10. Gautsch J. W., Wilson M. C. Delayed de novo methylation in teratocarcinoma suggests additional tissue-specific mechanisms for controlling gene expression. Nature. 1983 Jan 6;301(5895):32–37. doi: 10.1038/301032a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Grez M., Akgün E., Hilberg F., Ostertag W. Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9202–9206. doi: 10.1073/pnas.87.23.9202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jackson S. P., Tjian R. Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1781–1785. doi: 10.1073/pnas.86.6.1781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kempler G., Freitag B., Berwin B., Nanassy O., Barklis E. Characterization of the Moloney murine leukemia virus stem cell-specific repressor binding site. Virology. 1993 Apr;193(2):690–699. doi: 10.1006/viro.1993.1177. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Lund A. H., Duch M., Lovmand J., Jørgensen P., Pedersen F. S. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication. J Virol. 1993 Dec;67(12):7125–7130. doi: 10.1128/jvi.67.12.7125-7130.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Löwer R., Löwer J., Tondera-Koch C., Kurth R. A general method for the identification of transcribed retrovirus sequences (R-U5 PCR) reveals the expression of the human endogenous retrovirus loci HERV-H and HERV-K in teratocarcinoma cells. Virology. 1993 Feb;192(2):501–511. doi: 10.1006/viro.1993.1066. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Martin G. R. Teratocarcinomas and mammalian embryogenesis. Science. 1980 Aug 15;209(4458):768–776. doi: 10.1126/science.6250214. [DOI] [PubMed] [Google Scholar]
  22. Petersen R., Kempler G., Barklis E. A stem cell-specific silencer in the primer-binding site of a retrovirus. Mol Cell Biol. 1991 Mar;11(3):1214–1221. doi: 10.1128/mcb.11.3.1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Prince V. E., Rigby P. W. Derivatives of Moloney murine sarcoma virus capable of being transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site. J Virol. 1991 Apr;65(4):1803–1811. doi: 10.1128/jvi.65.4.1803-1811.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Stewart C. L., Stuhlmann H., Jähner D., Jaenisch R. De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4098–4102. doi: 10.1073/pnas.79.13.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES