Stem cell factor binding to retrovirus primer binding site silencers

M. Yamauchi, B. Freitag, C. Khan, B. Berwin, Eric Barklis

Research output: Contribution to journalArticle

26 Citations (Scopus)

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°C, but not at 4°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.

Original languageEnglish (US)
Pages (from-to)1142-1149
Number of pages8
JournalJournal of Virology
Volume69
Issue number2
StatePublished - 1995

Fingerprint

Retroviridae
stem cell factor
Stem Cell Factor
binding sites
Binding Sites
Lysine
Murine leukemia virus
lysine
Moloney murine leukemia virus
Embryonal Carcinoma Stem Cells
Transcriptional Silencer Elements
DNA
carcinoma
stem cells
Stem Cells
Spumavirus
Mason-Pfizer monkey virus
Visna-maedi virus
cells
Visna maedi virus

ASJC Scopus subject areas

  • Immunology

Cite this

Yamauchi, M., Freitag, B., Khan, C., Berwin, B., & Barklis, E. (1995). Stem cell factor binding to retrovirus primer binding site silencers. Journal of Virology, 69(2), 1142-1149.

Stem cell factor binding to retrovirus primer binding site silencers. / Yamauchi, M.; Freitag, B.; Khan, C.; Berwin, B.; Barklis, Eric.

In: Journal of Virology, Vol. 69, No. 2, 1995, p. 1142-1149.

Research output: Contribution to journalArticle

Yamauchi, M, Freitag, B, Khan, C, Berwin, B & Barklis, E 1995, 'Stem cell factor binding to retrovirus primer binding site silencers', Journal of Virology, vol. 69, no. 2, pp. 1142-1149.
Yamauchi M, Freitag B, Khan C, Berwin B, Barklis E. Stem cell factor binding to retrovirus primer binding site silencers. Journal of Virology. 1995;69(2):1142-1149.
Yamauchi, M. ; Freitag, B. ; Khan, C. ; Berwin, B. ; Barklis, Eric. / Stem cell factor binding to retrovirus primer binding site silencers. In: Journal of Virology. 1995 ; Vol. 69, No. 2. pp. 1142-1149.
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