A variant binding sequence for transcription factor EBP-80 confers increased promoter activity on a retroviral long terminal repeat

Miriam Falzon, Edward L. Kuff

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The cloned long terminal repeats (LTRs) of mouse intracisternal A-particle (IAP) proviral elements differ in their promoter activity. In this study, the LTR from a recently transposed IAP element (rc-mos) is shown to be a more effective promoter both in vivo and in vitro than the LTR from a randomly cloned genomic element (MIA14). These LTRs differ in nucleotide sequence in certain previously defined protein-binding domains. In particular, the MIA14 LTR contains two domains, designated Enh1 and Enh2, with sequence homology to the SV40 enhancer core motif, while in rc-mos the Enh2 position is occupied by a variant sequence which lacks core homology. EBP-80 is a general enhancer core-binding protein originally isolated by virtue of its affinity for the MIA14 Enh2 sequence (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). We now find by quantitative binding studies, binding competition, and UV cross-linking that EBP-80 from both human and mouse cells binds to the "Enh2" motif of rc-mos more strongly than to the Enh2 of MIA14. In vitro transcription from both LTRs is strongly enhanced by addition of EBP-80 showing that binding is related to function. The rc-mos LTR remains the more effective promoter in the presence of added EBP-80. Reciprocal substitution of the Enh2 domains in the two LTRs by site-directed mutagenesis shows that the rc-mos variant confers a 3-fold increment in in vivo promoter activity. The rc-mos motif or a closely related sequence is found in the cloned LTRs of many expressed and/or recently transposed IAP elements. EBP-80 is identified as a cellular transcription factor whose heightened levels in certain mouse cells might result in preferential expression of IAP elements containing this sequence motif.

Original languageEnglish (US)
Pages (from-to)13084-13090
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number22
StatePublished - Aug 5 1990
Externally publishedYes

Fingerprint

Terminal Repeat Sequences
Transcription Factors
Intracisternal A-Particle Genes
Mutagenesis
Transcription
Sequence Homology
Site-Directed Mutagenesis
Protein Binding
Carrier Proteins
Substitution reactions
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

A variant binding sequence for transcription factor EBP-80 confers increased promoter activity on a retroviral long terminal repeat. / Falzon, Miriam; Kuff, Edward L.

In: Journal of Biological Chemistry, Vol. 265, No. 22, 05.08.1990, p. 13084-13090.

Research output: Contribution to journalArticle

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abstract = "The cloned long terminal repeats (LTRs) of mouse intracisternal A-particle (IAP) proviral elements differ in their promoter activity. In this study, the LTR from a recently transposed IAP element (rc-mos) is shown to be a more effective promoter both in vivo and in vitro than the LTR from a randomly cloned genomic element (MIA14). These LTRs differ in nucleotide sequence in certain previously defined protein-binding domains. In particular, the MIA14 LTR contains two domains, designated Enh1 and Enh2, with sequence homology to the SV40 enhancer core motif, while in rc-mos the Enh2 position is occupied by a variant sequence which lacks core homology. EBP-80 is a general enhancer core-binding protein originally isolated by virtue of its affinity for the MIA14 Enh2 sequence (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). We now find by quantitative binding studies, binding competition, and UV cross-linking that EBP-80 from both human and mouse cells binds to the {"}Enh2{"} motif of rc-mos more strongly than to the Enh2 of MIA14. In vitro transcription from both LTRs is strongly enhanced by addition of EBP-80 showing that binding is related to function. The rc-mos LTR remains the more effective promoter in the presence of added EBP-80. Reciprocal substitution of the Enh2 domains in the two LTRs by site-directed mutagenesis shows that the rc-mos variant confers a 3-fold increment in in vivo promoter activity. The rc-mos motif or a closely related sequence is found in the cloned LTRs of many expressed and/or recently transposed IAP elements. EBP-80 is identified as a cellular transcription factor whose heightened levels in certain mouse cells might result in preferential expression of IAP elements containing this sequence motif.",
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