A variant binding sequence for transcription factor EBP-80 confers increased promoter activity on a retroviral long terminal repeat.
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 promotor 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.
Falzon
M
M
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Kuff
E L
EL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D014644:Genetic Variation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase