A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation.
The human immunodeficiency virus (HIV-1) long terminal repeat (LTR) contains two binding sites for NF-kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF-kappa B to the viral enhancer. In this report, we show that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein-protein interaction must occur between NF-kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF-kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross-linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF-kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF-kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF-kappa B and Sp1 is required for inducible HIV-1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.
Perkins
N D
ND
Howard Hughes Medical Institute, University of Michigan Medical Center, Department of Internal Medicine, Ann Arbor 48109-0650.
Edwards
N L
NL
Duckett
C S
CS
Agranoff
A B
AB
Schmid
R M
RM
Nabel
G J
GJ
D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D046911:Macromolecular Substances; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate