Isolation and characterization of a protein fraction that binds to enhancer core sequences in intracisternal A-particle long terminal repeats.
The U3 region of mouse intracisternal A-particle (IAP) long terminal repeats (LTRs) contains several nuclear protein-binding domains. Two of these contain sequences with homology to the SV40 enhancer core. We refer to these two domains as Enh1 and Enh2. The Enh2 domain is an important determinant of promoter activity in vivo. We report here the isolation of nuclear fractions from human 293 and mouse MOPC-315 cells which interact with Enh1 and Enh2. Purification was achieved via DNA-affinity chromatography on a multimerized oligonucleotide representing the Enh2 region from the LTR of the mouse genomic IAP element, MIA14. Glycerol gradient sedimentation suggested a native Mr of approximately 80-100 for the binding component(s) in both crude and affinity-purified fractions. UV cross-linking showed that the binding activity involved two polypeptides within this size range. The affinity-isolated fraction from each cell line was highly purified, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in vitro binding analysis. Exonuclease III footprinting showed that the two polypeptides interacted preferentially with the Enh1 and Enh2 domains within a 139-base pair segment from the MIA14 LTR. The polypeptides interacted in a sequence-specific manner with oligonucleotides representing these domains within the IAP LTR and with oligonucleotides containing the enhancer core sequence from SV40 and polyoma virus. Equilibrium binding studies indicated that the apparent dissociation constants for the polypeptides binding to the enhancer core sequence from MIA14, SV40, and polyoma virus were similar. Therefore, this affinity-purified fraction may represent a novel enhancer core-binding component which is distinct from the previously characterized rat CCAAT/enhancer-binding protein, C/EBP.
Falzon
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Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Kuff
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005090:Exodeoxyribonucleases; D007426:Genes, Intracisternal A-Particle; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011519:Proto-Oncogenes; D012091:Repetitive Sequences, Nucleic Acid
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III