Abstract
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.
Highlights
DNase I and exonuclease I11 footprinting have shown that nuclear extracts from a variety of mouse and primate cell lines contain protein(s) that interact with the Enh2 region of the MIA14 LTR [7].TWOcell lines, 293 and MOPC-315, are rich in componentswith EnhZ affinity
The cell line 293 was used to evaluate the functionarlole of the Enh2 domain
We have used sequence-specific DNA-affinity chromatography to isolate from 293 cells a protein fractionthat interacts preferentially with nucleotide sequences homologous to the SV40 enhancer core
Summary
Vol 264, No 36, Issue ofDecember 25, pp. 2P1r9i1n5te-2d1i9n22U,1.9S8.9A. Isolation and Characterizationof a Protein Fraction That Bindsto Enhancer Core Sequencesin Intracisternal A-particle Long Terminal Repeats*. The abbreviations used are: IAP, intracisternal A-particle; LTR, long terminal repeats; hp, base pair(s); Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; CAT, chloramphenicol acetyltransferase; EBP, enhancer-binding protein. The plasmidpMIAcat3’L(del) was derived from pMIAcat-3’L by deleting the LTR region upstream of the PuuII restriction site at nucleotide position 48 (Fig. l a ) All of these constructs contain theCAT gene downstream of the Isolation of Enhancer Core Binding Protein. The active fraction from the heparin-Sepharose column (0.3 M KC1 eluate, 6070 mg of protein) was preincubated on ice for 10 min with 100 pg of sheared calf thymus DNA, asnonspecific competitor, andloaded onto the oligonucleotide affinitycolumn. For quantitation of protein enrichment, binding activity was determined by the gel retardation assay, in the presence of0.2 ng (8 fmol) of Enh oligonucleotide as probe and 100 ng of poly(dI-dC). G A ladders used to calibrate exonuclease I11 footprints were generated by the method of Maxam and Gilbert [25]
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