At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.     

Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemia virus enhancer.

Binding sites for six distinct nuclear factors on the 75-base-pair repeat of the Moloney murine leukemia virus enhancer have been identified by an electrophoretic mobility shift assay combined with methylation interference. Three of these factors, found in WEHI 231 nuclear extracts, which we have named LVa, LVb, and LVc (for leukemia virus factors a, b, and c) have not been previously identified. Nuclear factors that bind to the conserved simian virus 40 corelike motif, the NF-1 motif, and the glucocorticoid response element were also detected. Testing of multiple cell lines showed that most factors appeared ubiquitous, except that the NF-1 binding factor was found neither in nuclear extracts from MEL cells nor in the embryonal carcinoma cell lines PCC4 and F9, and core-binding factor was relatively depleted from MEL and F9 nuclear extracts.     

The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence

We have purified a protein (TEF-1) that specifically binds to two sequence unrelated motifs (GT-IIC and Sph) of the simian virus 40 (SV40) enhancer. TEF-1 binds cooperatively to templates containing tandem but not inverted or spaced repeats of its cognate motifs. This cooperative binding correlates with the ability of the tandem repeats to generate enhancer activity in vivo. In contrast, TEF-1 and a second SV40 enhancer binding protein, TEF-2, bind independently to templates containing the cognate motifs of both proteins (GT-I and either GT-IIC or Sph motifs) even though these motifs cooperate in enhancer activity in vivo. These results allow us to distinguish different classes of enhancer factors.     

Distinct factors bind the AP-1 consensus sites in gibbon ape leukemia virus and simian virus 40 enhancers.

We have demonstrated that the gibbon ape leukemia virus (GALV) enhancer AP-1 element and the simian virus 40 AP-1 enhancer element bind different factors in HeLa nuclear extracts. A 39-kilodalton HeLa nuclear protein and the c-fos protein bind to the GALV element. Antibodies to c-fos abolish binding to the GALV AP-1 site. In contrast, anti-c-fos immunoglobulin fails to inhibit formation of the simian virus 40-specific complex from extracts of HeLa cells. Thus, AP-1-binding complexes are subject to compositional variation at different binding sites.     

One cell-specific and three ubiquitous nuclear proteins bind in vitro to overlapping motifs in the domain B1 of the SV40 enhancer.

We have used the gel retardation assay to investigate the binding of nuclear proteins to the domain B1 of the SV40 enhancer, which contains the GT-II motif. Four proteins (GT-IIA, GT-IIB alpha, GT-IIB beta and GT-IIC) were detected, three of which were present in nuclear extracts from several cell lines. The fourth protein (GT-IIC) showed a clear cell-specificity, being absent from the lymphoid cell extracts tested. The results of methylation interference assays and of the binding of the proteins to mutated templates indicate that the domain B1 contains three distinct, but overlapping, protein-binding motifs (GT-IIA, B and C). The cell-specific binding of protein GT-IIC in vitro correlates with the in vivo enhancer activity of its cognate motif, strongly suggesting that this protein acts as a positive trans-acting enhancer factor. Two of the proteins also recognize other enhancer motifs; protein GT-IIB alpha binds to the microE3 motif present in the immunoglobulin heavy chain enhancer; protein GT-IIC binds to an enhancer motif of the polyomavirus mutant PyEC9.1 adapted to growth in F9 embryonal carcinoma cells, but not to the corresponding wild-type sequence.     

Specific nuclear proteins interact with the Rous sarcoma virus internal enhancer and share a common element with the enhancer located in the long terminal repeat of the virus.

We have documented that the Rous sarcoma virus (RSV) internal enhancer functions in the nontransformed Baby Hamster Kidney (BHK) cell line. The sequences within this region were assayed for their ability to bind to specific factors present in BHK nuclear extracts using the gel retardation assay and DNAse I footprinting. At least two sequences within the internal enhancer which can specifically bind nuclear factors in vitro have been identified. These regions are located between nucleotides 813-850 and 856-877. These sites map within the overall region of the internal enhancer which has been shown to be essential for enhancer activity and within the specific region which can function as an orientation independent enhancer. Using the DNase I footprinting and binding data to design an oligonucleotide, we have demonstrated that an oligonucleotide extending from nucleotides 804-877 will substitute efficiently as an enhancer. We also demonstrate that the SV40 enhancer does not compete for the factors which bind to the RSV internal enhancer, whereas an oligonucleotide to the binding site for EFII in the LTR can compete for factor binding to the internal enhancer.     

Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.