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.     

Characterization of a delayed early serum response region.

The proliferin (PLF) gene promoter provides a relatively simple model system for the study of growth-regulated gene expression in mouse cells. The promoter elements required for this serum-induced regulation have been identified and include an AP-1 site as well as an adjacent element comprised of three imperfect repeats that are similar in sequence to the simian virus 40 (SV40) Sph motif. Distinct protein complexes bound independently to the AP-1 and Sph elements, and both of these juxtaposed sites could be occupied simultaneously. Furthermore, serum stimulation of mouse fibroblasts resulted in similar increases in protein binding to the AP-1 and Sph elements. Consistent with this increase in AP-1 and Sph binding activity, the PLF AP-1 and Sph elements were independently able to confer serum responsiveness to a minimal promoter, and together these two elements acted synergistically in response to serum. Although several members of the AP-1 family were able to activate the PLF gene promoter in transient cotransfection experiments, the predominant AP-1 components interacting with the PLF gene promoter in serum-stimulated cells were Fra-1, JunB, and JunD. Analysis of the Sph element revealed that mutation of Sph repeats I or III abolished serum responsiveness of the PLF gene promoter, and mutation of Sph repeat III decreased protein binding to this element. Although the Sph element is similar in sequence to the SV40 element, the PLF Sph-binding factor is distinct from TEF-1, the factor that binds to the SV40 Sph motif.     

Mapping the transcriptional transactivation function of simian virus 40 large T antigen

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.