Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.     

Interaction of nuclear factor EF-1A with the polyomavirus enhancer region.

Enhancer factor 1A (EF-1A) is a mammalian nuclear protein that previously was shown to bind cooperatively to the repeated core enhancer element I sequence in the adenovirus E1A enhancer region. We now have characterized three binding sites for EF-1A in the polyomavirus A2 (Py) enhancer region. Site 1 resides in the Py A enhancer domain, and sites 2 and 3 reside in the Py B enhancer domain. EF-1A binding to Py site 1 is independent of cooperation with other EF-1A sites or the adjacent binding sites for PEA-1 and PEA-2, two murine nuclear factors that bind in the Py A enhancer domain. EF-1A binding to Py sites 2 and 3, in contrast, is cooperative, similar to the situation previously observed with binding sites in the adenovirus E1A enhancer region. In a transient replication assay, EF-1A site 1 functions synergistically with the PEA-1 and PEA-2 sites in the A enhancer domain to enhance Py replication. The functional cooperativity observed with the EF-1A, PEA-1, and PEA-2 sites in vivo does not reflect cooperative DNA binding interactions, as detected in vitro. Py EF-1A site 1 alone is capable of weakly stimulating Py replication. EF-1A site 1 overlaps with the binding sites for the murine nuclear protein PEA-3 and the ets family of oncoproteins.     

Complex functional interactions at the early enhancer of the PQ strain of BK virus.

BK virus is a human papovavirus that latently infects a majority of the world's population. There are more than 30 strains of the virus, most of which differ in the structure of the early enhancer region. The enhancer of the progenitor strain, WW, from which the other strains can be derived, consists of four conserved DNA domains, P, Q, R, and S. Rearrangement of the enhancer occurs upon passage in tissue culture and is thought to occur during virus replication. The strain under study, PQ, was selected upon passage of the Gardner strain (PPPQS) in the permissive cell line, Vero. Mutational analysis of the entire enhancer region demonstrates the importance of five cis-acting sequences: DNA sites B, C, and F, which have homology to the NF-1 protein binding sequence; one purine-rich motif designated A; and site D, which is similar to an SP-1 protein binding site. Two sites, B and C, appear to have a negative influence on gene activity. To study the functional interactions in more detail, promoter-enhancer constructions that contain different combinations of the five DNA sites linked to the chloramphenicol acetyltransferase gene were tested for early gene activity. The results reveal that the proteins binding to the enhancer functionally cooperate with each other. The effects of making mutations at the DNA sites are very similar to the effects of using excess enhancer DNA sequences to titrate the proteins that bind to the cis-acting DNA sites (in vivo competition). Moreover, the effects of changing the spacing between the DNA sites also demonstrate that there are cooperative interactions among the proteins that bind to the PQ strain enhancer. DNA sites B, C, and F are clearly protected from DNase I digestion by Vero cell nuclear proteins. In addition, mutation of each DNA site alters its sensitivity to DNase I in the presence of Vero cell proteins. Interestingly, mutation of site B affects protein binding to site B as well as to sites A, C, D, and F. These results suggest that cooperative functional and physical interactions occur at the early enhancer of the PQ strain.     

Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.     

DNA-binding properties of the E1A-associated 300-kilodalton protein.

One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.