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.     

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.     

Repression of enhancer activity by the adenovirus E1A tumor protein(s) is mediated through a novel HeLa cell nuclear factor.

We have examined the mechanism for the host cell-dependent repression of enhancer activity by the adenovirus early region 1A (E1A) proteins. The enhancer used in this study, from the human BK virus P2, functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2. In addition, enhancer activity can be stimulated by the E1A gene products in these cells. However, cis-enhancer activity is repressed in the HeLa cell line, and we demonstrate here that further repression can be induced by the E1A proteins. We show that the binding site for the negative regulatory factor involved in cis-repression, designated BK virus enhancer factor 1 (BEF-1), is also required for E1A-induced repression. Using gel mobility retardation assays, we demonstrated a 4-fold increase in active BEF-1 in nuclear extracts containing the E1A proteins. However, the E1A proteins did not change the binding pattern or the strength of binding of BEF-1 to its target sequence. BEF-1 was identified as a 98-kDa nuclear factor, and phosphorylation was shown to be important for DNA binding. Three potential nuclear factor 1 (NF-1) sites are present in the BEF-1-binding site. Using a known NF-1 site as competitor DNA in a gel mobility retardation assay, we provide evidence that BEF-1 may be a newly identified NF-1 family member. In addition, the sequence TGA present in the repressor-binding site was shown to be essential for high affinity binding of BEF-1. Overall, our data demonstrate that an enhancer can be repressed by the trans-activation of a negative regulatory factor.     

Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family.

The cDNA encoding adenovirus E1A enhancer-binding protein E1A-F was isolated by screening a HeLa cell lambda gt11 expression library for E1A-F site-specific DNA binding. One cDNA clone produced recombinant E1A-F protein with the same DNA binding specificity as that endogenous to HeLa cells. Sequence analysis of the cDNA showed homology with the ETS-domain, a region required for sequence-specific DNA binding and common to all ets oncogene members. Analysis of the longest cDNA revealed about a 94% identity in amino acids between human E1A-F and mouse PEA3 (polyomavirus enhancer activator 3), a recently characterized ets oncogene member. E1A-F was encoded by a 2.5kb mRNA in HeLa cells, which was found to increase during the early period of adenovirus infection. In contrast, ets-2 mRNA was significantly reduced in infected HeLa cells. The results indicate that E1A enhancer binding protein E1A-F is a member of the ets oncogene family and is probably a human homologue of mouse PEA3.     

A cellular protein binds to a conserved sequence in the adenovirus type 2 enhancer.

A sensitive gel retention assay has been utilized to detect proteins from uninfected Hela nuclei which interact with the adenovirus type 2 enhancer. This assay has been employed to monitor fractionation of nuclear extracts. Three enhancer binding factors were resolved by chromatography on DEAE-Sepharose and one of the factors was further purified by chromatography on heparin-Sepharose. DNase protection experiments have shown that the heparin-Sepharose fraction contains a factor which binds predominantly to the conserved sequence GTGGAAATTT present at position 160 in the adenovirus type 2 genome and found in many viral and cellular enhancers. Protection of this sequence from DNase I digestion was abolished by competition with a synthetic duplex oligonucleotide spanning bases 144-181. This region corresponds to the sequence defined by Hen et al. as possessing enhancer function. Competition experiments indicated that the enhancer binding factor also bound, albeit with reduced affinity, to multiple sites in the Ela upstream region located between positions 192 and 353. Within the sequences which compete are regions with homology to the high affinity site at position 160. The enhancer binding factor also binds with high affinity to sequences within the SV40 enhancer demonstrating that this factor interacts with sequences common to both the adenovirus and SV40 enhancers.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

Characterization of an enhancer upstream from the muscle-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The muscle-type isozyme of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is encoded by a mRNA transcribed from the M promoter of a 55-kb gene, which also produces the liver-type isozyme from an alternative promoter. By transient transfection and in vitro protein-DNA binding assays we have delineated, within 4.7 kb of 5' flanking sequence, the M promoter proper and an enhancer located between -1615 and -1809. This enhancer stimulated up to 12-fold the activity of the promoter in the context of an intact 5' flanking sequence and close to 900-fold the activity of the minimal (+41 to -40) M promoter cloned directly downstream from it. A functional dissection of the enhancer by site-directed mutagenesis and use of oligonucleotides suggested that its activity involves the cooperative effect of six binding sites for trans-acting factors clustered within 150 bp. These sites contain either an EF-1A/E4TF1 motif (also known to bind the ets oncogene product) or a Sp1 motif, or both. The activity of the enhancer could be demonstrated in L6 myoblasts and myocytes and in FTO2B hepatoma cells. When left within the intact 5' flanking sequence, however, enhancer activity was inhibited upon differentiation of myoblasts into myocytes.