Expression of a 91-kilodalton PEA3-binding protein is down-regulated during differentiation of F9 embryonal carcinoma cells.

Proteins binding to the PEA3 enhancer motif (AGGAAG) activate the polyomavirus early promoter and help comprise the viral late mRNA initiator element (W. Yoo, M. E. Martin, and W. R. Folk, J. Virol. 65:5391-5400, 1991). Because many developmentally regulated cellular genes have PEA3 motifs near their promoter sequences, and because Ets family gene products activate the PEA3 motif, we have studied the expression of PEA3-binding proteins and Ets-related proteins during differentiation of F9 embryonal carcinoma cells. An approximately 91-kDa protein (PEA3-91) was identified in F9 cell nuclear extracts by UV cross-linking to a radiolabeled PEA3 oligonucleotide probe, and expression of PEA3-91 was down-regulated after differentiation of F9 cells to parietal endoderm. The c-ets-1 gene product binds to a sequence in the murine sarcoma virus long terminal repeat that is similar to the PEA3 motif (cGGAAG), but PEA3-91 was not cross-linked to this Ets-1-binding motif, nor did antiserum which recognizes murine c-ets-1 and c-ets-2 proteins have any effect on PEA3-binding activity in mobility shift assays. Furthermore, c-ets-1 mRNA was not detected in undifferentiated or differentiated F9 cells, and c-ets-2 mRNA levels remained high after differentiation. Antiserum against the Drosophila Ets-related E74A protein, however, recognized an approximately 92-kDa protein in F9 cells whose expression during differentiation varied in a manner identical to that of PEA3-91. These data suggest that PEA3-91 is not the product of the ets-1 or ets-2 genes but is likely to be the product of a murine homolog of the Drosophila E74 gene.     

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.     

PEA3 and AP-1 are required for constitutive IL-8 gene expression in hepatoma cells

Interleukin-8 (IL-8) mRNA was constitutively expressed in human hepatoma cell line, HepG2 and in human hepatocellular carcinoma (HCC), which often form hypervascular tumors. The sequence 5'-AGGAAG-3' at -137 to -132 bp of IL-8 promoter was shown to be polyomavirus enhancer A binding protein-3 (PEA3) binding site, which can cooperate with activator protein-1 (AP-1). Both PEA3 and AP-1 are essential for constitutive IL-8 expression in HepG2 cells, determined by promoter assays. Moreover, PEA3 and IL-8 proteins coexisted in HCC tissues, but not in uninvolved liver tissues. It is possible PEA3 may have important roles in tumor progression and in angiogenesis in HCC.     

Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.     

Structural diversity and nuclear protein binding sites in the long terminal repeats of feline leukemia virus.

The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between -155 and -244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications.     

Two different factors bind to the alpha-domain of the polyoma virus enhancer, one of which also interacts with the SV40 and c-fos enhancers.

Two nuclear factors from mouse 3T6 cells bind to a 22-bp segment constituting the alpha-domain of the polyoma virus enhancer. Binding of each factor can be competed out selectively by the appropriate double-stranded oligonucleotide, indicating that this binding is not strictly cooperative. Sequence homology between the two binding sites and the similar size of the protected regions may indicate that both factors, PEA1 and PEA2, are closely related. The binding site of PEA1 is centered on a sequence showing strong homology to the SV40 enhancer, the binding site of PEA2 is located immediately adjacent to it and shows a strong homology to the c-fos enhancer. Surprisingly, both SV40 and c-fos enhancers interact with PEA1, probably due to the presence of an extra base pair relative to c-fos in the PEA2 site. Factor PEA1 is probably identical to the recently described activator protein 1 (AP1).