Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer.

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.     

Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.     

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.     

Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.     

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).     

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.     

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)