We analyzed the ability of cyclic AMP-response element binding proteins (CREBs) to interact with the CRE sequences derived from different genes and examined the role of sequences flanking the core CRE element in rendering cAMP-responsiveness to the enhancer. We were able to detect reproducibly, sing the Southwestern blotting technique, five major CREB factors of molecular weights 56, 47, 40, and 36-34 kDa which were present in various rat tissues and cultured cells. The 34-40 kDa proteins (CREB-327/341) were able to bind to the CRE of cAMP-inducible genes (somatostatin, c-fos, E2A), but not to genes whose expression is not controlled by cAMP (glucagon, parathyroid hormone). The novel 47 kDa CREB had a high specificity for the core octameric CRE sequence and it bound equally well to the consensus CRE of cAMP-inducible and noninducible genes. On the other hand, the 47 kDa CREB did not bind at all to the phorbol ester response element (TRE), whereas the 56 kDa protein, reminiscent of the CRE-BP1 protein, could bind to both elements. A computer aided sequence analysis of cAMP-inducible gene promoters revealed the presence of an additional conserved element starting 4-6 nucleotides 3\' to the octomer with the consensus C/GAGA/C. We have shown this element to be essential for maximal cAMP-responsiveness of the enhancer in transient expression assays of CRE-CAT plasmid constructs indicating that the functional interaction of CREB proteins with the cAMP-inducible enhancer involves an additional 8-10 base pairs immediately downstream from the CRE core element.

Two regions of the Epstein-Barr virus BZLF1 trans-activator protein have sequence similarity to the c-fos protein. Part of the similarity corresponds to the region of c-fos which is similar to the DNA binding domain of c-jun and GCN-4. The structure of the exon which contains this region in c-fos and BZLF1 is also highly conserved between the two genes. Complete BZLF1 protein and a C terminal fragment were prepared either as purified fusion proteins or by in vitro translation from a BZLF1 cDNA. Gel retardation and DNase footprinting assays using these proteins show that BZLF1 is a sequence specific DNA binding protein capable of binding to a target sequence which contains a consensus AP-1 site.

We have demonstrated that the gibbon ape leukemia virus (GALV) enhancer AP-1 element and the simian virus 40 AP-1 enhancer element bind different factors in HeLa nuclear extracts. A 39-kilodalton HeLa nuclear protein and the c-fos protein bind to the GALV element. Antibodies to c-fos abolish binding to the GALV AP-1 site. In contrast, anti-c-fos immunoglobulin fails to inhibit formation of the simian virus 40-specific complex from extracts of HeLa cells. Thus, AP-1-binding complexes are subject to compositional variation at different binding sites.

We have compared the binding properties of c-JUN, JUN B, and JUN D in the absence or in the presence of c-FOS, FOS B, and FRA-1 to different AP-1 and CRE-containing oligonucleotides. The results demonstrate that for a given AP-1-containing oligonucleotide the binding affinities of the different JUN proteins are always c-JUN greater than JUN D greater than JUN B. The three JUN proteins have the capacity to bind to a CRE consensus sequence with very high affinity. We have found that c-JUN, JUN B, and JUN D bind with different affinities to different oligonucleotides containing an identical AP-1 or CRE binding site, implying that the adjacent sequences influence the stability of the JUN/DNA complexes. Interestingly, an AP-1-containing oligonucleotide which binds the JUN proteins with high affinity can be converted to a CRE-containing oligonucleotide which will also bind the different JUNs very efficiently. The heterodimers formed between the different JUN and FOS proteins have an enhanced binding activity compared to the JUN:JUN homodimers. In all cases the half-lives of the JUN:FOS/DNA complexes are longer than those of the JUN:JUN/DNA complexes. The most stable complexes were obtained in the presence of FOS B, followed by FRA-1 and c-FOS.