DNA binding activities of three murine Jun proteins: stimulation by Fos

Three members of the Jun/AP-1 family have been identified in mouse cDNA libraries: c-Jun, Jun-B, and Jun-D. We have compared the DNA binding properties of the Jun proteins by using in vitro translation products in gel retardation assays. Each protein was able to bind to the consensus AP-1 site (TGACTCA) and, with lower affinity, to related sequences, including the cyclic AMP response element TGACGTCA. The relative binding to the oligonucleotides tested was similar for the different proteins. The Jun proteins formed homodimers and heterodimers with other members of the family, and they were bound to the AP-1 site as dimers. When Fos translation product was present, DNA binding by Jun increased markedly, and the DNA complex contained Fos. The C-terminal homology region of Jun was sufficient for DNA binding, dimer formation, and interaction with Fos. Our general conclusion is that c-Jun, Jun-B, and Jun-D are similar in their DNA binding properties and in their interaction with Fos. If there are functional differences between them, they are likely to involve other activities of the Jun proteins.     

Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity

Association of Fos and Jun with the AP-1 site results in a conformational change in the basic amino acid regions that constitute the DNA-binding domain. We show that Fos and Jun induce a corresponding alteration in the conformation of the DNA helix. Circular permutation analysis indicated that both Fos-Jun heterodimers and Jun homodimers induce flexure at the AP-1 site. Phasing analysis demonstrated that Fos-Jun heterodimers and Jun homodimers induce DNA bends that are directed in opposite orientations. Fos-Jun heterodimers bend DNA toward the major groove, whereas Jun homodimers bend DNA toward the minor groove. Fos and Jun peptides encompassing the dimerization and DNA-binding domains bend DNA in the same orientations as the full-length proteins. However, additional regions of both proteins influence the magnitude of the DNA bend angle. Thus, despite the amino acid sequence similarity in the basic region Fos-Jun heterodimers and Jun homodimers form topologically distinct DNA-protein complexes.     

Selective DNA bending by a variety of bZIP proteins.

We have investigated DNA bending by bZIP family proteins that can bind to the AP-1 site. DNA bending is widespread, although not universal, among members of this family. Different bZIP protein dimers induced distinct DNA bends. The DNA bend angles ranged from virtually 0 to greater than 40 degrees as measured by phasing analysis and were oriented toward both the major and the minor grooves at the center of the AP-1 site. The DNA bends induced by the various heterodimeric complexes suggested that each component of the complex induced an independent DNA bend as previously shown for Fos and Jun. The Fos-related proteins Fra1 and Fra2 bent DNA in the same orientation as Fos but induced smaller DNA bend angles. ATF2 also bent DNA toward the minor groove in heterodimers formed with Fos, Fra2, and Jun. CREB and ATF1, which favor binding to the CRE site, did not induce significant DNA bending. Zta, which is a divergent member of the bZIP family, bent DNA toward the major groove. A variety of DNA structures can therefore be induced at the AP-1 site through combinatorial interactions between different bZIP family proteins. This diversity of DNA structures may contribute to regulatory specificity among the plethora of proteins that can bind to the AP-1 site.     

The basic region of Fos mediates specific DNA binding.

The DNA-binding domains of the members of the Fos and Jun families of proteins consist of a basic region followed by a dimerizing segment with heptad repeats of leucine. Fos-Jun heterodimers and Jun alone, but not Fos alone, bind to the symmetrical sequences TGACTCA (AP-1 site) or TGACGTCA (cAMP response element or CRE). We set out to test the hypothesis that in the Fos-Jun heterodimer the basic region of Fos confers specific DNA-binding properties equivalent to the contribution of the basic region of Jun. Fos-Jun chimeric proteins were prepared consisting of the basic region of one protein joined to the leucine repeat of the other. Heterodimers with mixed Fos and Jun leucine repeat segments showed high affinity binding to the AP-1 site or CRE whether they contained two basic regions from Jun, two basic regions from Fos, or one from each source. Heterodimers with two Fos basic regions showed somewhat greater affinity for the CRE and AP-1 site than the heterodimer with two Jun basic regions. The DNA sequence specificity and the purine and phosphate DNA contact sites for each heterodimer were similar. We conclude that in the Fos-Jun heterodimer the basic region of Fos contributes specific DNA-binding properties equivalent to those of Jun. Our results support a model in which the Fos and Jun basic regions of the Fos-Jun heterodimer each interact with symmetrical DNA half sites.     

Ten ERK-related proteins in three distinct classes associate with AP-1 proteins and/or AP-1 DNA.

We have identified seven ERK-related proteins ("ERPs"), including ERK2, that are stably associated in vivo with AP-1 dimers composed of diverse Jun and Fos family proteins. These complexes have kinase activity. We designate them as "class I ERPs." We originally hypothesized that these ERPs associate with DNA along with AP-1 proteins. We devised a DNA affinity chromatography-based analytical assay for DNA binding, the "nucleotide affinity preincubation specificity test recognition" (NAPSTER) assay. In this assay, class I ERPs do not associate with AP-1 DNA. However, several new "class II" ERPs do associate with DNA. p41 and p44 are ERK1/2-related ERPs that lack kinase activity and associate along with AP-1 proteins with AP-1 DNA. Class I ERPs and their associated kinase activity thus appear to bind AP-1 dimers when they are not bound to DNA and then disengage and are replaced by class II ERPs to form higher order complexes when AP-1 dimers bind DNA. p97 is a class III ERP, related to ERK3, that associates with AP-1 DNA without AP-1 proteins. With the exception of ERK2, none of the 10 ERPs appear to be known mitogen-activated protein kinase superfamily members.