Jun DNA-binding is modulated by mutations between the leucines or by direct interaction of fos with the TGACTCA sequence

Mutations between the leucines of the "leucine zipper" domain of Jun D can either decrease (Asn 301 to Ala) or increase (Thr 307, Ala 308, to Glu, Val) homodimer formation and specific binding to DNA even though such changes do not modify the predicted alpha-helical structure of this region. As shown previously, addition of Fos strongly increases the affinity of Jun for DNA by forming a heterodimer. The jun down mutation (Asn 301 to Ala) also diminishes DNA binding by the Fos-Jun D heterodimer. These data strongly support the coiled coil conformation of this region where residues adjacent to the leucines are also important for dimer formation. Ultraviolet cross-linking experiments have shown that both Fos and Jun directly contact the TGACTCA palindromic sequence defined as a TPA (12-O-tetradecanoyl phorbol-13-acetate) response element or TRE. Both Jun homodimers and Jun-Fos heterodimers bind this TRE as well as the cAMP responsive element (CRE or TGACGTCA) with comparable affinities. While strong c-Jun or Jun D binding requires a perfect palindrome, Jun-Fos complexes can also efficiently recognize sequences where the right half of the palindrome is less conserved (TGACTAA or TGACGCA).     

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.     

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.     

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.     

Asymmetrical recognition of the palindromic AP1 binding site (TRE) by Fos protein complexes.

Fos and Jun proteins form a tight complex which binds specifically to the AP1 recognition sequence, a palindromic DNA element also referred to as the TPA responsive element (TRE). To elucidate the mechanism of Fos-Jun interaction with the TRE we have performed UV cross-linking studies using oligonucleotides where thymines were replaced with bromouracil. Our results indicate that both Fos and Jun directly contact the TRE but that the interaction of Fos and Jun with thymines in structurally equivalent positions in the two half sites of the TRE is different. In addition, we have carried out a comprehensive mutagenesis study of the TRE by introducing all possible point mutations plus thymine----uracil substitutions into the palindromic TRE core sequences and the adjacent nucleotides on both sides. The results of this analysis clearly show that the palindromic TRE is asymmetrical with respect to binding of Fos-Jun. We also show that a Fos protein complex with a homodimeric DNA binding site binds considerably less efficiently to TRE mutants with a perfect dyad symmetry compared with the binding to the wild-type TRE. This demonstrates that the asymmetrical recognition of the TRE is not due to the heterodimeric nature of the Fos/Jun complex but directly related to an asymmetry in the TRE sequence. The methyl groups of all four thymine residues within the TRE seem to be functionally crucial since thymine----uracil substitutions strongly reduce or abolish binding to Fos/Jun. The relevance of structurally equivalent methyl groups in the TRE core sequence is different, lending further support to the conclusion that the TRE is asymmetrical.     

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.