Control of the orientation of Fos-Jun binding and the transcriptional cooperativity of Fos-Jun-NFAT1 complexes

Heterodimeric transcription regulatory proteins can bind to palindromic rec ognition elements in two opposite orientations. We have developed a gel-bas ed fluorescence resonance energy transfer assay for quantifying heterodimer orientation preferences. Fos-Jun heterodimers bind in opposite orientation s to, AP-1 sites with different flanking sequences. The effects of individu al amino acid and base pair substitutions on heterodimer binding orientatio n were quantified. Base pairs at positions +/-6 and +/-10 relative to the c enter of the AP-1 site were the principal determinants of Fos-Jun binding o rientation. Amino acid residues of opposite charge adjacent to the basic re gions of Fos and Jun had independent effects on heterodimer orientation. Ex change of these amino acid residues between the basic region-leucine zipper domains of Fos and Jun reversed the binding orientation. Heterodimers form ed by full-length Fos and Jun exhibited the same changes in binding orienta tion in response to amino acid and base pair substitutions. The preferred o rientation of heterodimer binding affected the stability of Fos-Jun-NFAT1 c omplexes at composite regulatory elements. Changes in heterodimer orientati on preference altered the transcriptional activity and the promoter selecti vity of Fos-Jun-NFAT1 complexes. Consequently, the orientation of Fos-Jun b inding can influence transcriptional activity by altering cooperative inter actions with other transcription regulatory proteins.