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 recognition elements in two opposite orientations. We have developed a gel-based fluorescence resonance energy transfer assay for quantifying heterodimer orientation preferences. Fos-Jun heterodimers bind in opposite orientations to AP-1 sites with different flanking sequences. The effects of individual amino acid and base pair substitutions on heterodimer binding orientation were quantified. Base pairs at positions ±6 and ±10 relative to the center of the AP-1 site were the principal determinants of Fos-Jun binding orientation. Amino acid residues of opposite charge adjacent to the basic regions of Fos and Jun had independent effects on heterodimer orientation. Exchange of these amino acid residues between the basic region-leucine zipper domains of Fos and Jun reversed the binding orientation. Heterodimers formed by full-length Fos and Jun exhibited the same changes in binding orientation in response to amino acid and base pair substitutions. The preferred orientation of heterodimer binding affected the stability of Fos-Jun-NFAT1 complexes at composite regulatory elements. Changes in heterodimer orientation preference altered the transcriptional activity and the promoter selectivity of Fos-Jun-NFAT1 complexes. Consequently, the orientation of Fos-Jun binding can influence transcriptional activity by altering cooperative interactions with other transcription regulatory proteins.

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