Background: Heterologous transcription factors bound to adjacent sites in eukaryotic promoters often exhibit cooperative behavior. In most instances, the molecular basis for this cooperativity is poorly understood. Our efforts have been directed toward elucidation of the mechanism of cooperativity between NFAT and AP-1, two proteins that coordinately direct expression of the T-cell growth factor interleukin-2 (IL-2).
Results: We have previously shown that NFAT1 orients the two subunits of AP-1, c-Jun and c-Fos, on DNA through direct protein-protein interactions. In the present study, we have constructed cJun-cFos chimeric proteins and determined their orientation using a novel affinity-cleavage technology based on chemical ligation. We find that, in the presence of NFAT, the chimeric heterodimer binds in such a way as to preserve the orientation of the AP-1 leucine zipper, but not that of the basic region.
Conclusions: Protein-protein interactions between NFAT and the leucine zipper of AP-1 enable the two proteins to bind DNA cooperatively and coordinately regulate the IL-2 promoter. The chemical ligation technology presented here provides a powerful strategy for affinity cleavage studies, including those using recombinant proteins.