Transcription factors of the NFAT family control the activation of genes encoding cytokines and their receptors in response to antigenic stimulation of immune cells. Localized in the cytoplasm of resting cells, NFAT translocates to the nucleus upon dephosphorylation by the Ca²⁺-activated Ser/Thr phosphatase calcineurin. The clinically important immunosuppressive agents FK506 and cyclosporin A block nuclear translocation of NFAT by directly inhibiting the phosphatase activity of calcineurin. The pronounced toxicity of these agents has created impetus for the development of new drugs that suppress T cell activation through inhibition of novel protein targets, foremost among which is NFAT.

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The transcription factors NFAT and AP-1 coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein-protein interactions with NFAT in the ternary NFAT / AP-1 / DNA complex.  We have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the Rel Insert Region (RIR) of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, Asn-541). These results were confirmed and extended through in vitro binding assays.
We have also shown that NFAT orients the two subunits of AP-1, c-Jun and c-Fos, on DNA through direct protein-protein interactions.  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.
Activation of gene transcription in eukaryotes requires the cooperative assembly of an initiation complex containing many protein subunits. The necessity that these components contact each other and the promoter/enhancer in defined ways suggests that their spatial arrangement might influence the activation response. Indeed, growing evidence indicates that DNA architecture can profoundly affect transcriptional potency. Much less is known about the influence of protein architecture on transcriptional activation. Here, we examine the architectural dependence of activator function through the analysis of matched pairs of AP-1DNA complexes differing only in their orientation.

• Zhou, P.; Sun, L. J.; Dötsch, V.; Wagner, G.; Verdine, G. L. "Solution Structure of the Core NFATC1/DNA Complex," Cell 1998, 92, 687-696. Download pdf file
• Wolfe, S. A.; Zhou, P.; Dötsch, V.; Chen, L.; You, A.; Ho, S. N.; Crabtree, G. R.; Wagner, G.; Verdine, G. L. "Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc," Nature 1997, 385, 172-176.
• Sun, L. J.; Peterson, B. R.; Verdine, G. L. "Dual Role of the NFAT Insert Region in DNA Recognition and Cooperative Contacts to AP-1," Proc. Natl. Acad. Sci. USA 1997, 94, 4919-4924. Download pdf file
• Peterson, B. R.; Sun, L. J.; Verdine, G. L. "A Critical Arginine Residue Mediates Cooperativity in the Contact Interface Between NFAT and AP-1," Proc. Nat. Acad. Sci. USA 1996, 93, 13671-13676. Download pdf file
• Erlanson, D. A.; Chytil, M.; Verdine, G. L. "The Leucine Zipper Domain Controls the Orientation of AP-1 in the NFAT•AP-1•DNA Complex," Chem. & Biol. 1996, 3, 981-991. Download pdf file
• Chen, L.; Oakley, M. G.; Glover, J. N. M.; Jain, J.; Dervan, P. B.; Hogan, P. G.; Rao, A.; Verdine, G. L. "Only One of the Two DNA-bound Orientations of AP-1 Found in Solution Cooperates with NFATp," Curr. Biol. 1995, 5, 882-889.
• Chytil, M.; Verdine, G. L. "The Rel Family of Eukaryotic Transcription Factors," Curr. Op. Struct. Biol. 1996, 6, 91-100.

NFATC1-DBD* utilizes a combination of direct and indirect readout mechanisms involving both major and minor groove contacts to achieve DNA sequence specificity. The 5'-end of the recognition site (GAGGAAAA) is recognized mainly through major groove contacts made by residues of the DNA recognition loop (ßA-ßB loop). Several of these key contacts are very similar to those made by the homologous DNA recognition loop of NF-B p50. The side chain guanidinium groups of Arg-441 and Arg-439 are positioned to hydrogen bond to G1 and G2, respectively, and are buttressed by the carboxylate of Glu-445 . The side chain of Tyr-442 is brought close to T3' and T4' through a hydrophobic contact between the phenyl ring and the T3' methyl group, and a hydrogen bond between the hydroxyl and the DNA backbone phosphate located between T4' and T5' (T4'pT5').  At the extreme 5'-end of the site, G(-2) is recognized through an interaction with Arg-448.  Positions 4 and 5 in the poly(A) stretch of the NFAT site (GAGGAAAA) are both highly conserved among known NFAT sites and are selected at a high frequency in PCR site selection experiments using NFATC2. The basis for such exquisite sequence specificity at these positions is not obvious from the structural data alone. No residue of NFATC1-DBD* is close enough to base pair 4 to make a sequence-specific interaction. Although the side chains of Ser-588 and Arg-590 are in the vicinity of T5', they do not appear to lie within optimal contact distance. Even if side chains of the protein do contact T5', these interactions do not appear to contribute to sequence specificity: switching base pairs 5 and 4 from A·T to I·C, which drastically alters major groove functionality but leaves minor groove functionality unperturbed, has little if any effect on the strength of NFATC1-DNA interactions. Taken together, these data suggest that NFATC1 (and NFATC2) recognizes A4 and A5 by sensing the sequence-dependent deformability of the poly(A) stretch, a mechanism known as "indirect readout."  Finally, a residue of the insert region, Arg-555, projects its side chain into the minor groove at the extreme 3'-end of the site to contact T6' and T7.

The DNA recognition elements of many transcription factors are disordered in the absence of DNA and undergo an induced folding transition upon interaction with DNA. Such behavior appears to be especially common among transcription factors that contain predominantly a-helical DNA recognition domains, such as members of the bZIP and basic helix-loop-helix families. Even though the core DNA recognition domain of NFATC1 comprises predominantly ?structure, the structure presented here reveals that it, too, undergoes an induced folding transition upon interaction with DNA. Specifically, NMR relaxation measurements on unliganded NFAT-DBD revealed that the Ig domain adopts a well-defined three-dimensional structure, with the exception of the ßA-B and ßG'-H loops, which are disordered. In the binary solution structure, both of these loops become ordered upon interaction with DNA, and indeed, both contribute directly to the DNA-contact interface. Especially striking is the structural transition of the insert region from a completely disordered loop to a compact module containing an a helix (aA). This helix appears to be stabilized directly through a hydrogen-bonding interaction between two of its N-terminal amide protons (Asn-541 and Ser-542) and a DNA backbone phosphate (T5'pT6'), and the overall helix is aligned so as to permit a favorable interaction of its helix dipole with DNA.
The comparison of the binary solution structure of NFAT with the X-ray structure of the ternary complex thus suggests that whole-domain structural remodeling facilitates the cooperative assembly of a higher order transcriptional complex. It is now recognized that the generation of a transcriptional response in eukaryotic cells requires the cooperative promoter assembly of a particle known as an enhanceosome, which may contain a dozen or more individual DNA-binding subunits. Most if not all of these subunits participate in enhanceosome assembly on multiple promoters, amongst which there is great variation in physical location and relationship of cooperating protein partners. Given such stereochemical diversity in enhanceosome assembly, the requirement that these proteins be capable of physically contacting each other while remaining anchored to DNA imposes a formidable geometric challenge. Whole-domain structural remodeling serves to decrease the geometric precision required for cooperative interactions on DNA and may thus be generally associated with the process of transcriptional activation in eukaryotic cells.

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