Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1

Abstract

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. 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. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here 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 RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

Figure 1

Activation of lacZ reporter gene transcription by NFAT acting alone and in cooperation with AP-1. (A) A model of the NFAT2-DBD (23) bound to a consensus NFAT site. AD denotes the B42 activation domain, which is fused to the N terminus of the NFAT2-DBD. (B) A composite model of the NFAT2-DBD, bound to a polypurine tract in ARRE2, adjacent to AP-1 bound to the nonconsensus AP-1 site. Cooperative recruitment of c-Jun/c-Fos-AD to DNA by NFAT activates reporter gene expression (25). In the cooperativity assay the B42 activation domain is fused to the N terminus of c-Fos. (C) Reporter gene expression driven by NFAT-AD binding alone to a consensus NFAT site in yeast. (D) ARRE2-linked reporter assay detects assembly of the cooperative AP-1/NFAT/ARRE2 complex in yeast (25). Transcriptional activity refers to units of β-gal activity.

Figure 2

The effects of alanine-scanning mutations on NFAT2 activity in vivo. Sequence of the NFAT2 RIR, with positions mutated to alanine denoted below. To the right of the sequences are shown the transcriptional activity of wild-type and mutant NFAT2-RHR proteins in the cooperative ARRE2 reporter assay, and (for selected examples) in the NFAT-AD reporter assay. The standard error in the measurements of transcriptional activity assays are estimated at ± 25%.

Figure 3

EMSA analysis of NFAT and AP-1 DNA-binding activity in vitro. (A–C) Wild-type, E545A, and T551A mutant NFAT2 proteins bind with essentially identical affinities to the ARRE2 (K_d ≈ 16 nM). (B and C) The E545A and T551A mutants show a substantially diminished ability to cooperate with NFAT2. From the data in A, the effective K_d of AP-1 for the wild-type NFAT2-RHR/DNA complex is estimated at 50 nM. From the data in B and C, the effective K_d of AP-1 for the NFAT2(E545A)/ARRE2 complex is estimated at 130 nM, and for the NFAT2(T551A)/ARRE2 complex is 160 nM. Nonspecific binding of proteins to DNA was observed at high protein concentrations (lane 6 of both A and B).

Figure 4

EMSA analysis of NFAT DNA-binding activity in vitro. (A) The effective K_d of wild-type NFAT2-RHR for ARRE2 is estimated at 20 nM. (B and C) The K538A and R540 NFAT2 mutants bind ARRE2 with substantially lower affinity. From the data in A–C, the effective K_d of the wild-type, K538A and T551A mutants for ARRE2 is ≈20 nM, ≈165 nM, and ≈170 nM, respectively. Nonspecific binding of proteins to DNA was observed at high protein concentrations (lane 5 and 6 of A).

Figure 5

(A) Model of the ternary NFAT/AP-1/DNA complex (23). The solution structure of the NFAT2-DBD (23) was superimposed with x-ray coordinates of DNA-bound p50 (30) by rigid body fitting of their respective β-barrels. The overall conformation of the NFAT RIR in the model was derived from a non DNA-bound structure and is likely to differ from that in the actual complex. The x-ray coordinates of the AP-1 bZip domain (27) were used to assemble the heterodimer over the nonconsensus AP-1 site in ARRE2, with positional and orientational information derived from affinity cleaving experiments (28). (B) An expanded view of the of the NFAT2 RIR and the junction of AP-1 between the basic and leucine-zipper domains. The side chains of three residues identified as being most critical for cooperativity (NFAT E545, NFAT T551, and c-Jun R285) are illustrated. (C) The view shown in B rotated by 180° about the vertical axis. The side chains of two NFAT residues critical for specific DNA recognition (K538 and R540) are shown. (D) Sequence alignment of the insert region of the four NFAT isoforms. Conserved residues identified here as being important for DNA recognition and cooperative binding with AP-1 are boxed.