SummaryNuclear factor of activated T cells-family proteins (NFAT1/NFATp, NFATc, NFAT3, and NFAT4/NFATx/NFATc3) play a key role in the transcription of cytokine genes and other genes during the immune response. We have defined the mechanisms of transactivation by NFAT1. NFAT1 possesses two transactivation domains whose sequences are not conserved in the other NFAT-family proteins, and a conserved DNA-binding domain that mediates the recruitment of cooperating nuclear transcription factors even when it is expressed in the absence of other regions of the protein. The activity of the NH2-terminal transactivation domain is modulated by an adjacent regulatory region that contains several conserved sequence motifs represented only in the NFAT family. Our results emphasize the multiple levels at which NFAT-dependent transactivation is regulated, and predict significant differences in the architecture of cooperative transcription complexes containing different NFAT-family proteins.T he nuclear factor of activated T cells (NFAT) 1 is a calcium-regulated transcription factor that is a major target for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (1-3). The DNA-binding specificity of NFAT is conferred by a novel family of transcription factors, of which four members (NFAT1/NFATp, NFATc, NFAT3, and NFATx/NFAT4/NFATc3, each containing several isoforms) have so far been described (4-9). NFAT1 protein is expressed in several classes of immune system cells (10-12), consistent with the detection of nuclear NFAT DNA-binding activity in activated T, B, mast, and NK cells (13)(14)(15)(16)(17)(18)(19). The distribution of the other NFAT-family proteins has not yet been assessed at the protein level; however the mR.NAs encoding these proteins differ in their cell and tissue distributions and their representation in resting versus activated cells (5-7), suggesting that each protein may subserve a different function. NFATc mlLNA is expressed at high levels in skeletal muscle and in activated lymphoid cells, NFATx/ NFAT4 mP,.NA is predominantly expressed in the thymus, and NFAT3 mR.NA is most strongly expressed outside the immune system (5-7).An important component of transactivation by NFATfamily proteins is their cooperation in the nucleus with Fos and Jun (20,21). This cooperation is mediated by interac1Abbreviations used in this paper: CAT, chloramphenicol acetyltransferase; CsA, cyclosporin A; DBD, DNA-binding domain; hGH, human growth hormone; NFAT, nuclear factor of activated T cells; NHP,., NFAT homology region; TAD, transactivation domain. tion of the highly conserved DNA-binding domains (DBDs) of NFAT-family proteins with Fos-Jun and Jun-Jun dimers (6,7,22); the bZIP (basic region-leucine zipper) regions of Fos and Jun suffice for this interaction (21), although an acidic region of Fos has also been implicated (23). Overexpression of Fos-and Jun-family proteins in T cells greatly augments transcription driven by tandem copies of the IL-2 and IL-4 promoter NFAT sites in activated T cells (20,(24)(25)(26). It has al...