At eukaryotic promoters, multi-faceted protein-protein and protein-DNA interactions can result in synergistic transcriptional activation. NFAT and AP-1 proteins induce interleukin-2 (IL-2) transcription in stimulated T cells, but the contributions of individual members of these activator families to synergistically activating IL-2 transcription is not known. To investigate the combinatorial regulation of IL-2 transcription we tested the ability of different combinations of NFATc2, NFATc1, cJun, and cFos to synergistically activate transcription from the IL-2 promoter. We found that NFATc2 and cJun are exclusive in their ability to synergistically activate human IL-2 transcription. Protein-protein interaction assays revealed that in the absence of DNA, NFATc2, but not NFATc1, bound directly to cJun/cJun dimers, but not to cFos/cJun heterodimers. A region of NFATc2 C-terminal of the DNA binding domain was necessary and sufficient for interaction with cJun in the absence of DNA, and this same region of NFATc2 was required for the synergistic activation of IL-2 transcription in T cells. Moreover, expression of this C-terminal region of NFATc2 specifically repressed the synergistic activation of IL-2 transcription. These studies show that a previously unidentified interaction between human NFATc2 and cJun is necessary for synergistic activation of IL-2 transcription in T cells.

NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAF II ) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein ( Transcription is a highly regulated process of RNA synthesis, the initiation of which is a primary control point in gene expression. In eukaryotes, genes encoding mRNA are transcribed by RNA polymerase II, a multiprotein enzyme. Reconstitution of an in vitro transcription system with purified RNA polymerase II proved to be insufficient for RNA synthesis from promoters, propelling the identification of a phylogenetically conserved set of general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that are necessary for basal transcription (reviewed in reference 45). The assembly of RNA polymerase II and the general transcription factors on promoter DNA leads to the formation of stable nucleoprotein complexes (preinitiation complexes) that are transcriptionally competent in vitro. Preinitiation complex formation can be regulated by the action of activators and repressors. The activities of these distinct regulatory proteins can promote (activators) or repress (repressors) the recruitment of general transcription factors and RNA polymerase II onto promoters, resulting in promoter-specific augmentation or suppression of transcription.Eukaryotic organisms have evolved elaborate mechanisms for the deployment of RNA polymerase II onto specialized promoter elements that can modulate basal promoter strength. Many RNA polymerase II promoters contain one or more of three identified core promoter elements that direct accurate transcription initiation (5): the TATA box, the initiator element, and the downstream promoter element. In a temporal scheme, TFIID is the first basal factor to bind to a core promoter, serving as a nucleation center for the rest of the transcriptional apparatus (60). The architecture of TFIID is also phylogenetically conserved, and it appears that TFIID complexes in yeast, Drosophila melanogaster, and humans have a similar complement of subunits (6). TFIID is a sequence-specific DNA binding general transcription factor consisting of TATA-binding protein (TBP) and 10 to 13 TATA-associated factors (TAF II s) that together can recognize the TATA box, initiator, and downstream promoter elements.

Background: NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp.