Human TAF II 130 Is a Coactivator for NFATp

Abstract

ABSTRACT 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 (TBP) alone was insufficient in supporting activated transcription. In vitro interaction assays revealed that human TAF II 130 (hTAF II 130) and its Drosophila melanogaster homolog dTAF II 110 bound specifically and reproducibly to immobilized NFATp. Sequences contained in the C-terminal domain of NFATp (amino acids 688 to 921) were necessary and sufficient for hTAF II 130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAF II 250, and hTAF II 130 supported NFATp-activated transcription, demonstrating the ability of hTAF II 130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAF II 130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAF II 130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.