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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 (TAFII) 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 TAFII130 (hTAFII130) and itsDrosophila melanogaster homolog dTAFII110 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 hTAFII130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAFII250, and hTAFII130 supported NFATp-activated transcription, demonstrating the ability of hTAFII130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAFII130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAFII130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.
ACKNOWLEDGMENTS
We thank Tim Hoey, Naoko Tanese, Robert Tjian, and Anne Whalen for reagents and advice. J.A.G. is grateful to Robert Tjian and Tom Cech for their generous support, especially during the early stages of this work.
This research was supported by a Public Health Service grant, GM-55235, from the National Institutes of Health. J.A.G. is currently a Pew Scholar in the Biomedical Sciences and was a Special Fellow of the Leukemia Society of America during the early stages of this work. L.J.K. was supported in part by an NIH Predoctoral Training Grant, T32 GM08345, and by Beverly Sears Dean's Small Grants (University of Colorado).