Abstract

Combining in vitro DNA binding studies and functional transcription assays in the Xenopus oocyte, we have tested the presence and functional state of transcription factors controlling the interleukin-2 (IL-2) promoter through the NFAT binding site. In naive T-helper cells, the IL-2 gene is repressed by a silencer. After first mitogenic stimulation, this silencer becomes undetectable while an activator is newly synthesized. In resting memory cells, the activator has low DNA- binding affinity and is located in the cytoplasm. However, no silencer is formed. Upon renewed cellular activation, this pre-existing activator is again targeted to the nucleus and regains function in promoting transcription. Cyclosporin A and FK506 act on two distinct levels of the IL-2 control mechanism. They prevent nuclear transport and reactivation of the performed activator in memory cells and, in naive cells, they render the silencer resistant to displacement by the activator. DNA-binding of silencer and activator from T-helper, and NFAT-1 from Jurkat cells, requires the same three G residues, but cross- linking analyses show differences in their constituent subunits. Supershift experiments show that the activator contains fra-2 and junD, whereas the silencer reacts with none of the antibodies tested.

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