In vitro transcription faithfully reflecting T-cell activation requirements.
T-cell activation is a complex process mediated by cell membrane molecules including the T-cell antigen receptor (TCR), adhesive molecules, and cytokine receptors that collectively produce an increase in intracellular Ca2+, and activation of protein kinase C that initiate a genetic program resulting in immunologic function and irreversible differentiation. To understand how these cell membrane events are translated into a genetic regulatory cascade resulting in T-cell function, we have developed an in vitro transcription system, derived from Jurkat T-cells, which demonstrates inducible, cell-type-specific transcription following T-cell stimulation. Nuclear extracts from cells stimulated with phorbol 12-myristate 13-acetate and ionomycin, which activate protein kinase C and mimic physiological activation through the T-cell antigen receptor, transcribe an interleukin-2 (IL-2) enhancer (-326 to +24) template 5-fold more efficient than nuclear extracts from resting T-cells and severalfold more efficient than extracts from Jurkat cells treated with phorbol 12-myristate 13-acetate or ionomycin alone. Further results demonstrate that in vitro transcription of the IL-2 enhancer is T-cell specific since nuclear extracts from rat liver and stimulated HeLa cells are unable to induce IL-2 transcription. The activation-dependent, T-cell-specific in vitro transcription system described here should facilitate the dissection of signals that emanate from the T-cell surface resulting in IL-2 transcription and T-cell activation.
Flanagan
W M
WM
Howard Hughes Medical Institute, Beckman Center, Stanford University, California 94305.
Crabtree
G R
GR
D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate