Characterization of nuclear protein binding to the interferon-gamma promoter in quiescent and activated human T cells.
Nuclear protein binding to the human interferon-gamma (IFN-gamma) promoter was investigated to determine the structural basis for the control of gene expression during T cell activation. DNase I footprinting of gel-shift complexes demonstrated that proteins bind to two downstream (-124 to -114 and -36 to -30) and one upstream (-534 to -486) element in the IFN-gamma gene promoter. Treatment of human peripheral blood lymphocytes or continuous T cell tumors with phorbol 12-myristate 13-acetate (PMA) plus phytohemagglutinin or calcium ionophore results in a pattern of response that is similar when using either the upstream or downstream elements. Upon induction of T cells, the lower mobility gel-shift band disappears. Yet the equivalent band which is also present in non-T cells is unperturbed after PMA + calcium ionophore treatment. The higher mobility band which is modified upon induction is restricted to the T cell lineage. Upstream and downstream elements share similar protein-binding motifs as indicated by the homology of footprinted sequences, the similarity of protein-binding patterns, and the ability of these elements to compete against each other in gel-shift protein-binding assays. Protein binding to the downstream elements appears to be interactive, since both sites are required for complex formation. When either of the two downstream elements is disrupted by site-directed mutagenesis, the higher mobility gel-shift band is diminished by an amount that is consistent with the reduction in reporter (chloramphenicol acetyltransferase) gene expression. Therefore, proteins in the ubiquitous gel-shift band appear to be associated with the inactive state of IFN-gamma, while the modified band is closely associated with the positive regulation of IFN-gamma gene expression.
Brown
D A
DA
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Kondo
K L
KL
Wong
S W
SW
Diamond
D J
DJ
D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus; D002872:Chromosome Deletion; D016572:Cyclosporine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism*; D009928:Organ Specificity; D010835:Phytohemagglutinins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009687:Nuclear Proteins; D010835:Phytohemagglutinins; D007371:Interferon-gamma; D016572:Cyclosporine; D004247:DNA; D013755:Tetradecanoylphorbol Acetate