Constitutive and inducible protein/DNA interactions of the interferon-gamma promoter in vivo in [corrected] CD45RA and CD45R0 T helper subsets.
Interferon-gamma (IFN-gamma) is a key cytokine of T lymphocytes with major regulatory functions in the immune system. To determine and compare protein/DNA interactions at the native IFN-gamma locus in T cells, we analyzed the human IFN-gamma promoter by ligation-mediated polymerase chain reaction (LM-PCR) techniques. Accordingly, Jurkat T cells and primary CD45RA and CD45R0 CD4+ T cell subsets isolated from peripheral blood using immunomagnetic beads were cultured and analyzed by LM-PCR. Constitutive and inducible protein/DNA interactions of the IFN-gamma promoter in vivo were detected in all T cells tested. Interestingly, an inducible footprint between -183 and -196 was consistently observed in Jurkat T cells and CD45RA and CD45R0 T helper subsets upon stimulation with phorbol 12-myristate 13-acetate+phytohemagglutinin (PMA+PHA) that was highly sensitive to treatment with corticosteroids. This novel target site, denoted the C-site, was shown by several criteria, including cell distribution studies, stimulation experiments, supershift assays, and cross-competition electrophoretic mobility shift assays to bind the transcription factor AP-1. Mutation of the C-site that prevented AP-1 binding to this site was sufficient strikingly to reduce inducible promoter activity in primary CD45R0 T cells. In summary, the data demonstrate that IFN-gamma gene transcription in primary T cells is regulated in vivo at the level of constitutive and inducible protein/DNA interactions. We propose a model where basal transcription is maintained by binding of various transcription factors to the IFN-gamma promoter, whereas PMA+PHA-inducible IFN-gamma transcription in CD45R0 T cells is associated with binding of AP-1 to the C-site.
Barbulescu
K
K
Department of Medicine, University of Mainz, Germany.
Meyer zum Büschenfelde
K H
KH
Neurath
M F
MF
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000032:analysis*; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009698:Nucleoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D010835:Phytohemagglutinins / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000276:immunology*; D016329:Sp1 Transcription Factor / Q000096:biosynthesis; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects
D005938:Glucocorticoids; D009698:Nucleoproteins; D010835:Phytohemagglutinins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D007371:Interferon-gamma; D004247:DNA; D017493:Leukocyte Common Antigens; D013755:Tetradecanoylphorbol Acetate