Constitutive and inducible protein/DNA interactions of the interferon-gamma promoter in vivo in [corrected] CD45RA and CD45R0 T helper subsets

Eur J Immunol. 1997 May;27(5):1098-107. doi: 10.1002/eji.1830270509.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites / genetics
  • DNA / metabolism*
  • DNA Footprinting
  • Genes, Reporter
  • Glucocorticoids / pharmacology
  • Humans
  • Interferon-gamma / genetics*
  • Jurkat Cells
  • Leukocyte Common Antigens / analysis*
  • Lymphocyte Activation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleoproteins / biosynthesis*
  • Nucleoproteins / genetics*
  • Nucleoproteins / metabolism
  • Phytohemagglutinins / pharmacology
  • Promoter Regions, Genetic / immunology*
  • Sp1 Transcription Factor / biosynthesis
  • T-Lymphocytes, Helper-Inducer / drug effects
  • T-Lymphocytes, Helper-Inducer / immunology*
  • T-Lymphocytes, Helper-Inducer / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription Factor AP-1 / metabolism
  • Transcription Factors / drug effects

Substances

  • Glucocorticoids
  • Nucleoproteins
  • Phytohemagglutinins
  • Sp1 Transcription Factor
  • Transcription Factor AP-1
  • Transcription Factors
  • Interferon-gamma
  • DNA
  • Leukocyte Common Antigens
  • Tetradecanoylphorbol Acetate