Gamma interferon (IFN-gamma), a cytokine produced by activated T- lymphocytes and natural killer (NK) cells, has several effects on its target cells including the induction of antiviral activity, the modulation of immune cell function (monocyte differentiation, macrophage and NK cell activation, immunoglobulin class-switching, and increased MHC class I and II expression), and the regulation of cell growth. The overall aim of this project is to investigate the mechanisms by which IFN-gamma elicits such a diverse group of cellular responses. As a first step in identifying the components of the intracellular signalling system(s) used by IFN-gamma, this proposal describes the study of the transcriptional regulation of a gene that is expressed in murine macrophages in response to IFN-gamma. The mRNA encoded by this gene, designated mig, is strongly induced by IFN- gamma, but not by a number of other macrophage activators, including alpha- and beta-interferon and endotoxin. Transcriptional activation of the mig gene by IFN-gamma has been confirmed in nuclear run-on experiments. Sequence analysis of mig cDNA clones has revealed that the protein encoded by the mig mRNA represents a new member of a family of growth and chemotactic factors that includes, among others, the gro protein/melanoma growth stimulatory activity (MGSA), connective tissue activating peptide III, platelet factor 4, and a monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8). It is likely, therefore, that the regulation of mig gene expression may be important in the biologic responses to IFN-gamma.
The specific aims of this proposal are to: further define the cis-acting element(s) responsible for the induction of mig mRNA by IFN-gamma, identify and characterize the trans-acting factors that bind to the IFN-gamma- responsive element(s) and regulate mig transcription; purify these trans- acting factors and obtain their cDNA clones. Toward these ends, genomic DNA clones that include the region of the mig gene immediately 5' to the transcription start site have been isolated and sequenced. Genomic fragments from the promoter region have been inserted into vectors containing the reporter gene chloramphenicol acetyltransferase (CAT) and have been found to be selectively induced by IFN-gamma. The 5' end of a novel IFN-gamma-responsive element has been localized to within 2 base pairs by deletion analysis. The genomic fragment that includes this sequence confers IFN-gamma-inducibility a heterologous promoter. Gel electrophoresis mobility shift assays with this mig genomic fragment demonstrate the formation of a new DNA-protein complex in cells treated with IFN-gamma. The proposed studies should provide important information regarding the molecular basis for the regulation of gene expression in response to IFN-gamma. Since it has been reported that the actions of IFN- gamma are highly dependent on the expression of new mRNAs and proteins, the proposed studies are particularly relevant to our understanding of the mechanisms by which IFN-gamma exerts its antiviral, antineoplastic, and immunomodulatory properties.
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