Promoter analysis of an interferon-inducible gene associated with macrophage activation.
We have investigated the regulation of an activation-associated guanylate-binding protein gene (mGBP-1/mag-1) in murine macrophage cell lines in response to the cytokine IFN-gamma. One of the cell lines utilized (RAW 264.7) acquires the ability to kill tumor cells after IFN-gamma and LPS treatment, whereas the other (WEHI-3) does not. We previously have demonstrated that mGBP-1 is induced by IFN-gamma in RAW 264.7 but not WEHI-3 cells. Here we present information concerning the cloning, sequencing, and initial characterization of the upstream region of the mGBP-1 gene as a first step towards understanding the differential control of this gene in RAW 264.7 versus WEHI-3 cells. Genomic fragments encompassing a portion of the mGBP-1 5' flanking region were inserted into vectors containing a luciferase reporter gene. 928 bp of upstream sequence were found to be sufficient for IFN-gamma-mediated induction of luciferase activity in the RAW 264.7 cell line. Furthermore, sequences within 100 bp of the major transcription initiation site conferred strong IFN-gamma responsiveness to the reporter gene. A perfect match to the interferon-stimulated response element (ISRE) was present within this region, and was shown to be essential for interferon-induced expression. An oligonucleotide corresponding to the mGBP-1 ISRE bestowed interferon-inducible expression on a heterologous minimal promoter. Site-specific mutation of the ISRE within the 106-bp upstream region eliminated interferon inducibility of this construct. Taken together, the results indicate the ISRE is necessary and sufficient for IFN-gamma induction of the mGBP-1 gene. Transient transfection assays carried out with the WEHI-3 cell line indicated that all promoter constructs were transcriptionally inactive in these cells, including the ISRE-minimal promoter construct. The inability of the WEHI-3 cell line to utilize an ISRE after IFN-gamma induction may underlie the functional differences exhibited by the two cell lines after IFN-gamma stimulation.
Nicolet
C M
CM
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison 53706.
Paulnock
D M
DM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000276:immunology; D017930:Genes, Reporter; D015698:Genomic Library; D007371:Interferon-gamma / Q000502:physiology*; D008262:Macrophage Activation / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
D007371:Interferon-gamma; D019204:GTP-Binding Proteins