We have investigated events following treatment of cells with interferon‐gamma (IFN‐gamma) that lead to the immediate transcriptional activation of an inducible gene. A gamma‐interferon activation factor (GAF) was activated in the cytoplasm of human fibroblasts immediately after IFN‐gamma treatment and bound to a newly identified target DNA sequence, the gamma‐interferon activation site (GAS). The time course of activation of GAF was different in fibroblasts and HeLa cells and correlated well with IFN‐gamma‐induced transcriptional activation in both cell types. IFN‐gamma‐dependent activation of GAF also occurred in enucleated cells (cytoplasts), showing that an inactive cytoplasmic precursor is converted to the active factor. These findings support the concept that ligand‐specific signals originating at the cell surface are transmitted through latent cytoplasmic proteins which are activated to bind specific DNA sites and then move to the nucleus to activate the transcription of specific sets of genes.

The gene encoding a 67-kDa cytoplasmic guanylate-binding protein (GBP) is transcriptionally induced in cells exposed to interferon of either type I (alpha interferon [IFN-a] or INF-,) or type II (IFN--y 20,30,31). A DNA-binding protein termed ISGF-3 is responsible for the IFN-x-dependent activation through the ISRE. This conclusion is based on a variety of evidence, including the parallel induction of ISGF-3 and transcription even in the absence of ongoing protein synthesis (19,20), and on in vitro transcriptional activation of an ISRE-containing promoter by ISGF-3 (13a). Other factors bind to the ISRE, but their role, if any, in IFN-regulated transcription is unknown (7,19,20,34,36 Given a clearly defined DNA site that is relevant for transcriptional induction by IFN--y, proteins must be found and defined that fulfill the requirements of transcriptional activators. Blanar et al. (2) have proposed that an IFN-yinducible ISRE-binding factor, which they termed IBP-1, mediates the response of the H2-Kb gene to IFN-y. On the basis of its apparent molecular weight, ISRE-binding characteristics, and IFN--y inducibility, IBP-1 is probably identical to the previously described ISGF-2 (also called IRF-1) (25,28). Is ISGF-2 (IRF-1, IBP-1) the factor responsible for the response of ISRE-containing genes to IFN--y? In this report, we describe the cloning and characterization of the promoter of the guanylate-binding protein (GBP) gene, which is inducible by both IFN types (3,8,21

Interferon‐alpha (IFN alpha) and interferon‐gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell‐surface receptors to induce expression of largely non‐overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10‐fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis‐acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10‐fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The promoter of the gene encoding a cytoplasmic guanylate-binding protein (GBP) contains two overlapping elements: the interferon stimulation response element (ISRE), which mediates alpha interferon (IFN-at)-dependent transcription, and the IFN-y activation site (GAS), which is required for IFN-y-mediated stimulation. The ISRE binds a factor called ISGF-3 that is activated by IFN-a but not by IFN-'y. The GAS binds a protein that is activated by IFN-,y, which we have termed GAF (IFN--y activation factor; T. Decker,

Transcriptional regulation of the gene encoding a guanylate‐binding protein (GBP) by the two interferon (IFN) types was studied. GBP gene transcription was regulated by alpha IFN in a manner identical to that of previously described IFN‐stimulated genes (ISGs): rapid induction, without a need for protein synthesis, followed by a protein synthesis‐dependent suppression of transcription to basal levels within 6 h. Transcriptional induction by gamma IFN was equally rapid and independent of ongoing protein synthesis but remained at elevated levels for greater than 24 h. Experiments employing combined treatments with IFNs of both types revealed that induction of the GBP gene by gamma IFN overrides the alpha IFN‐induced active repression and reverses the alpha IFN‐induced repressed state. Moreover, the alpha IFN‐mediated repression of ISG54, a gene normally responsive to only alpha IFN, is also reversed by gamma IFN. Induction of GBP by gamma IFN is presumably mediated by a factor different from the recently described activator Interferon Stimulated Gene Factor 3 (ISGF3) because induction of this factor was not observed upon treatment of cells with gamma IFN. Finally, a complex set of reinforcing or synergistic effects were observed when induction of the GBP gene was evoked by a combined treatment with the two IFN types.