IFN-gamma induces a p91/Stat1 alpha-related transcription factor with distinct activation and binding properties.

The 5' flanking region of the mig gene, a member of the chemokine family of small m.w. chemoattractant and growth regulatory factors, contains an IFN-gamma-responsive enhancer, gamma RE-1, consisting of an extended imperfect palindrome. In this report we show that a novel factor, gamma RF-1, which binds to the gamma RE-1 element, is rapidly activated in a variety of primary cell types and tumor cell lines treated with IFN-gamma. Our data indicate that gamma RF-1 is present in a latent form in unstimulated cells and its DNA-binding activity is dependent upon tyrosine phosphorylation. UV cross-linking studies revealed that gamma RF-1 consists of at least two proteins of approximately 95 and 130 kDa, which interact with the gamma RE-1 element. A comparison of gamma RF-1 and GAF, an IFN-gamma-activated transcription factor containing the p91/Stat1 alpha protein (Stat, signal transducer and activator of transcription), showed that these two factors exhibited differences in electrophoretic mobility, responsiveness to IFN-alpha, and kinetics of activation. Using anti-Stat Ab, however, we found that one or more subunits of gamma RF-1 are antigenically related to p91/Stat1 alpha. Our results indicate, therefore, that gamma RF-1 and GAF are distinct IFN-gamma-responsive transcription factors and probably contain closely related members of the Stat protein family.

A unique palindromic element mediates gamma interferon induction of mig gene expression.

To define the molecular mechanisms involved in the action of gamma interferon (IFN-gamma), we have analyzed the transcriptional regulation of the mig (monokine induced by gamma interferon) gene, a member of the platelet factor 4-interleukin-8 cytokine family that is expressed in murine macrophages specifically in response to IFN-gamma. Analysis of mig/CAT chimeric constructs transiently transfected into the RAW 264.7 mouse monocytic cell line revealed a unique IFN-gamma-responsive element (gamma RE-1). The sequence of this cis regulatory element defined by deletion analysis contains an imperfect inverted repeat extending 27 bp. Examination of mig/CAT constructs with mutations in gamma RE-1 revealed that the palindromic positions in the element were essential for activity. Consistent with its function as an enhancer, a single copy of gamma RE-1 conferred IFN-gamma inducibility to a heterologous (herpes simplex virus thymidine kinase) promoter. Exonuclease III protection assays demonstrated symmetrical protection of a mig promoter fragment centered about the gamma RE-1 palindromic sequence. Using the gel electrophoretic mobility shift assay, we identified a factor (gamma RF-1) present in nuclear extracts prepared from IFN-gamma-stimulated RAW 264.7 cells which binds to gamma RE-1. The activation of gamma RF-1 occurred rapidly (within 1 min) in response to IFN-gamma and was independent of protein synthesis. Similar to the expression of mig mRNA, the formation of gamma RF-1 was selectively induced by IFN-gamma and not IFN-alpha. The regulation of gene expression through gamma RF-1 and gamma RE-1 may explain the preferential activation of a subset of interferon-inducible genes by IFN-gamma.