Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons.

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-alpha] or type II (IFN-gamma). The promoter of the GBP gene was cloned and found to contain an IFN-alpha-stimulated response element, which mediated the response of the GBP gene to IFN-alpha. On the basis of transfection experiments with recombinant plasmids, two different elements were delineated. Both were required to obtain the maximal response of the GBP gene to IFN-gamma: the IFN-alpha-stimulated response element and an overlapping element termed the IFN-gamma activation site. Different proteins that act on each element were investigated, and their possible involvement in IFN-gamma-induced transcriptional regulation is discussed.

Interferon regulatory factor 1 is required for mouse Gbp gene activation by gamma interferon.

Full-scale transcriptional activation of the mouse Gbp genes by gamma interferon (IFN-gamma) requires protein synthesis in embryonic fibroblasts. Although the Gbp-1 and Gbp-2 promoters contain binding sites for transcription factors Stat1 and IFN regulatory factor 1 (IRF-1), deletion analysis revealed that the Stat1 binding site is dispensable for IFN-gamma inducibility of Gbp promoter constructs in transfected fibroblasts. However, activation of the mouse Gbp promoter by IFN-gamma requires transcription factor IRF-1. Transient overexpression of IRF-1 cDNA in mouse fibroblasts resulted in high-level expression of Gbp promoter constructs. Unlike wild-type cells, IRF-1% embryonic stem cells lacking functional transcription factor IRF-1 contained very low levels of Gbp transcripts that were not increased in response to differentiation or treatment with IFN-gamma. Treatment of IRF-1% mice with IFN-gamma resulted in barely detectable levels of Gbp RNA in spleens, lungs, and livers, whereas such treatment induced high levels of Gbp RNA in the organs of wild-type mice. These observations suggest two alternative pathways for transcriptional induction of genes in response to IFN-gamma: immediate response that results from activation of preformed Stat1 and delayed response that results from induced de novo synthesis of transcription factor IRF-1.

Macrophage precursor cells produce perforin and perform Yac-1 lytic activity in response to stimulation with interleukin-2.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2-treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunohistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor's granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided.

The interferon-inducible GBP1 gene: structure and mapping to human chromosome 1.

We have isolated DNA clones containing the interferon (IFN)-inducible guanylate-binding protein-1-encoding gene (GBP1) from a human genomic library. Two overlapping phage clones contained the entire GBP1 gene. The 2880 bp corresponding to the GBP1 cDNA were subdivided into eleven exons which were interrupted by a total of 9500 bp of intron DNA. All exon/intron junctions contained consensus splice donor and acceptor sequences. Using hybrid rodent cell lines containing human chromosomes, GBP1 was mapped to human chromosome 1. In addition to GBP1, we detected and partially characterized a novel gene, GBP3, with a structure related to GBP1 and a high degree of sequence homology to both GBP1 and GBP2. Our data suggest that the GBP family of genes contains members other than the previously characterized GBP1 and GBP2.

Transcriptional induction of IFN-gamma-responsive genes is modulated by DNA surrounding the interferon stimulation response element.

The 9/27 and GBP mRNAs are both inducible by Interferon-gamma (IFN-gamma). The promoters of both genes contain an Interferon Stimulation Response Element (ISRE), but while the GBP gene is strongly induced transcriptionally by IFN-gamma the response of the 9/27 promoter is very weak. We investigated the molecular basis for this difference. The different IFN-gamma-responsiveness was found to have more than one reason. First, 9/27 promoter DNA was unable to bind the Gamma Interferon Activation Factor (GAF) with a single high affinity site. It efficiently competed for the association of the GAF with the GBP promoter but this competition was due to the presence of two low affinity sites, the ISRE and an ISRE-like sequence, suggesting that the GAS and ISRE, though both having clear preferences for specific proteins, may nevertheless share a certain degree of structural homology. Second, the 9/27 and GBP ISREs differed markedly in their affinities for regulatory proteins (ISGFs 1,2,3) and the GBP ISRE was more potent in mediating IFN-gamma-induced promoter activity in transient transfection. Third and most importantly, however, the strong difference between the IFN-gamma response of the two promoters was mainly due to the sequences surrounding the ISRE: the positive-acting GAS on one side and sequences with silencing properties 5' and 3' of the 9/27 ISRE on the other side. The data thus show mechanisms to both up- and down-regulate the activity of the ISRE.

Amino-terminal signal transducer and activator of transcription (STAT) domains regulate nuclear translocation and STAT deactivation.

The first approximately 100 amino acids of the STAT (signal transducer and activator of transcription) family of transcription factors share a high degree of sequence similarity. To determine whether they encode a functionally conserved domain, amino-terminal chimeric STATs were created. These chimeric STATs share a number of properties with wild-type Stat1, including a predominately cytoplasmic pattern of expression in unstimulated cells. Upon stimulation with ligand, the chimeric STATs rapidly become tyrosine-phosphorylated, dimerize, and are able to bind DNA. They are also able to heterodimerize with coexpressed wild-type Stat1. Yet in contrast to wild-type Stat1, the chimeric STATs exhibit a marked defect in deactivation. Moreover, the persistence of active chimeras correlates directly with an inability to translocate to the nucleus. The defects both in nuclear translocation and in deactivation are rescued by heterodimerization with coexpressed wild-type Stat1. This study indicates that STAT amino termini provide a signal that is important for nuclear translocation and, subsequently, deactivation. It also suggests that deactivation may depend on a prior nuclear localization event.

Functionally distinct isoforms of STAT5 are generated by protein processing.

The interleukin-3 family of cytokines, which play an important role in the development of myeloid lineages, transduce signals through the JAK-STAT pathway. Previous studies demonstrate that this process entails the activation of four distinct isoforms of STAT5, where two shorter isoforms are activated in a distinct population of cells. We now demonstrate that the shorter isoforms represent carboxy-terminal truncations. Moreover, these truncations are not generated by RNA processing, but by a specific proteolytic activity. Consistent with the notion that truncated STAT5 isoforms transduce distinct signals, they fail to promote the activation of several known interleukin-3 target genes. These studies suggest that the activity of a specific protease may play a critical role in defining the biological responses transduced by STAT5.

A novel interferon-alpha-regulated, DNA-binding protein participates in the regulation of the IFP53/tryptophanyl-tRNA synthetase gene.

We have investigated the transcriptional response of the IFP53/tryptophanyl-tRNA synthetase gene to interferon-alpha (IFN-alpha). A single gamma-interferon activation site (GAS) in proximity to the transcription start sites was found to mediate the response of the IFP53 gene to IFN-alpha. This DNA element bound two distinct protein factors, alpha-interferon activation factor 1 (AAF1) and AAF2, which were rapidly activated in the cytoplasm of IFN-alpha-treated HeLa cells. AAF1, like the gamma-interferon activation factor, bound to the GAS from different IFN-responsive promoters and contained the 91-kDa ISGF3 protein (p91). However, in complexes with the IFP53 or Ly6A/E GAS, p91 was the only ISGF3 protein, whereas in the case of the GBP GAS, the 48-kDa protein (p48) was also present. AAF2 was found to preferentially bind to the IFP53 GAS, but not at all to the GBP GAS, and contained no ISGF3 protein. Therefore, GAS-binding regulatory factors in the IFN-alpha response can either consist of proteins found in ISGF3 or be formed by distinct proteins that are similarly linked to IFN-alpha-induced signal transduction.

IL-10-induced factors belonging to the p91 family of proteins bind to IFN-gamma-responsive promoter elements.

Expression of the gene encoding the high affinity IgG receptor (Fc gamma RI) is stimulated by IFN-gamma through a promoter element designated gamma-IFN activation site (GAS). This sequence binds a transcription factor designated gamma-IFN activation factor (GAF). GAF-GAS complexes contain an IFN-regulated 91-kDa protein (p91). In mouse peritoneal macrophages, IL-4 and IL-10 influenced both basal and IFN-gamma-induced expression of Fc gamma RI in opposite ways: IL-10 was stimulatory and IL-4 repressed Fc gamma RI expression. IL-4 or IL-10 did not affect the activation of GAF by IFN-gamma, but both activated the binding of latent, receptor-activated factors (RAFTs) to the Fc gamma RI GAS. RAFTs-IL-4 and -IL-10 migrated similarly in electrophoretic mobility shift assays but could be distinguished through their specificities for different GAS sequences and their reactivity with anti-p91 antisera. These experiments also revealed two distinct RAFTs-IL-10 to be members of the p91 family of proteins. The data suggest GAS-related elements to integrate signals from IFN-gamma-, IL-4- and IL-10-activated signaling paths.

The gene encoding IFP 53/tryptophanyl-tRNA synthetase is regulated by the gamma-interferon activation factor.

We have obtained genomic DNA encoding the interferon-gamma (IFN-gamma)-inducible IFP 53/tryptophanyl-tRNA synthetase. Comparison with several different IFP 53 cDNA clones revealed a complex pattern of alternatively spliced 5'-untranslated regions. The interferon-responsive region within the IFP 53 promoter was found to contain a gamma-interferon activation site (GAS) but not the interferon-stimulated response element and to bind the gamma-interferon activation factor (GAF). GAF.GAS complexes contained the IFN-regulated 91-kDa protein. Competition experiments defined the GAS boundaries and showed that GAF binding to the IFP 53 GAS could be prevented by an excess of the IFN-gamma response regions of several other IFN-gamma-inducible genes. We thus provide evidence for a central role of GAS.GAF in gene transcription mediated by IFN-gamma and suggest a consensus sequence defining more precisely the requirements for GAF binding to DNA.

Characterization of the Stat5 protease.

Immature myeloid cells have been shown to transduce signals through a carboxyl-terminally truncated isoform of Stat5. This functionally distinct signal transducer and activator of transcription isoform is generated through a unique protein-processing event. Evaluation of numerous cell lines has determined that there is a direct correlation between the expression of truncated Stat5 and protease activity. Moreover, protease activity is found only in the myeloid and not in lymphoid progenitors. To further characterize the protease small quantities have been purified to near homogeneity. Studies on this purified material indicate that the protease has an apparent molecular mass of approximately 25 kDa and is active over a wide range of pH values. The protease will also cleave both activated (i.e. tyrosine-phosphorylated) and inactivate Stat5. Although this activity is sensitive to phenylmethylsulfonyl fluoride, it is notably not sensitive to several other serine protease inhibitors. Additional studies have led to the identification of the unique site where the protease cleaves Stat5. Mutagenesis of this site renders Stat5 resistant to cleavage. Consistent with the model that Stat5 cleavage is important for early myeloid development, introduction of a "non-cleavable" isoform of Stat5 into FDC-P1 cells (a myeloid progenitor line) leads to significant phenotypic changes.