Chris Schindler

Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways.

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon‐stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN‐alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C‐terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild‐type cells completely restored the response to both IFN‐alpha and ‐gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN‐alpha response of all genes tested so far. It failed, however, to restore the IFN‐gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN‐alpha and ‐gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.

STF-IL-4: a novel IL-4-induced signal transducing factor.

The mechanism by which interleukin‐4 (IL‐4) regulates the expression of particular genes is unknown. We have determined that IL‐4 induces a DNA binding factor (termed STF‐IL‐4) which has a strong affinity for an IFN‐gamma activation site (GAS). Interestingly, STF‐IL‐4 also binds to the IL‐4 responsive promoter for the Ig heavy chain germline epsilon transcript. The IL‐4 dependent activation of STF‐IL‐4 is rapid, does not require protein synthesis and results in the sequential appearance of binding activity first in the cytoplasm and then later in the nucleus. Activation of STF‐IL‐4 is sensitive to tyrosine kinase inhibitors and the active factor is tyrosine phosphorylated. This pattern of activation is similar to the activation of interferon‐induced transcription factors. STF‐IL‐4 appears to be a new member of a growing family of cytokine‐induced transcriptional regulators.

The genomic structure of the murine ICSBP gene reveals the presence of the gamma interferon-responsive element, to which an ISGF3 alpha subunit (or similar) molecule binds.

ICSBP, a member of the interferon regulatory factor family, is expressed predominantly in lymphoid tissues and is induced by gamma interferon (IFN-gamma). We have studied the genomic organization of the murine ICSBP gene and its 5 upstream region. The murine ICSBP gene (Icsbp) is present as a single copy on chromosome 8 and consists of nine exons. Transcription initiates at two juxtaposed sites downstream from the TATA and CAAT boxes and produces two species of ICSBP mRNA (3.0 and 1.7 kb), presumably by differential usage of poly(A)+ signals. A sequence from -175 to -155 was identified to be an IFN response region that conferred IFN-gamma induction upon a heterologous promoter in lymphoid cell line EL4. This region includes a motif, TTCNNGGAA, designated the palindromic IFN response element (pIRE), to which an IFN-gamma-inducible, cycloheximide-sensitive factor(s) binds. A similar palindromic motif was found in the upstream region of the murine IRF-1 gene, the IFN-gamma activation site of the guanylate-binding protein gene and the IFN-gamma-responsive region of the Fc receptor type I gene, all of which competed with the pIRE for factor binding in gel mobility shift assays. We show that the pIRE binding factor reacts with the antibody against the 91-kDa subunit of ISGF3 alpha recently shown to bind to the IFN-gamma activation site. These results suggest that this factor is related to the IFN-gamma activation factor and contains the 91-kDa subunit of ISGF3 alpha. Taken together, pIRE represents an IRE that is distinct from the classical IFN-stimulated response element and that is capable of conferring IFN-gamma induction through the binding of the 91-kDa ISGF3 alpha subunit (or an antigenically similar molecule).

Early events in signalling by interferons

The heterogeneity of the biological responses to alpha/beta and gamma interferons, the degree of overlap, synergy and antagonism appear to reflect functional interactions between these two signalling pathways. Recent biochemical and genetic experimental approaches have identified some of the effector proteins involved in interferon signalling, and have advanced our understanding of the crosstalk between these signalling networks.

Regulation of IFN‐α/β genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN‐α/β

Background: Efficient production of interferons (IFNs) in virally infected cells is an essential aspect of the host defence. The transcription factor complex ISGF3 (IFN‐stimulated gene factor 3) was originally identified as a critical mediator of the IFN signal; it is formed upon IFN receptor (IFNR) stimulation and binds to ISREs (IFN‐stimulated response elements) to activate IFN‐inducible genes. It has recently been shown that the DNA binding component of ISGF3, p48 (ISGF3γ) also binds to virus‐inducible elements in the IFN‐α/β genes, suggesting a potential new role of p48 in IFN production.

Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages.

Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.

Interleukin-6-induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin-6 target gene induction

The cytokine interleukin‐6 (IL‐6) rapidly activates a latent cytoplasmic transcription factor, acute‐phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immediate‐early gene induction by IL‐6 and, as we show here, stimulation of the promoters of the genes for α 2‐macroglobulin, Jun‐B, and intercellular adhesion molecule‐1 (ICAM‐1) are blocked by the serine/threonine kinase inhibitor H7. We now show that IL‐6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL‐6 signal transduction leading to target gene induction.

Cytokines and Growth factors signal through tyrosine phosphorylation of a family of related transcription factors

The ability of cytokines to activate distinct but overlapping sets of genes defines their characteristic biological response. We now show that IFN gamma, IL-3, IL-4, IL-6, erythropoietin, EGF, and CSF-1 activate differing members of a family of latent cytoplasmic transcription factors. Although these factors have distinct physical and functional properties and exhibit different patterns of expression, they share many important features, including recognition of a related set of enhancer elements, rapid activation, tyrosine phosphorylation, and cross-reactivity to antibodies against p91, a cytoplasmic signaling protein activated by IFN alpha, IFN gamma, and IL-6. These shared features point to either parallel or common patterns of signal transduction. A general model of cytokine signal transduction is presented, in which receptor-associated tyrosine kinases activate ligand-specific members of a family of signal-transducing factors. Once activated, these factors carry their signals to the nucleus, where they bind a family of related enhancer elements.

A factor induced by differentiation signals in cells of the macrophage lineage binds to the gamma interferon activation site.

Rapid transcriptional induction of genes in response to gamma interferon (IFN-gamma) is mediated by the IFN-gamma activation site (GAS) and its cognate protein, the IFN-gamma activation factor (GAF). We describe a GAS-associated, differentiation-induced factor (DIF) as a potential molecular link between the activities of IFN-gamma and of growth and differentiation factors. DIF DNA binding was activated by colony-stimulating factor 1 in murine macrophages and also during tetradecanoyl phorbol acetate-induced differentiation or IFN-gamma treatment in myeloid U937 cells. IFN-gamma activation of DIF decreased significantly upon monocytic differentiation. DIF binding to DNA was inhibited by antiphosphotyrosine antibodies and could be induced by treatment of U937 cells with vanadate. Unlike GAF, DIF-DNA complexes did not contain the 91-kDa protein (p91) from ISGF-3. DIF bound with high affinity to GAS from the promoters of the IFP 53/tryptophanyl-tRNA synthetase and Fc gamma RI genes, intermediate affinity to the Ly6A/E GAS, and low affinity to the guanylate-binding protein GAS. DIF may belong to a family of cytokine- or growth factor-induced factors binding with variable affinities to GAS-related elements: the interleukin-6-responsive acute-phase response factor associated with GAS from different IFN-inducible promoters but with a different preference of binding compared with DIF. The sis-inducible element of the c-fos promoter bound GAF but not DIF. However, the sis-inducible element could be changed by point mutation to compete for GAF and DIF binding. Our data show DIF to be a novel DNA-binding protein which is activated in response to differentiating signals. Moreover, they suggest that a family of cytokine- or growth factor-regulated proteins integrates and coordinates the responses to cytokines and to growth and differentiation factors by binding to GAS-related elements.