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).

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1364 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1364-1373 ◽

Cited By ~ 37

Author(s):

A Eilers ◽

M Baccarini ◽

F Horn ◽

R A Hipskind ◽

C Schindler ◽

...

Keyword(s):

Growth Factor ◽

Dna Binding ◽

Binding Protein ◽

Trna Synthetase ◽

Gamma Interferon ◽

U937 Cells ◽

Gamma Activation ◽

Ifn Gamma ◽

Differentiation Factors ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1364-1373.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1364-1373

Author(s):

A Eilers ◽

M Baccarini ◽

F Horn ◽

R A Hipskind ◽

C Schindler ◽

...

Keyword(s):

Growth Factor ◽

Dna Binding ◽

Binding Protein ◽

Trna Synthetase ◽

Gamma Interferon ◽

U937 Cells ◽

Gamma Activation ◽

Ifn Gamma ◽

Differentiation Factors ◽

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.

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In vitro activation of the transcription factor gamma interferon activation factor by gamma interferon: evidence for a tyrosine phosphatase/kinase signaling cascade

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1634-1640.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1634-1640

Author(s):

K Igarashi ◽

M David ◽

D S Finbloom ◽

A C Larner

Keyword(s):

Tyrosine Phosphatase ◽

Gamma Interferon ◽

Cell Surface Receptor ◽

Signaling Cascade ◽

Kinase Signaling ◽

Gamma Activation ◽

Ifn Gamma ◽

Genistein Treatment ◽

Activation Factor

Although it has been well documented that the biological activities of gamma interferon (IFN-gamma) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-gamma signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-gamma activates the formation of the IFN-gamma activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-gamma. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-gamma. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-gamma was added. The addition of either agent 5 min after IFN-gamma had no effect. These results provide the first evidence for an IFN-gamma-regulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.

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In vitro activation of the transcription factor gamma interferon activation factor by gamma interferon: evidence for a tyrosine phosphatase/kinase signaling cascade.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1634 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1634-1640 ◽

Cited By ~ 33

Author(s):

K Igarashi ◽

M David ◽

D S Finbloom ◽

A C Larner

Keyword(s):

Tyrosine Phosphatase ◽

Gamma Interferon ◽

Cell Surface Receptor ◽

Signaling Cascade ◽

Kinase Signaling ◽

Gamma Activation ◽

Ifn Gamma ◽

Genistein Treatment ◽

Activation Factor

Although it has been well documented that the biological activities of gamma interferon (IFN-gamma) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-gamma signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-gamma activates the formation of the IFN-gamma activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-gamma. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-gamma. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-gamma was added. The addition of either agent 5 min after IFN-gamma had no effect. These results provide the first evidence for an IFN-gamma-regulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3579 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3579-3586 ◽

Cited By ~ 61

Author(s):

A Eilers ◽

D Georgellis ◽

B Klose ◽

C Schindler ◽

A Ziemiecki ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Serine Phosphorylation ◽

Gamma Activation ◽

Mobility Shift ◽

Two Dimensional Gel Electrophoresis ◽

Ifn Gamma ◽

Activation Site

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.182 ◽

1991 ◽

Vol 11 (1) ◽

pp. 182-191 ◽

Cited By ~ 177

Author(s):

D J Lew ◽

T Decker ◽

I Strehlow ◽

J E Darnell

Keyword(s):

Binding Protein ◽

Gene Promoter ◽

Maximal Response ◽

Type I ◽

Gene Encoding ◽

Response Element ◽

Ifn Gamma ◽

Ifn Alpha ◽

Transcriptional Induction ◽

Guanylate Binding Protein

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.182-191.1991 ◽

1991 ◽

Vol 11 (1) ◽

pp. 182-191

Author(s):

D J Lew ◽

T Decker ◽

I Strehlow ◽

J E Darnell

Keyword(s):

Binding Protein ◽

Gene Promoter ◽

Maximal Response ◽

Type I ◽

Gene Encoding ◽

Response Element ◽

Ifn Gamma ◽

Ifn Alpha ◽

Transcriptional Induction ◽

Guanylate Binding Protein

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.

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