Component Of ISGF3 Research Articles

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

STAT1 as a potential prognosis marker for poor outcomes of early stage colorectal cancer with microsatellite instability

Proteomic analyses indicate that STAT1 protein (signal transducer and activator of transcription 1 or transcription factor ISGF-3 components p91/p84) is upregulated in some colorectal cancers. This study examined 736 colorectal cancer patients for the expression of STAT1 protein in tissue specimens, including 614 early stage patients and 122 advanced stage patients. Tissue microarrays were constructed, and STAT1 expression was examined by immunohistochemistry and scored semi-quantitatively. Among all cases, 9% of cases displayed high levels of cytoplasmic expression of STAT1 and 15% of cases had positive nuclear expression. Based on statistical analyses of a cohort of 559 early stage patients with survival data and no neoadjuvant therapy, we found that high levels of cytoplasmic expression of STAT1 correlated with shorter survival time in early stage colorectal cancer, particularly of the microsatellite instability (MSI) subtype. Additional analysis of a 244-case cohort of colorectal cancers from the Cancer Genome Atlas found that STAT1 gene expression correlated positively with PD-L1 (CD274) and PD-1 (PDCD1) but had no correlation with KRAS or BRAF mutation status. STAT1 expression showed no clear correlation with any of the 4 clinical diagnostic markers of mismatch repair, MLH1, MSH2, MSH6, and PMS2, suggesting its potential as an independent outcome marker for MSI cancers. Our findings suggest that STAT1 may be used as a potential prognostic protein marker for stratifying the outcome risk of early stage MSI colorectal cancer.

Fine-Tuning of Type I Interferon Response by STAT3

Type I interferon (IFN-I) is induced during innate immune response and is required for initiating antiviral activity, growth inhibition, and immunomodulation. STAT1, STAT2, and STAT3 are activated in response to IFN-I stimulation. STAT1, STAT2, and IRF9 form ISGF3 complex which transactivates downstream IFN-stimulated genes and mediates antiviral response. However, the role of STAT3 remains to be characterized. Here, we review the multiple actions of STAT3 on suppressing IFN-I responses, including blocking IFN-I signaling, downregulating the expression of ISGF3 components, and antagonizing the transcriptional activity of ISGF3. Finally, we discuss the evolution of the suppressive activity of STAT3 and the therapeutic potential of STAT3 inhibitors in host defense against viral infections and IFN-I-associated diseases.

Non-redundant ISGF3 Components Promote NK Cell Survival in an Auto-regulatory Manner during Viral Infection

SUMMARYNatural killer (NK) cells are innate lymphocytes that possess adaptive features, including antigen-specific clonal expansion and long-lived memory responses. Although previous work demonstrated that type I interferon (IFN) signaling is crucial for NK cell expansion and memory cell formation following mouse cytomegalovirus (MCMV) infection, the global transcriptional mechanisms underlying type I IFN-mediated responses remained to be determined. Here, we demonstrate that among the suite of transcripts induced in activated NK cells, IFN-α is necessary and sufficient to promote expression of its downstream transcription factors STAT1, STAT2, and IRF9, via an auto-regulatory, feedforward loop. Similar to STAT1 deficiency, we show that STAT2- or IRF9-deficient NK cells are defective in their ability to expand following MCMV infection, in part because of diminished survival rather than an inability to proliferate. Thus, our findings demonstrate that individual ISGF3 components are crucial cell-autonomous and non-redundant regulators of the NK cell response to viral infection.

Unphosphorylated ISGF3 drives constitutive expression of interferon-stimulated genes to protect against viral infections.

Interferon (IFN)-stimulated genes (ISGs) are antiviral effectors that are induced by IFNs through the formation of a tripartite transcription factor ISGF3, which is composed of IRF9 and phosphorylated forms of STAT1 and STAT2. However, we found that IFN-independent ISG expression was detectable in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. The constitutive expression of ISGs was mediated by the unphosphorylated ISGF3 (U-ISGF3) complex, consisting of IRF9 together with unphosphorylated STAT1 and STAT2. Under homeostatic conditions, STAT1, STAT2, and IRF9 were found in the nucleus. Analysis of a chromatin immunoprecipitation sequencing data set revealed that STAT1 specifically bound to the promoters of ISGs even in the absence of IFNs. Knockdown of STAT1, STAT2, or IRF9 by RNA interference led to the decreased expression of various ISGs in Huh7.5 human liver cells, which was confirmed in mouse embryonic fibroblasts (MEFs) from STAT1-/-, STAT2-/-, or IRF9-/- mice. Furthermore, decreased ISG expression was accompanied by increased replication of hepatitis C virus and hepatitis E virus. Conversely, simultaneous overexpression of all ISGF3 components, but not any single factor, induced the expression of ISGs and inhibited viral replication; however, no phosphorylated STAT1 and STAT2 were detected. A phosphorylation-deficient STAT1 mutant was comparable to the wild-type protein in mediating the IFN-independent expression of ISGs and antiviral activity, suggesting that ISGF3 works in a phosphorylation-independent manner. These data suggest that the U-ISGF3 complex is both necessary and sufficient for constitutive ISG expression and antiviral immunity under homeostatic conditions.

ID: 242 : Combinatorial association and abundance of ISGF3 components dictate selectivity and kinetics of interferon responses

IFN α induces ISG expression by phosphorylating STAT1 and STAT2. STAT1 homodimers facilitate transcriptional responses by directly activating ISGs containing GAS element. Combinatorial association of STAT1 and STAT2 with the additional DNA binding protein IRF9 (forming ISGF3) induce ISRE-containing ISGs. Evidence is accumulating for the existence of a STAT2/IRF9-dependent, STAT1-independent IFN α signaling pathway that generates ISGF3-like response. However, no detailed insight exists in the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9 dependent IFN α signaling as compared to ISGF3 and how WT- and alternative responses are dictated. In hST2-U3C IFN α -induced expression of classical ISGs correlated with the kinetics of STAT2 phosphorylation, and the presence of STAT2/IRF9 complex. Expression profiling of IFN α treated 2fTGH and hST2-U3C identified 120 known antiviral ISRE-containing ISGs commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9 directed expression profile of these ISGs was prolonged as compared to the transient response mediated by ISGF3. ChIP-seq analysis confirmed binding of STAT2 to the ISG promoters, in IFN α -dependent STAT1-independent manner. STAT2/IRF9 was able to generate a sustained antiviral response upon EMCV and VSV. Interestingly, IFN α -treated NB4 cells that display transient phosphorylation of STAT1 and prolonged phosphorylation of STAT2, revealed transient STAT1-mediated ISG expression and prolonged expression of STAT2/IRF9-dependent genes. Our results prove that IFN α -activated STAT2/IRF9 induces prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. We provide additional evidence that in the presence of STAT1, abundance and phosphorylation kinetics of ISGF3 components dictates the nature of IFN α responses.

25: STAT2 and IRF9-dependent IFN-I signaling restores ISRE-mediated transcription and anti-viral activity independent of STAT1

Type I Interferons (IFN-I) mediate cellular responses to virus infection. IFN-I induce IFN stimulated gene (ISG) expression by phosphorylating STAT1 and STAT2, and together with interferon regulatory factor (IRF)9, form the transcription complex ISGF3 that binds to the interferon-stimulated response element (ISRE) in ISG promoters. As a component of ISGF3 it is clear that STAT2 plays an essential role in the transcriptional responses to IFN-I with a strong dependence on STAT1. Previously, we showed that STAT2 also forms homodimers that interact with IRF9 (STAT2-IRF9) to activate transcription of ISRE containing ISGs in response to IFN-I. Indeed, evidence is accumulating for the existence of a STAT1-independent IFN-I signaling pathway, where STAT2-IRF9 can substitute the role of ISGF3. Here, we provide further insight in the transcriptional regulation and the biological implications of STAT2-IRF9 dependent IFN-I signaling. In human STAT1 KO cells overexpressing human STAT2 (U3C-STAT2) we observed that in response to IFN-I STAT2 homodimers interact with IRF9 to regulate ISG transcription. The IFN-I-induced phosphorylation profile of STAT2 in U3C-STAT2 was prolonged as compared to WT cells (2fTGH), which corresponded with the expression pattern of OAS2 that also depended on IRF9. Subsequent microarray analysis of IFN-I treated 2fTGH and U3C-STAT2 extended our initial observations and identified more than 60 known antiviral ISGs commonly up-regulated in both cell types. The expression profile of these ISGs was delayed and prolonged in U3C-STAT2 as opposed to the early and transient response in 2fTGH. Moreover, U3C-STAT2 were able to restore an antiviral response upon EMCV and VSV infection that was comparable to the response in 2fTGH. Together, our results strongly suggest that an alternative IFN-I-mediated, STAT2-IRF9 dependent signaling pathway exists that can generate an antiviral response without STAT1 and could be beneficial for example against viruses that directly block STAT1 and impair the formation of ISGF3.

Multiple Functions of the IKK-Related Kinase IKKε in Interferon-Mediated Antiviral Immunity

IKKepsilon is an IKK (inhibitor of nuclear factor kappaBkinase)-related kinase implicated in virus induction of interferon-beta (IFNbeta). We report that, although mice lacking IKKepsilon produce normal amounts of IFNbeta, they are hypersusceptible to viral infection because of a defect in the IFN signaling pathway. Specifically, a subset of type I IFN-stimulated genes are not activated in the absence of IKKepsilon because the interferon-stimulated gene factor 3 complex (ISGF3) does not bind to promoter elements of the affected genes. We demonstrate that IKKepsilon is activated by IFNbeta and that IKKepsilon directly phosphorylates signal transducer and activator of transcription 1 (STAT1), a component of ISGF3. We conclude that IKKepsilon plays a critical role in the IFN-inducible antiviral transcriptional response.

Interferon-resistant Human Melanoma Cells Are Deficient in ISGF3 Components, STAT1, STAT2, and p48-ISGF3γ

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation. However, analysis of ISGF3 transcription factor components, STAT1, STAT2, and p48-ISGF3gamma, revealed that their expression and activation correlated with cellular IFN responsiveness. The analysis was extended to also include IFN-sensitive primary melanocytes, three additional IFN-resistant melanoma cell lines, and seven cell cultures recently established from melanoma patient biopsies. It was consistently observed that the most marked difference in ISGF3 was a lack of STAT1 in the resistant versus the sensitive cells. Transfection of the IFN-resistant MM96 cell line to express increased levels of STAT1 protein partially restored IFN responsiveness in an antiviral assay. We conclude that a defect in the level of STAT1 and possibly all three ISGF3 components in IFN-resistant human melanoma cells may be a general phenomenon responsible for reduced cellular responsiveness of melanomas to IFNs.

PHOSPHOLIPASE C γ1 OVEREXPRESSION AND ACTIVATION INDUCED BY INTERFERON BETA IN HUMAN T LYMPHOCYTES: AN ISGF3-INDEPENDENT RESPONSE

Interferons exert their antiviral, antiproliferative and immunoregulatory activities by stimulating the expression of several genes. Such genes disclose a common element within their promoters, defined Interferon Stimulated Response Element (ISRE), which binds a nuclear factor(s) translocated from the cytoplasm to the nucleus (ISGF3) after the binding of interferon (IFN) to the specific receptor. Here we report the induction of the synthesis and of the hydrolytic activity of phospholipase C gamma 1 (PLC γ1) in human T lymphocytes by IFN-β. The increased level of PLC γ1 becomes evident after 90 min of IFN-β treatment and is still detectable after 24 h. Neither the PLC γ1 overexpression induced by IFN nor the increased hydrolytic activity of the enzyme appear to be affected by pretreatment of the cells with the protein tyrosine kinase inhibitor genistein, which is known to prevent the association of ISGF3 components. These results suggest that in human T lymphocytes IFN-β can activate other transcription factor(s) distinct from ISGF3 to regulate PLC γ1 expression. In addition, the ability of this enzyme to hydrolyse PIP2, also in the presence of genistein, implies the possibility that this enzyme can exert its hydrolytic activity independently of protein tyrosine kinase activation.

Role of protein kinase C and tyrosine kinase activity in IFN-gamma-induced expression of the class II MHC gene.

Astrocytes are induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. Our previous studies demonstrated that IFN-gamma-initiated signaling events important for class II expression include activation of protein kinase C (PKC) and the Na+/H+ antiporter. We have extended these studies and found that protein tyrosine kinase (PTK) activity is also required for class II expression. Treatment of astrocytes with inhibitors specific for PKC and PTK blocked INF-gamma-induced class II gene transcription, mRNA expression, and protein expression. Immunoblotting and immunoprecipitation experiments demonstrated that IFN-gamma induced tyrosine phosphorylation of the p91 component of ISGF3, which is blocked by preincubation of cells with PTK inhibitors. Treatment of astrocytes with IFN-gamma and either PKC and PTK inhibitors changed the mobility and intensity of a nuclear factor, IFN-gamma-enhanced factor X, which binds to the X box of the class II MHC promoter. Taken together, these data provide evidence that activation of both PTK and PKC is required for IFN-gamma-induced expression of the class II gene.

Interferon gamma-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3.

A 39-nt DNA sequence, the interferon gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF3, the IFN-alpha-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-alpha can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-alpha does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.

Modulation of interferon signaling in human fibroblasts by phorbol esters.

Phorbol esters activate the expression of a variety of early-response genes through protein kinase C-dependent pathways. In addition, phorbol esters may promote cell growth by the inhibition of expression of cellular gene products regulated by antiproliferative agents such as interferons (IFN)s. In human diploid fibroblasts, phorbol 12-myristate 13-acetate (PMA) selectively inhibits the IFN-alpha-induced cellular gene ISG54. Using transient transfection assays, we have delineated two elements in the promoter of this gene that are necessary for the inhibitory actions of PMA. These elements include (i) the IFN-stimulated response element (ISRE) which is necessary for IFN-alpha-induced cellular gene expression, and (ii) an element located near the site of transcription initiation. IFN-alpha treatment resulted in the rapid induction of ISGF3, a multisubunit transcription factor which binds to the ISRE. PMA caused a substantial reduction in IFN alpha-induced ISGF3 in both nuclear and cytoplasmic extracts, as determined by electrophoretic mobility shift assays with the ISRE as a probe. In vitro reconstitution experiments revealed that IFN-alpha activation of the ISGF3 alpha component of ISGF3 was not affected by PMA. Further experiments were consistent with the possibility that PMA regulated the activity of a cellular factor which competed with ISGF3 gamma for binding of the activated ISGF3 alpha polypeptides. Electrophoretic mobility shift assays using the cap site of ISG54 as a probe demonstrated the formation of a specific complex whose DNA binding activity was not affected by treatment of cells with PMA or IFN-alpha. Competitive inhibition studies were consistent with the DNA-protein complex at the cap site of ISG54 containing proteins with DNA binding sites in common with those which also interact with the ISRE. These data suggest a unique regulatory mechanism by which phorbol esters can modulate IFN signaling.

Modulation of interferon signaling in human fibroblasts by phorbol esters.

Phorbol esters activate the expression of a variety of early-response genes through protein kinase C-dependent pathways. In addition, phorbol esters may promote cell growth by the inhibition of expression of cellular gene products regulated by antiproliferative agents such as interferons (IFN)s. In human diploid fibroblasts, phorbol 12-myristate 13-acetate (PMA) selectively inhibits the IFN-alpha-induced cellular gene ISG54. Using transient transfection assays, we have delineated two elements in the promoter of this gene that are necessary for the inhibitory actions of PMA. These elements include (i) the IFN-stimulated response element (ISRE) which is necessary for IFN-alpha-induced cellular gene expression, and (ii) an element located near the site of transcription initiation. IFN-alpha treatment resulted in the rapid induction of ISGF3, a multisubunit transcription factor which binds to the ISRE. PMA caused a substantial reduction in IFN alpha-induced ISGF3 in both nuclear and cytoplasmic extracts, as determined by electrophoretic mobility shift assays with the ISRE as a probe. In vitro reconstitution experiments revealed that IFN-alpha activation of the ISGF3 alpha component of ISGF3 was not affected by PMA. Further experiments were consistent with the possibility that PMA regulated the activity of a cellular factor which competed with ISGF3 gamma for binding of the activated ISGF3 alpha polypeptides. Electrophoretic mobility shift assays using the cap site of ISG54 as a probe demonstrated the formation of a specific complex whose DNA binding activity was not affected by treatment of cells with PMA or IFN-alpha. Competitive inhibition studies were consistent with the DNA-protein complex at the cap site of ISG54 containing proteins with DNA binding sites in common with those which also interact with the ISRE. These data suggest a unique regulatory mechanism by which phorbol esters can modulate IFN signaling.

Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3.

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)