P-STAT1 mediates higher-order chromatin remodelling of the human MHC in response to IFNgamma.

Transcriptional activation of the major histocompatibility complex (MHC) by IFNgamma is a key step in cell-mediated immunity. At an early stage of IFNgamma induction, chromatin carrying the entire MHC locus loops out from the chromosome 6 territory. We show here that JAK/STAT signalling triggers this higher-order chromatin remodelling and the entire MHC locus becomes decondensed prior to transcriptional activation of the classical HLA class II genes. A single point mutation of STAT1 that prevents phosphorylation is sufficient to abolish chromatin remodelling, thus establishing a direct link between the JAK/STAT signalling pathway and human chromatin architecture. The onset of chromatin remodelling corresponds with the binding of activated STAT1 and the chromatin remodelling enzyme BRG1 at specific sites within the MHC, and is followed by RNA-polymerase recruitment and histone hyperacetylation. We propose that the higher-order chromatin remodelling of the MHC locus is an essential step to generate a transcriptionally permissive chromatin environment for subsequent activation of classical HLA genes.

Signaling through a mutant IFN-gamma receptor.

Activation of STAT1 and the IFN-gamma response are thought to be mediated exclusively through the Y440 motif of the human IFNGR1 receptor subunit. Contrary to this accepted dogma, here it is shown that IFNGR1 with a mutant (Y440F) motif, when stably expressed in IFNGR1-negative human fibroblasts at levels similar to wild type, can sustain a substantial IFN-gamma response. The mutant receptor supports selective induction of IFN-gamma-inducible genes but is notably defective in the CIITA, class II HLA, suppressor of cytokine signaling and antiviral responses. Remarkably, similar selective defects are observed in human fibrosarcoma cells expressing a mutant JAK1. The phenotypes are novel and appear distinct from those observed in response to the inhibition of known additional pathways. Data from different cell types further emphasizes the importance of cellular background in determining the response.

Role of tyrosine 441 of interferon-gamma receptor subunit 1 in SOCS-1-mediated attenuation of STAT1 activation.

Suppressor of cytokine signaling (SOCS)-1, the key negative regulator of interferon (IFN)-gamma-dependent signaling, is induced in response to IFNgamma. SOCS-1 binds to and inhibits the IFNgamma receptor-associated kinase Janus-activated kinase (JAK) 2 and inhibits its function in vitro, but the mechanism by which SOCS-1 inhibits IFNgamma-dependent signaling in vivo is not clear. Upon stimulation, mouse IFNgamma receptor subunit 1 (IFNGR1) is phosphorylated on several cytoplasmic tyrosine residues, and Tyr(419) is required for signal transducer and activator of transcription (STAT) 1 activation in mouse embryo fibroblasts. However, the functions of the other three cytoplasmic tyrosine residues are not known. Here we show that Tyr(441) is required to attenuate STAT1 activation in response to IFNgamma. Several tyrosine to phenylalanine mutants of IFNGR1, expressed at normal levels in stable pools of IFNGR1-null cells, were analyzed for the phosphorylation of STAT1 during a 48-h period, and antiviral activity in response to IFNgamma was also measured. Stronger activation of STAT1 was observed in cells expressing all IFNGR1 variants mutated at Tyr(441), and, consistently, stronger antiviral activity was also observed in these cells. Furthermore, constitutive overexpression of SOCS-1 inhibited IFNgamma-dependent signaling only in cells expressing IFNGR1 variants that included the Tyr(441) mutation. Mutation of Tyr(441) also blocked the ability of SOCS-1 to bind to IFNGR1 and JAK2 in response to IFNgamma and the normal down-regulation of STAT1 activation and antiviral activity. These results, together with data from the literature, suggest a model in which, in response to IFNgamma, phosphorylation of Tyr(441) creates a docking site for SOCS-1, which then binds to JAK2 within the receptor-JAK complex to partially inhibit JAK2 phosphorylation. Furthermore, the virtually complete blockade of STAT1 phosphorylation by overexpressed SOCS-1 in this experiment suggests that the binding of SOCS-1 to Tyr(441) also blocks the access of STAT1 to Tyr(419) and that this effect may be the principal mechanism of inhibition of downstream signaling.

The JAK/STAT pathway is not sufficient to sustain the antiproliferative response in an interferon-resistant human melanoma cell line.

The mechanism of resistance of malignant melanoma to treatment with interferon-alpha is unknown, and currently there is no reliable method of predicting response. Signalling via the JAK/STAT pathway is known to mediate many interferon-regulated events and has been implicated in mediating the antiproliferative response. The objective of this study was to determine whether defects in JAK/STAT signalling may be responsible for interferon resistance. The in vitro response to interferon was determined in a panel of established melanoma cell lines, and the components and functioning of the JAK/STAT pathway were examined in sensitive and resistant cell lines. Two melanoma cell lines, characterized as sensitive (MM418) and resistant (MeWo) to the antiproliferative effect of interferon, were both shown by Western blotting to possess all the protein components of the JAK/STAT pathway, and were shown to be capable of producing functional transcription factors using an electrophoretic mobility shift assay and a ribonuclease protection assay of known interferon-induced genes. In addition, both cell lines had intact antiviral and HLA upregulation responses. These data suggest that there is no defect in the JAK/STAT pathway per se in the MeWo cell line, and that the substantial resistance to interferon must be mediated through components either downstream or additional to this signalling pathway. Others have shown JAK/STAT defects to be responsible for interferon resistance in some melanoma cell lines. However, our results highlight the likely heterogeneity in the mechanisms leading to interferon resistance both in cell lines and tumours, and suggest that a clinical assay based on analysis of components of the JAK/STAT pathway may have only limited use as a predictor of interferon response.

The antiviral response to gamma interferon.

A role for alpha/beta interferon (IFN-alpha/beta) in the IFN-gamma antiviral response has long been suggested. Accordingly, possible roles for autocrine or double-stranded-RNA (dsRNA)-induced IFN-alpha/beta in the IFN-gamma response were investigated. Use was made of wild-type and a variety of mutant human fibrosarcoma cell lines, including mutant U5A cells, which lack a functional IFN-alpha/beta receptor and hence an IFN-alpha/beta response. IFN-gamma did not induce detectable levels of IFN-alpha/beta in any of the cell lines, nor was the IFN-gamma response per se dependent on autocrine IFN-alpha/beta. On the other hand, a number of responses to dsRNA [poly(I). poly(C)] and encephalomyocarditis virus were greatly enhanced by IFN-gamma pretreatment (priming) of wild-type cells or of mutant cells lacking an IFN-alpha/beta response; these include the primary induction of dsRNA-inducible mRNAs, including IFN-beta mRNA, and, to a lesser extent, the dsRNA-mediated activation of the p38 mitogen-activated protein (MAP) kinase(s). IFN-gamma priming of mRNA induction by dsRNA is dependent on JAK1 and shows biphasic kinetics, with an initial rapid (<30-min) response being followed by a more substantial effect on overnight incubation. The IFN-gamma-primed dsRNA responses appear to be subject to modulation through the p38, phosphatidylinositol 3-kinase, and ERK1/ERK2 MAP kinase pathways. It can be concluded that despite efficient priming of IFN-beta production, the IFN-alpha/beta pathways play no significant role in the primary IFN-gamma antiviral response in these cell-virus systems. The observed IFN-gamma priming of dsRNA responses, on the other hand, will likely play a significant role in combating virus infection in vivo.