| dc.description.abstracteng | Toxoplasma gondii is an obligate intracellular parasite which infects up to 30% of humans world-wide. To control T. gondii, the host mounts a strong IFN-γ-driven immune response via the signal transducer and activator of transcription (STAT)-1 pathway. The parasite, on the other side, largely silences STAT1-mediated gene transcription and thereby counteracts multiple host resistance functions. The mechanism of this immune evasion strategy is only incompletely understood. In this study, the native chromatin within the host cell was shown to play a decisive role in the T. gondii-mediated inhibition of IFN-γ-regulated gene transcription. Luciferase reporter assays revealed that the parasite inhibits the cytokine-induced transcription of reporter constructs which were stably transfected into the host’s genome but does not significantly diminish transcription of constructs which are transcribed from transiently transfected plasmids. In addition, T. gondii abrogates the IFN-γ-induced transcriptional activity of both, the CIITA promoter IV and a minimal GAS-containing promoter in a chromatin-dependent manner. This suggests a common molecular mechanism of the parasite to inhibit both, primary and secondary IFN-γ response genes. Consistently, the parasite profoundly impairs the epigenetic landscape around several IFN-γ-responsive promoters, as determined by chromatin immunoprecipitation assays. Kinetical analyses revealed that T. gondii abolishes IFN-γ-triggered enrichment of multiple histone modifications which were here identified to be indicative for active IFN-γ-stimulated transcription, i.e. H4ac, H3K9ac and H3K4me3. In contrast, H3S10p and under distinct conditions also some of the other chromatin marks are also induced by the parasite. The data nevertheless suggests that T. gondii specifically inhibits IFN-γ-dependent epigenetic regulation in infected macrophages. Consistently, in silico analyses revealed a correlation between a strong repression of IFN-γ-induced transcription by T. gondii and the absence of CpG islands. Thus, the majority of genes whose expression is heavily counteracted by T. gondii appear to have a more closed promoter state under resting conditions. DNA binding studies by electrophoretic mobility shift assays (EMSA) showed that T. gondii diminishes the formation of IFN-γ-induced canonical STAT1-DNA binding complexes and instead induces modified complexes with lower electrophoretic mobility. The data suggests that the parasite effector T. gondii inhibitor of STAT1-dependent transcription (TgIST) previously identified to bind to STAT1 dimers, i.e. the gamma interferon-activated factor (GAF), also targets and even preferentially binds to the transcriptionally more relevant 2xGAF complex, thereby inducing a non-canonical parasite-modified 2xGAF complex. 2xGAF complexes are stabilised by reciprocal N-terminal STAT1 interactions and the cooperative DNA binding is critical for efficient IFN-γ-induced gene transcription. Interestingly, TgIST binding to STAT1 is largely abolished when cooperative DNA binding is prevented by a phenylalanine to alanine substitution in the N-terminal domain of STAT1. However, analysis of the mRNA level of several IFN-γ-responsive genes indicated that the T. gondii-induced transcriptional inhibition does not solely depend on STAT1 cooperativity. Binding of TgIST
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requires the amino-terminus, but not the C-terminal domain of STAT1, as determined by cell free in vitro co-incubation studies of extracts from infected cells and deletion mutants of STAT1. Remarkably, binding of TgIST to STAT1 profoundly modulates the DNA binding activity of the transcription factor. Oligonucleotide precipitation as well as EMSA competition experiments showed that the parasite-induced non-canonical 2xGAF complex exhibits strongly increased DNA binding activity towards DNA with high affinity GAS motifs. Furthermore, binding of TgIST also facilitates strong promiscuous STAT1 binding to imperfect nonGAS sequences as determined by EMSA and pulldown assays. This suggests that T. gondii infection dramatically diminishes the demands of the transcription factor for sequence specificity of its DNA recognition site. Although the pool of total STAT1 is reduced in parasite-infected cells after IFN-γ stimulation as compared to non-infected cells, Western blot analyses and immunofluorescence microscopy revealed that T. gondii increases and prolongs the nuclear accumulation of the transcription factor. Nuclear STAT1 from parasite-infected cells appears to be fully activated as revealed by the phosphorylation of the signature residues Tyr701 and Ser727. Moreover, nuclear STAT1 from T. gondii-infected cells is to a significant extent strongly bound to DNA as determined by protein extraction combined with an additional DNase treatment or cell extraction with harsh detergent containing lysis buffer and subsequent Western blotting. Disruption of IFN-γ signal transduction with the kinase inhibitor staurosporine further unravelled that STAT1 remains significantly longer in the nuclei of infected cells, suggesting that the nuclear export is decelerated. In conclusion, T. gondii interferes with the epigenetic regulation of promoter activity in response to IFN-γ. In addition, parasite infection impairs the IFN-γ signal transduction by altering the binding activities of STAT1 to DNA and consequently reducing the pool of activation-competent STAT1. These mechanisms may contribute to the T. gondii-mediated inhibition of IFN-γ responsiveness, thus facilitating intracellular survival and the establishment of persistent infection. | de |