<i>Toxoplasma</i> infection leads to defective binding of components of chromatin-remodelling complexes to GAS-containing oligonucleotides <i>in vitro</i> and to the IFN-γ responsive CIITA promoter IV in infected cells.

<p>(A) RAW264.7 macrophages were infected with <i>T. gondii</i> for 24 hours, or were left non-infected and stimulated with IFN-γ during the final 30 min. Complete cellular lysates were used to isolate GAS-binding protein complexes by a pull-down assay. Precipitates were resolved by isoelectric focussing and SDS-PAGE, and proteins were visualized by silver staining. Protein spots that were specifically identified in the pull-downs from IFN-γ-treated, non-infected MΦ are indicated. (B) GAS-binding proteins that were pulled down from lysates of non-infected, but not <i>Toxoplasma</i>-infected MΦ after stimulation with IFN-γ were analysed by mass spectrometry. The three hits of highest probability for each of the four protein spots are shown; probability scores greater than 61 are considered significant. (C) RAW264.7 macrophages infected or not with <i>T. gondii</i> for 24 hours were stimulated with IFN-γ during the final 30 min, or were left non-stimulated. Complete cellular extracts or proteins that had been pulled-down from the extracts, by using a biotin-conjugated GAS-containing oligonucleotide and immobilized streptavidin, were subsequently analysed by immunoblotting using antibodies directed against STAT1, β-actin and BRG-1. Nc: negative control: pull-down assay without lysate. (D) Nuclear and cytosolic extracts were prepared from RAW264.7 macrophages infected with <i>T. gondii</i>, and stimulated with IFN-γ as described in (C). After separation of the extracts by SDS-PAGE, STAT1, β-actin, GAPDH and BRG-1 were detected by immunoblotting. (E) <i>Toxoplasma</i>-infected and non-infected (n.i.) RAW264.7 macrophages were stimulated with IFN-γ for 2 hours, or were left non-stimulated. After cross-linking DNA-protein complexes and shearing, chromatin was immunoprecipitated using an anti-BRG-1 antibody and protein A agarose. After DNA isolation from immunoprecipitates or from input lysates, fragments of the CIITA promoter IV (pIV) or β-actin were amplified by real-time PCR. The IFN-γ-induced binding of BRG-1 was calculated according to the ratio (E_ChIP)^{ΔCPChIP(unstimulated – IFN-γ-treated)}/(E_input)^{ΔCPinput(unstimulated – IFN-γ-treated)}. Results are means ± S.E.M. (n = 2), significant differences were identified by Student's <i>t</i>-test (*, <i>p</i><0.05).