Fig. 7. A recently established line of murine fibroblasts expressing a fusion protein between IRF1 and the ligand-binding domain of the human estrogen receptor (IRF1-hER) was used (1, 2). In this cell line, IRF1 is constitutively expressed, but its transcription factor activity is strictly controlled by estrogen. This allowed us to determine whether IRF1 binding to the gbp2 promoter requires tyrosine-phosphorylated STAT1 dimers. (A) IRF1-hER cells and wild type fibroblasts were left untreated or treated with 1 mM b-estradiol, 10 ng/ml (240 units/ml) IFN-g or both for 2 h. Chromatin was isolated and the immunoprecipitation was performed with STAT1C-terminal and anti-IRF1 antibodies. DNA recovered from the immunoprecipitates was analyzed by PCR for proximal and distal gbp2 promoter fragments as indicated. Left shows that STAT1 binding to gbp2 promoter chromatin occurred after IFN-g treatment as expected, but not upon estrogen treatment. Therefore, this experimental system was suitable to study effects of IRF1 activity on gbp2 expression in absence of STAT1 activity. ChIP with antibodies to IRF1 showed that in a significant fraction of the analyzed cells the gbp2 promoter was occupied with the IRF1-hER fusion protein in absence of exogenous stimuli. Estrogen enhanced binding »2-fold, as determined by scanning densitometry of the image, and a weak synergism with IFN-g was noted (»3-fold the amount produced by IFN-g alone). The specificity of the IRF1 ChIP was confirmed by precipitating DNA from WT fibroblasts, with significant amounts of gbp2 promoter DNA amplified only after IFN-g treatment. Together with the results obtained in cells expressing STAT1S727A, the result in A establishes that IRF1 associates with the gbp2 promoter in absence of detectable nuclear STAT1 dimers, and their recruitment of histone acetyl transferase activity. (B) To monitor gbp2 mRNA expression, the cells were stimulated with 1 mM b-estradiol, 10 ng/ml (240 units/ml) IFN-g or both for the indicated periods, and total RNA was extracted and reverse-transcribed. Inducibility of gbp2 mRNA was detected by real-time PCR and normalized to endogenous GAPDH expression. gbp2 expression in these cells showed a low but reproducible induction of its mRNA by estrogen alone. IFN-g produced much higher levels of gbp2 expression. As seen with IRF1-hER binding, simultaneous treatment with IFN-g and estrogen produced a somewhat more than additive effect over the individual treatments.

1. Kirchhoff S, Schaper F, Hauser H (1993) Nucleic Acids Res 21:2881-2889.

2. Kroger A, Dallugge A, Kirchhoff S, Hauser H (2003) Oncogene 22:1045-1056.