IFN-gamma and its receptor subunit IFNGR1 are recruited to the IFN-gamma-activated sequence element at the promoter site of IFN-gamma-activated genes: evidence of transactivational activity in IFNGR1.

We have shown previously that IFN-gamma and one of its receptor subunits, IFNGR1, are translocated to the nucleus, together with STAT1alpha as one macromolecular complex, via the classical importin-dependent pathway. In this study, we have identified the nuclear targets of IFN-gamma and IFNGR1. By chromatin immunoprecipitation followed by PCR, IFN-gamma, its receptor subunit IFNGR1, and STAT1alpha were found to be associated with the IFN-gamma-activated sequence (GAS) in the promoter of two of the genes stimulated by IFN-gamma. Immunoprecipitated chromatin also showed the association of the IFN-gamma, IFNGR1, and STAT1alpha on the same DNA sequence. Examination of nuclear extracts from WISH cells treated with IFN-gamma revealed the specific binding of IFN-gamma, IFNGR1, and STAT1alpha to biotinylated GAS nucleotide sequence. Association of IFN-gamma, IFNGR1, and STAT1alpha with the GAS promoter was also demonstrated by EMSA. Transfection with a GAS-luciferase gene together with the IFNGR1 and nonsecreted IFN-gamma resulted in enhanced reporter activity. In addition, IFNGR1 fused to the yeast GAL4 DNA binding domain resulted in enhanced transcription from a GAL4 response element, suggesting the presence of a trans activation domain in IFNGR1. Our observations put IFN-gamma and its receptor subunit, IFNGR1, in direct contact with the promoter region of IFN-gamma-activated genes with associated increased activity, thus suggesting a transcriptional/cotranscriptional role for IFN-gamma/IFNGR1 as well as a possible role in determining the specificity of IFN-gamma action.