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.
Ahmed
Chulbul M I
CM
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA. ahmed1@ufl.edu
Johnson
Howard M
HM
D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3 / Q000502:physiology; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism*; D017471:Receptors, Interferon / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020218:Response Elements / Q000276:immunology; D050794:STAT1 Transcription Factor / Q000502:physiology; D015533:Transcriptional Activation / Q000276:immunology; D000096743:Interferon gamma Receptor
D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma; D019913:Nuclear Localization Signals; D021122:Protein Subunits; D017471:Receptors, Interferon; D050794:STAT1 Transcription Factor; D000096743:Interferon gamma Receptor; C079868:gamma interferon activation factor