Two gamma-interferon-activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM-1) gene are required for induction of transcription by IFN-gamma.
We describe the molecular features of the interferon (IFN)-gamma-mediated transcription of the human intercellular adhesion molecule (ICAM-1) gene. We identified putative IFN-gamma-activated sites (GAS) distributed throughout a large segment of the ICAM-1 promoter (4.0 kb region). Using computer-assisted search, these sequences were similar to potential IFN-gamma responsive elements that have a core sequence 5'-TTNCNNNAA-3'. In this report we show that in the ICAM-1 promoter a GAS site is located at -115 from the translation initiation site, and binds with strong affinity to IFN-gamma-activated Signal Transducers and Activators of Transcription (STAT1) homodimers. The same sequence is responsible for the IFN-gamma-mediated transcription of the ICAM-1 gene. Moreover, we present evidence that a more distal GAS element that maps at -2787 from the translation initiation site, binds IFN-gamma-activated STAT1 dimers with lower affinity. Multimeric copies of such GAS sequence inserted into a tkCAT minimal promoter can drive transcription, demonstrating that the -2787 bp GAS element has an independent functional activity upon binding of IFN-gamma-activated STAT1 proteins as documented by in vitro binding assays. Furthermore, using recombinant ICAM-CAT mutants, we show that, in vivo, the -2787 GAS, but not a mutagenized -2787 GAS site, when coupled to the more proximal -115 GAS element, has an additive effect in enhancing the IFN-gamma-mediated transcription of ICAM-1 promoter. Nevertheless, using a recombinant construct bearing the wild type -2787 GAS element and a mutagenized -115 GAS element, we could not detect any transcription after transfection of U937 recipient cells, suggesting that the -2787 bp GAS element is not sufficient as such for gene activation, but can cooperate with its cognate proximal sequence to give full function to the ICAM-1 promoter during the IFN-gamma response. Taken together these data provide evidence that two GAS sites are required for the full potential activity in the mechanism of ICAM-1 gene activation by IFN-gamma.
Tessitore
A
A
University of L'Aquila, Department of Experimental Medicine, Italy.
Pastore
L
L
Rispoli
A
A
Cilenti
L
L
Toniato
E
E
Flati
V
V
Farina
A R
AR
Frati
L
L
Gulino
A
A
Martinotti
S
S
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics* / Q000502:physiology; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D020298:U937 Cells