In vivo footprinting of the IRF-1 promoter: inducible occupation of a GAS element next to a persistent structural alteration of the DNA.

Abstract

GAS (gamma activated sequence) and GAS-like elements are found in a rapidly growing number of genes. Data from EMSA (electromobility shift assay) and transient transfection assays using heterologous promoter systems do not necessarily reflect transcriptional involvement and protein occupation of a binding site in vivo. This has been shown recently by in vivo footprinting of the NF-kappa B site at -40 in the interferon regulatory factor-1 (IRF-1) promoter. Here we show by in vivo footprinting using dimethylsulfate (DMS) that the GAS of the IRF-1 promoter, which also contains an overlapping putative NF-kappa B site, is occupied upon treatment with gamma-interferon (IFN gamma) but not with phorbol 12-myristate 13-acetate (PMA). Irrespective of induction, we detect a very strong DMS hypersensitivity at a guanosine just adjacent to GAS and a less persistent minor DMS hypersensitivity at a central cytosine. Our data confirm the crucial role of GAS in transcriptional activation by IFN gamma and are consistent with induced binding of p91 to GAS. In addition, our data suggest a major conformational distortion of the DNA at the GAS element of the IRF-1 promoter and that this GAS element is not involved in transcriptional activation by PMA.