Nucleic Acids Research, 1994, Vol. 22, No. 15 3033-3037
© 1994
MOLECULAR BIOLOGY |
In vivo footprinting of the IRF-1 promoter: inducible occupation of a GAS element next to a persistent structural alteration of the DNA
Institut für Biochemie, Am Klopferspitz 18a D-82152 Martinsried, Germany 1Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien Linke Bahngasse 11, A-1030 Vienna, Austria
*To whom correspondence should be addressed
Received May 10, 1994. Revised June 26, 1994. Accepted June 26, 1994.
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-xB site at - 4 0 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-xB site, is occupied upon treatment with 7-interferon (IFN7) 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 IFN7 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.
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