Identification of key elements that are responsible for heme-mediated induction of the avian heme oxygenase-1 gene

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is highly inducible by a large number of physical and chemical factors. In recent work, we had identified a metalloporphyin-responsive element (MPRE) that localized at - 3.7 kb upstream of the transcription start site of the chick HO-I gene. Here, we identify four additional heme-responsive elements (HeREs), which are "expanded" AP-1 sites, in the 5'-flanking region of the chick HO-I gene. These sites, located at - 4675, - 4599, - 3660, and - 3625 bp from the transcription start site of the gene, were necessary and sufficient for up-regulation of luciferase reporter gene expression in the presence of heme and cobalt protoporphyrin (CoPP), but not several other metalloporphyrins. Site-directed mutagenesis was carried out using pcHO7.1Luc or pcHO7.1-4.6Luc as templates. Single and multiple mutations of HeREs and MPRE significantly abrogated the heme- and CoPP-dependent up-regulation of reporter gene expression in transient or stable transfection experiments. Conclusions: The chick HO-I promoter region contains "expanded" AP-1 sites that are important for upregulation of the gene by heme and CoPP, but not other metalloporphyrins. These key regulatory elements consist of consensus AP-1 binding sites that have been extended by three base pairs. (C) 2004 Elsevier B.V. All rights reserved.