GENES: STRUCTURE AND REGULATION
Identification of Activating Transcription Factor 4 (ATF4) as an Nrf2-interacting Protein: IMPLICATION FOR HEME OXYGENASE-1 GENE REGULATION*

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Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE). Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist. By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. Furthermore, Nrf2·ATF4 dimers bound to an StRE sequence from the ho-1 gene. CdCl2, a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 minversus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl2-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells. A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl2. These results indicate that ATF4 regulates basal and CdCl2-induced expression of theho-1 gene in a cell-specific manner and possibly in a complex with Nrf2.

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Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101198200

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This work was supported by United States Public Health Service Grants DK-43135, HL-55330, HL-60234, and AI-42365.

Both authors contributed equally to this work and should be considered as first authors.

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Both laboratories contributed equally to this work.