Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation.
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. CdCl(2), 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 min versus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl(2)-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 CdCl(2). These results indicate that ATF4 regulates basal and CdCl(2)-induced expression of the ho-1 gene in a cell-specific manner and possibly in a complex with Nrf2.
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Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Gong
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Hu
B
B
Stewart
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Choi
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ME
Choi
A M
AM
Alam
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J
D051700:Activating Transcription Factor 3; D051701:Activating Transcription Factor 4; D000595:Amino Acid Sequence; D000818:Animals; D001940:Breast; D019256:Cadmium Chloride / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004847:Epithelial Cells; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D006801:Humans; D016350:Leucine Zippers; D008114:Liver Neoplasms, Experimental; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
C497109:ATF4 protein, human; D051700:Activating Transcription Factor 3; C497108:Atf3 protein, rat; C497110:Atf4 protein, rat; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; C495637:Nfe2l2 protein, rat; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051701:Activating Transcription Factor 4; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; D019256:Cadmium Chloride