Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.[Key Words: ARE; electrophile; Keap1; Nrf2; oxidative stress] Received August 14, 1998; revised version accepted November 10, 1998.Both electrophiles and reactive oxygen species (ROS) contribute to DNA damage and consequent development of malignancy, as well as to many other diseases (Miller 1970, Sims et al. 1974Ames 1983). To counteract these insults, terrestrial organisms have developed elaborate defense mechanisms (Bannai 1984;Prestera et al. 1993a;Primiano et al. 1997), which include the coordinated induction of a battery of specific genes that encode phase II detoxifying enzymes and oxidative stressinducible proteins (Buetler et al. 1995;Hayes and Pulford 1995). An illustration of how these defense mechanisms might work has been provided in model studies of carcinogenesis. Development of cancer in animals fed with strong chemical carcinogens is inhibited by the administration of low, but tolerable, concentrations of electrophilic agents (Wattenberg 1978). This protective phenomenon is referred to as the electrophile counterattack response (Prestera et al. 1993a), and is thought to be mediated by the induction of genes encoding phase II enzymes (Primiano et al. 1997) and antioxidative stress proteins. This coordinated response is principally regulated through cis elements, called antioxidant responsive elements (AREs; Rushmore et al. 1991) or electrophile responsive elements (EpREs;Friling et al. 1990), associated with these target genes.The ARE consensus sequence shows striking similarity to a binding motif referred to as the Maf recognition element (MARE), also known as the erythroid transcription factor NF-E2 binding sequence Motohashi et al. 1997). MAREs are specifically recognized by either homodimers of Maf family members or by heterodimeric proteins composed of CNC (Cap'n'Collar) and small Maf partners (Igarashi et al. 1994;Blank and Andrews ...

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Transcription factor NF-E2 is crucial for regulating erythroid-specific gene expression. Cloning of the NF-E2 p45 protein has revealed that it contains a basic region-leucine zipper (b-zip) domain which associates with another unidentified protein (of relative molecular mass 18,000) to form functional NF-E2. We show here that products of the maf proto-oncogene family, MafF, MafG and MafK (the small Maf proteins) which possess a b-zip DNA-binding domain but lack a canonical transactivation domain, directly control the DNA-binding properties of p45 by heterodimeric association with p45. Whereas homodimers of the small Maf proteins act as negative regulators, heterodimers composed of Maf and p45 support active transcription in vivo. These results indicate that one (or all) of the small Maf proteins is the second constituent chain required for NF-E2 activity, and that negative as well as positive regulation can be achieved through an NF-E2 site, depending on the equilibrium concentrations of p45 and the Maf proteins inside erythroid cells.

S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N-methyladenosine (mA) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the mA reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these mAs were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.

The maf oncogene encodes a bZip nuclear protein which recognizes sequences related to an AP-1 site either as a homodimer or as heterodimers with Fos and Jun. We describe here a novel maf-related gene, mafG, which shows extensive homology with two other maf-related genes, mafK and mafF. These three maf-related genes encode small basic-leucine zipper proteins lacking the trans-activator domain of v-Maf. Bacterially expressed small Maf proteins bind to DNA as homodimers with a sequence recognition profile that is virtually identical to that of v-Maf. As we have previously described, the three small Maf proteins also dimerize with the large subunit of NF-E2 (p45) to form an erythroid cell-specific transcription factor, NF-E2, which has distinct DNA-binding specificity. This study shows that the small Maf proteins can also dimerize among themselves and with Fos and a newly identified p45-related molecule (Ech) but not with v-Maf or Jun. Although the small Maf proteins preferentially recognize the consensus NF-E2 sequence as heterodimers with either NF-E2 p45, Ech, or Fos, these heterodimers seemed to be different in their transactivation potentials. Coexpression of Fos and small Mafs could not activate a promoter with tandem repeats of the NF-E2 site. These results raise the possibility that tissue-specific gene expression and differentiation of erythroid cells are regulated by competition among Fos, NF-E2 p45, and Ech for small Maf proteins and for binding sites.The maf oncogene was identified by structural analysis of the genome of the AS42 avian transforming retrovirus (26,40). It encodes a nuclear basic-leucine zipper (bZip) protein which can form a homodimer through its zipper structure (23). Recently, we reported that the v-Maf homodimer specifically recognizes two relatively long palindromic DNA sequences, TGCTGACTCAGCA and TGCTGACGTCAGCA, at roughly equal efficiency (24). The middle parts of the two consensus binding sequences for Maf are identical with two well-characterized binding sequences of the AP-1 transcription factor, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE; TGACTCA) and the cyclic AMP-responsive element (CRE; TGACGTCA), respectively. We therefore named the two types of recognition elements TRE-type Maf recognition elements (MAREs) and CRE-type MAREs. It was also recently revealed that Maf forms heterodimers with the two major components of AP-1, Fos and Jun (24, 27, 28). These heterodimers preferably bind to asymmetric DNA sequences consisting of the two consensus binding sequences of Maf homodimer and AP-1 (24). Thus, Maf and the two AP-1 components are suggested to interact with each other in a cooperative or inhibitory way in association with their recognition sequences by forming heterodimers of altered binding specificities.Like many other proto-oncogenes, the c-maf gene is a member of a gene family. To date, four maf-related genes, mafK, mafF, mafB, and nrl, have been reported (14,22,53). Their gene products are closely related to v-Maf especially in the structur...