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 ...

Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-Ela) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-Elb) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-Elc) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-Elc may play a unique regulatory role in the developing chicken brain and in T lymphocytes.

MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic ␤ cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.Insulin is the only polypeptide hormone that is essential for the regulation of blood glucose levels and is synthesized exclusively in ␤ cells of the islets of Langerhans in the pancreas. The molecular mechanisms that control ␤-cell-specific insulin gene transcription are well characterized. Three conserved cis-regulatory elements within the promoter, E1, A3, and RIPE3b/ C1, respectively, appear to be indispensable for proper insulin gene regulation (22,25). Islet-restricted transcription factors Beta2/NeuroD and Pdx1 bind to the E1 and A3 elements in vitro. Gene disruption experiments in mice have revealed that both Beta2 and Pdx1 play critical roles in insulin gene regulation as well as in islet development and function (1,8,21). Furthermore, mutations in both the Beta2 and Pdx1 genes have been identified within populations of patients with type II diabetes (18,29,30).The third regulatory element, RIPE3b/C1, has also been shown to play a critical role in ␤-cell-specific insulin gene transcription as well as in glucose-regulated expression. Previous studies identified a pancreatic ␤-cell-restricted factor, called the RIPE3b1 activator, that is enriched in response to glucose in pancreatic ␤-cell nuclear extracts. Very recently, four groups reported that the RIPE3b1 activator is a member of the Maf family of transcription factors, MafA (10,12,20,26). The large Maf proteins, MafA/L-Maf/SMaf1 (2, 9, 24), MafB (11), c-Maf (23), and Nrl (31), each contain a basic motif followed by a leucine zipper, and all four family members harbor acidic domains that act as transcriptional activation domains. Although a role for MafA in insulin gene regulation was hypothesized, in vivo tests of the hypothesis have not been reported. To elucidate MafA function in insulin gene regulation, we generated MafA-deficient mice. MATERIALS AND METHODSTargeted disruption of the mafA gene. mafA genomic clones were isolated from a 129/SvJ genomic library (Stratagene) using a partial mouse MafA cDNA as a probe. The targeting vector was constructed with the bacterial lacZ gene containing a nuclear loca...

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.Many transcription factors have been found to be members of highly related multifactor families, and thus their specificity of action must be addressed in order to ascertain their respective functions. Consequently, it is critical to determine which factor, from an array of factors with closely related DNA-binding motifs, acts upon a particular recognition site from a variety of sites with similar sequences. Equally important is the elucidation of the means by which this discrimination is achieved; a variety of mechanisms by which related transcription factors are targeted to distinct regulatory elements have been discovered. Differences in DNA-binding properties direct the zinc finger estrogen and progesterone receptors to their appropriate targets (7,44), as is the case with the Antennapedia-related homeodomain proteins Ultrabithorax and Deforned (8,9). The related retinoic acid, thyroid hormone, and vitamin D receptors exemplify a unique solution to the problem of differential target recognition by discriminating between the spacing and orientation of closely related binding sites of these obligate dimers (27,45

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The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.