Maf Proteins Research Articles

Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides

SummaryMaf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.

Poly(ADP-ribose) polymerase-1 modulates Nrf2-dependent transcription

The basic leucine zipper transcription factor Nrf2 has emerged as a master regulator of intracellular redox homeostasis by controlling the expression of a battery of redox-balancing antioxidants and phase II detoxification enzymes. Under oxidative stress conditions, Nrf2 is induced at the protein level through redox-sensitive modifications on critical cysteine residues in Keap1, a component of an E3 ubiquitin ligase complex that targets Nrf2 for proteasomal degradation. Poly(ADP-ribose) polymerase-1 (PARP-1) is historically known to function in DNA damage detection and repair; however, recently PARP-1 has been shown to play an important role in other biochemical activities, such as DNA methylation and imprinting, insulator activity, chromosome organization, and transcriptional regulation. The exact role of PARP-1 in transcription modulation and the underlying mechanisms remain poorly defined. In this study, we report that PARP-1 forms complexes with the antioxidant response element (ARE) within the promoter region of Nrf2 target genes and upregulates the transcriptional activity of Nrf2. Interestingly, PARP-1 neither physically interacts with Nrf2 nor promotes the expression of Nrf2. In addition, PARP-1 does not target Nrf2 for poly(ADP-ribosyl)ation. Instead, PARP-1 interacts directly with small Maf proteins and the ARE of Nrf2 target genes, which augments ARE-specific DNA-binding of Nrf2 and enhances the transcription of Nrf2 target genes. Collectively, these results suggest that PARP-1 serves as a transcriptional coactivator, upregulating the transcriptional activity of Nrf2 by enhancing the interaction among Nrf2, MafG, and the ARE.

BACH2: A marker of DNA damage and ageing

DNA damage and ageing share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. "Omics" technologies have permitted large-scale parallel measurements covering global cellular constituents and aided the identification of specific response pathways that change during ageing and after DNA damage. We have set out to identify genes with highly conserved response patterns through meta-analysis of mRNA expression datasets collected during natural ageing and accelerated ageing caused by a Transcription-Coupled Nucleotide Excision Repair (TC-NER) defect in a diverse set of organs and tissues in mice, and from in vitro UV-induced DNA damage in a variety of murine cells. The identified set of genes that show similar expression patterns in response to organ ageing (accelerated and normal), and endogenously and exogenously induced DNA damage, consists of genes involved in anti-oxidant systems and includes the transcription factor Bach2 as one of the most consistent markers. BACH2 was originally identified as a partner of the small Maf proteins and antagonist of the NRF2 anti-oxidant defence pathway and has been implicated in B-cell differentiation and immune system homeostasis. Although BACH2 has never before been associated with UV-induced damage or ageing, it shows a strong downregulation in both conditions. We have characterized the dynamics of Bach2 expression in response to DNA damage and show that it is a highly sensitive responder to transcription-blocking DNA lesions. Gene expression profiling using Affymetrix microarray analysis after siRNA-mediated silencing of Bach2 identified cell cycle and transcription regulation as the most significantly altered processes consistent with a function as transcription factor affecting proliferation.

Declining signal dependence of Nrf2‐MafS‐regulated gene expression correlates with aging phenotypes

Aging is a degenerative process characterized by declining molecular, cell and organ functions, and accompanied by the progressive accumulation of oxidatively damaged macromolecules. This increased oxidative damage may be causally related to an age-associated dysfunction of defense mechanisms, which effectively protect young individuals from oxidative insults. Consistently, older organisms are more sensitive to acute oxidative stress exposures than young ones. In studies on the Drosophila Nrf2 transcription factor CncC, we have investigated possible causes for this loss of stress resistance and its connection to the aging process. Nrf2 is a master regulator of antioxidant and stress defense gene expression with established functions in the control of longevity. Here, we show that the expression of protective Nrf2/CncC target genes in unstressed conditions does not generally decrease in older flies. However, aging flies progressively lose the ability to activate Nrf2 targets in response to acute stress exposure. We propose that the resulting inability to dynamically adjust the expression of Nrf2 target genes to the organism's internal and external conditions contributes to age-related loss of homeostasis and fitness. In support of this hypothesis, we find the Drosophila small Maf protein, MafS, an Nrf2 dimerization partner, to be critical to maintain responsiveness of the Nrf2 system: overexpression of MafS in older flies preserves Nrf2/CncC signaling competence and antagonizes age-associated functional decline. The maintenance of acute stress resistance, motor function, and heart performance in aging flies overexpressing MafS supports a critical role for signal responsiveness of Nrf2 function in promoting youthful phenotypes.

Mo1131 Genetic Polymorphism of MAFK (Rs4268033) Is Associated With the Susceptibility to Gastric Cancer in Japan

[Background] The small Maf proteins have emerged as crucial regulators of mammalian gene expression. Small Mafs do not contain an obvious transcriptional activation domain. However, it has been clear that small Maf proteins are regulated transcription factors, such as Nrf2 or Bach1. MafK, one of the small Maf proteins, is encoded by MAFK gene. Within 20kbp around MAFK gene, there are two linkage disequilibrium blocks, with above 0.05 of HWE p value and above 0.05 of minor allele frequency. In this study, we investigated an association between gastric cancer (UC) and each tag polymorphism, rs4268033G .A and rs3735656 (*910T.C), and the other one rs10226620 (*1506T.C). [Material and Methods] The study was performed in 334 patients with gastric cancer (GC cases) and 661 subjects with no evidence of gastric malignancies (controls) on upper gastro-duodenal endoscopy. We employed the PCR-SSCP method to detect the gene polymorphism. [Results] The mean age, male/female ratio and Helicobacter pylori (HP) positive ratio in GC cases were significantly higher than those in controls. The distributions of rs4268033, rs3735656 and rs10226620 in controls were 326GG, 284GA and 51AA (HWE p=0.35), 291TT, 304TC and 66CC (HWE p=0.33) and 304TT, 288TC and 69CC (HWE p=0.93) respectively, whereas the distributions in GC cases was 127GG, 163GA and 44AA, 142TT, 153TC and 39CC, 144TT, 154TC and 36CC, respectively. The allelic frequency of rs4268033 Awas significantly higher in GC cases than in controls ‘(37.6% vs. 29.2%, p=0.0002). Overall, rs4268033 minor variant had a significantly increased risk for the development of gastric cancer by logistic regression analysis after adjustment for gender, age and HP infection status (recessive genetic model: OR, 1.72 95%CI, 1.10-2.69; p=0.016 and dominant genetic model: OR, 1.61; 95%CI, 1.21-2.14; p=0.0009), whereas no significant association was found between gastric cancer susceptibility and rs3735656 or rs10226620. The rs4268033 minor allele was more strongly associated with intestinal type of gastric cancer (recessive genetic model: OR, 2.12; 95%CI, 1.27-3.54; p=0.0040 and dominant genetic model: OR, 1.66; 95%CI, 1.17-2.35; p=0.0042), and slightly associated with diffuse type of gastric cancer (dominant genetic model: OR, 1.50; 95%CI, 1.02-2.21; p=0.040). In addition, rs4268033 minor allele was significantly associated with non-cardiac gastric cancer (recessive genetic model: OR, 1.76; 95%CI, 1.11-2.77; p=0.015 and dominant genetic model: OR, 1.68; 95%CI, 1.252.25; p=0.0005). [Conclusion] Our results provided the first evidence that MAFK gene polymorphism is significantly associated with the susceptibility to gastric cancer development. The rs4268033 minor allele is closely associated with an increased risk for the development of gastric cancer.

Mo1367 The Impact of JC Virus Antibody Testing on Treatment Selection in Patients With Crohn's Disease Who Are Primary Non-Responders to Infliximab: A Decision Analysis

[Background] The small Maf proteins have emerged as crucial regulators of mammalian gene expression. Small Mafs do not contain an obvious transcriptional activation domain. However, it has been clear that small Maf proteins are regulated transcription factors, such as Nrf2 or Bach1. MafK, one of the small Maf proteins, is encoded by MAFK gene. Within 20kbp around MAFK gene, there are two linkage disequilibrium blocks, with above 0.05 of HWE p value and above 0.05 of minor allele frequency. In this study, we investigated an association between ulcerative colitis (UC) and each tag polymorphism, rs4268033G.A and rs3735656 (*910T.C), and the other one rs10226620 (*1506T.C). [Material and Methods] The studied population comprised 922 subjects, including patients with UC (UC cases, n=174), who were enrolled Fujita Health University Hospital, and the subjects without UC (controls, n=748). We employed the PCR-SSCP method to detect a gene polymorphism. [Results] In controls, mean age was 57.1 years old and male/female ratio was 438/310. In UC cases, mean age was 40.3 years old, mean age of onset was 33.0 years old and male/ female ratio was 98/76. The distributions of rs4268033, rs3735656 and rs10226620 in controls were 366GG, 324GA and 58AA (HWE p=0.25), 329TT, 346TC and 73CC (HWE p=0.21) and 345TT, 328TC and 75CC (HWE p=0.87) respectively, whereas the distributions in UC cases was 80GG, 66GA and 28AA, 80TT, 69TC and 25CC, 80TT, 72TC and 22CC, respectively. The genotype frequency of rs4268033 AA and allelic frequency of rs4268033A allele were significantly higher in UC cases than in controls (p=0.0013 and 0.045 by Fisher). By logistic regression analysis after adjustment for age and gender, rs4268033 AA and rs3735656 CC genotypes were significantly associated with the susceptibility to UC development (OR, 2.62; 95%CI, 1.54-4.48; p=0.0004 and OR, 1.79; 95%CI, 1.05-3.04; p=0.031, respectively). In addition, rs4268033 AA genotype was significantly associated with all phenotypes of UC (i.e. total colitis phenotype: OR, 2.58; 95%CI, 1.29-5.17; p=0.0073, not total colitis: OR, 2.39; 95%CI, 1.22-4.72; p=0.012). The rs3735656CC genotype was significantly associated with steroid-independent or not required phenotype of UC (OR, 1.87; 95%CI, 1.08-3.26; p=0.026). There was no significant association between rs10226620 and UC. [Conclusion] Our results provided the first evidence that MAFK gene polymorphisms were significantly associated with the susceptibility to UC development. In particular, rs4268033 was closely associated with an increased risk for the development of UC.

Mo1366 Genetic Polymorphisms of MAFK, Encoding Small Maf Protein MAFK, Are Associated With the Susceptibility to Ulcerative Colitis

[Background] The small Maf proteins have emerged as crucial regulators of mammalian gene expression. Small Mafs do not contain an obvious transcriptional activation domain. However, it has been clear that small Maf proteins are regulated transcription factors, such as Nrf2 or Bach1. MafK, one of the small Maf proteins, is encoded by MAFK gene. Within 20kbp around MAFK gene, there are two linkage disequilibrium blocks, with above 0.05 of HWE p value and above 0.05 of minor allele frequency. In this study, we investigated an association between ulcerative colitis (UC) and each tag polymorphism, rs4268033G.A and rs3735656 (*910T.C), and the other one rs10226620 (*1506T.C). [Material and Methods] The studied population comprised 922 subjects, including patients with UC (UC cases, n=174), who were enrolled Fujita Health University Hospital, and the subjects without UC (controls, n=748). We employed the PCR-SSCP method to detect a gene polymorphism. [Results] In controls, mean age was 57.1 years old and male/female ratio was 438/310. In UC cases, mean age was 40.3 years old, mean age of onset was 33.0 years old and male/ female ratio was 98/76. The distributions of rs4268033, rs3735656 and rs10226620 in controls were 366GG, 324GA and 58AA (HWE p=0.25), 329TT, 346TC and 73CC (HWE p=0.21) and 345TT, 328TC and 75CC (HWE p=0.87) respectively, whereas the distributions in UC cases was 80GG, 66GA and 28AA, 80TT, 69TC and 25CC, 80TT, 72TC and 22CC, respectively. The genotype frequency of rs4268033 AA and allelic frequency of rs4268033A allele were significantly higher in UC cases than in controls (p=0.0013 and 0.045 by Fisher). By logistic regression analysis after adjustment for age and gender, rs4268033 AA and rs3735656 CC genotypes were significantly associated with the susceptibility to UC development (OR, 2.62; 95%CI, 1.54-4.48; p=0.0004 and OR, 1.79; 95%CI, 1.05-3.04; p=0.031, respectively). In addition, rs4268033 AA genotype was significantly associated with all phenotypes of UC (i.e. total colitis phenotype: OR, 2.58; 95%CI, 1.29-5.17; p=0.0073, not total colitis: OR, 2.39; 95%CI, 1.22-4.72; p=0.012). The rs3735656CC genotype was significantly associated with steroid-independent or not required phenotype of UC (OR, 1.87; 95%CI, 1.08-3.26; p=0.026). There was no significant association between rs10226620 and UC. [Conclusion] Our results provided the first evidence that MAFK gene polymorphisms were significantly associated with the susceptibility to UC development. In particular, rs4268033 was closely associated with an increased risk for the development of UC.

Characterization of small Maf regulators in lens fiber cell differentiation and cataract formation

The MAF (musculoaponeurotic fibrosarcoma) gene family includes “large” and “small” MAF subgroups that encode bZIP transcriptional regulators. Although mutations in the large MAF gene “MAF” cause human congenital cataract, function of the small MAF genes MAFF, MAFK, and MAFG in the lens remains uncharacterized. iSyTE (integrated Systems Tool for Eye gene discovery), a tool that identifies genes associated with lens development and cataract, has predicted MAFG as a potential regulator in the lens. To test this prediction, we aimed to characterize mouse models carrying targeted mutant alleles of small Maf genes. In situ hybridization and Western blotting confirmed that Mafg mRNA and protein were expressed in the embryonic and adult mouse lens tissue. Furthermore, Mafg−/− ;Mafk+/− compound mutant mice exhibit lens defects and develop pre‐senile cataract by age six months. Expression profiling by mouse Illumina microarrays on 2‐month old Mafg−/−;Mafk+/− compound mutant and control lenses identified several differentially regulated genes in the lens. This analysis indicates that expression of the heat shock protein gene Hspb1 (also known as Hsp27) is significantly down‐regulated in Mafg−/−;Mafk+/− compound mutant lenses. Together, these results suggest a functional role for the small Maf proteins MAFG and MAFK in maintenance of lens transparency. This research was funded by a Fight For Sight Foundation grant.

Fuzzy Complex Formation between the Intrinsically Disordered Prothymosin α and the Kelch Domain of Keap1 Involved in the Oxidative Stress Response

Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTα–Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region 38NANEENGE45 of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding.

7,8-Dihydroxyflavone Suppresses Oxidative Stress-Induced Base Modification in DNA via Induction of the Repair Enzyme 8-Oxoguanine DNA Glycosylase-1

The modified guanine base 8-oxoguanine (8-oxoG) is abundantly produced by oxidative stress, can contribute to carcinogenesis, and can be removed from DNA by 8-oxoguanine DNA glycosylase-1 (OGG1), which acts as an 8-oxoG glycosylase and endonuclease. This study investigated the mechanism by which 7,8-dihydroxyflavone (DHF) inhibits oxidative stress-induced 8-oxoG formation in hamster lung fibroblasts (V79-4). DHF significantly reduced the amount of 8-oxoG induced by hydrogen peroxide (H2O2) and elevated the levels of OGG1 mRNA and protein. DHF increased the binding of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response element sequences in the upstream promoter region of OGG1. Moreover, DHF increased the nuclear levels of Nrf2, small Maf proteins, and the Nrf2/small Maf complex, all of which are decreased by H2O2 treatment. Likewise, the level of phosphorylated Akt, which activates Nrf2, was decreased by H2O2 treatment but restored by DHF treatment. The levels of OGG1 and nuclear translocation of Nrf2 protein were decreased upon treatment with PI3K inhibitor or Akt inhibitor, and DHF treatment did not restore OGG1 and nuclear Nrf2 levels in these inhibitor-treated cells. Furthermore, PI3K and Akt inhibitors abolished the protective effects of DHF in cells undergoing oxidative stress. These data indicate that DHF induces OGG1 expression via the PI3K-Akt pathway and protects cells against oxidative DNA base damage by activating DNA repair systems.

Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

Nuclear factor- (erythroid-derived 2) like 2 (NFE2L2, NRF2) is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN) to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq) identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE) in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1), and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA–DNA Complex Structure and Binding Specificities

MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA-DNA complex. MafA forms base-specific hydrogen bonds with the flanking G(-5)C(-4) and central C(0)/G(0) bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse-chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

Nrf2–MafG heterodimers contribute globally to antioxidant and metabolic networks

NF-E2-related factor 2 (Nrf2) is a key transcription factor that is critical for cellular defense against oxidative and xenobiotic insults. Nrf2 heterodimerizes with small Maf (sMaf) proteins and binds to antioxidant response elements (AREs) to activate a battery of cytoprotective genes. However, it remains unclear to what extent the Nrf2–sMaf heterodimers contribute to ARE-dependent gene regulation on a genome-wide scale. We performed chromatin immunoprecipitation coupled with high-throughput sequencing and identified the binding sites of Nrf2 and MafG throughout the genome. Compared to sites occupied by Nrf2 alone, many sites co-occupied by Nrf2 and MafG exhibit high enrichment and are located in species-conserved genomic regions. The ARE motifs were significantly enriched among the recovered Nrf2–MafG-binding sites but not among the Nrf2-binding sites that did not display MafG binding. The majority of the Nrf2-regulated cytoprotective genes were found in the vicinity of Nrf2–MafG-binding sites. Additionally, sequences that regulate glucose metabolism and several amino acid transporters were identified as Nrf2–MafG target genes, suggesting diverse roles for the Nrf2–MafG heterodimer in stress response. These data clearly support the notion that Nrf2–sMaf heterodimers are complexes that regulate batteries of genes involved in various aspects of cytoprotective and metabolic functions through associated AREs.

The Predicted Molecular Weight of Nrf2: It Is What It Is Not

The Predicted Molecular Weight of Nrf2: It Is What It Is Not

Identification of a putative glycosyltransferase responsible for the transfer of pseudaminic acid onto the polar flagellin of Aeromonas caviae Sch3N

Motility in Aeromonas caviae, in a liquid environment (in broth culture), is mediated by a single polar flagellum encoded by the fla genes. The polar flagellum filament of A. caviae is composed of two flagellin subunits, FlaA and FlaB, which undergo O-linked glycosylation with six to eight pseudaminic acid glycans linked to serine and threonine residues in their central region. The flm genetic locus in A. caviae is required for flagellin glycosylation and the addition of pseudaminic acid (Pse) onto the lipopolysaccharide (LPS) O-antigen. However, none of the flm genes appear to encode a candidate glycotransferase that might add the Pse moiety to FlaA/B. The motility-associated factors (Maf proteins) are considered as candidate transferase enzymes, largely due to their conserved proximity to flagellar biosynthesis loci in a number of pathogens. Bioinformatic analysis performed in this study indicated that the genome of A. caviae encodes a single maf gene homologue (maf1). A maf mutant was generated and phenotypic analysis showed it is both nonmotile and lacks polar flagella. In contrast to flm mutants, it had no effect on the LPS O-antigen pattern and has the ability to swarm. Analysis of flaA transcription by reverse transcriptase PCR (RT-PCR) showed that its transcription was unaltered in the maf mutant while a His-tagged version of the FlaA flagellin protein produced from a plasmid was detected in an unglycosylated intracellular form in the maf strain. Complementation of the maf strain in trans partially restored motility, but increased levels of glycosylated flagellin to above wild-type levels. Overexpression of maf inhibited motility, indicating a dominant negative effect, possibly caused by high amounts of glycosylated flagellin inhibiting assembly of the flagellum. These data provide evidence that maf1, a pseudaminyl transferase, is responsible for glycosylation of flagellin and suggest that this event occurs prior to secretion through the flagellar Type III secretion system.

Nrf2b, Novel Zebrafish Paralog of Oxidant-responsive Transcription Factor NF-E2-related Factor 2 (NRF2)

NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.

Embryonic Lethality and Fetal Liver Apoptosis in Mice Lacking All Three Small Maf Proteins

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.

The roles of ATF2 (activating transcription factor 2) in tumorigenesis

MAPK (mitogen-activated protein kinase) pathways are among the most frequently deregulated signalling events in cancer. Among the critical targets of MAPK activities are members of the AP-1 (activator protein 1) transcription factor, a dimeric complex consisting of Jun, Fos, Maf and ATF (activating transcription factor) family DNA-binding proteins. Depending on the cellular context, the composition of the dimeric complexes determines the regulation of growth, survival or apoptosis. JNK (c-Jun N-terminal kinase), p38 and a number of Jun and Fos family proteins have been analysed for their involvement in oncogenic transformation and tumour formation. These data are also emerging for the ATF components of the AP-1 factor. The aim of the present review is to provide an overview of the functions of two ATF family proteins, ATF2 and ATF7, in mammalian development and their potential functions in tumour formation.

Novel insights into the regulation of antioxidant-response-elementmediated gene expression by electrophiles: induction of the transcriptional repressor BACH1 by Nrf2

A central mechanism in cellular defence against oxidative or electrophilic stress is mediated by transcriptional induction of genes via the ARE (antioxidant-response element), a cis-acting sequence present in the regulatory regions of genes involved in the detoxification and elimination of reactive oxidants and electrophiles. The ARE binds different bZIP (basic-region leucine zipper) transcription factors, most notably Nrf2 (nuclear factor-erythroid 2-related factor 2) that functions as a transcriptional activator via heterodimerization with small Maf proteins. Although ARE activation by Nrf2 is relatively well understood, the mechanisms by which ARE-mediated signalling is down-regulated are poorly known. Transcription factor BACH1 [BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1] binds to ARE-like sequences, functioning as a transcriptional repressor in a subset of ARE-regulated genes, thus antagonizing the activator function of Nrf2. In the present study, we have demonstrated that BACH1 itself is regulated by Nrf2 as it is induced by Nrf2 overexpression and by Nrf2-activating agents in an Nrf2-dependent manner. Furthermore, a functional ARE site was identified at +1411 from the transcription start site of transcript variant 2 of BACH1. We conclude that BACH1 is a bona fide Nrf2 target gene and that induction of BACH1 by Nrf2 may serve as a feedback-inhibitory mechanism for ARE-mediated gene regulation.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.