Differential induction of mafF, mafG and mafK expression by electrophile-response-element activators.
ABSTRACT: The three small Maf proteins, MafF, MafG and MafK, have been implicated in a number of physiological processes, including development, differentiation, haematopoiesis and stress response. Here we report the constitutive expression of mafF, mafG and mafK in six human cell lines derived from various tissues (HepG2, IMR-32, K-562, HEK-293, RD and A549). The expression patterns of mafF, mafG and mafK varied widely among cell lines. Because small Maf proteins have been implicated in electrophile response element (EpRE)-mediated stress response, the ability of three EpRE activators [pyrrolidinedithiocarbamate (PDTC), phenylethyl isothiocyanate (PEITC) and t-butylhydroquinone (tBHQ)] to induce small Maf expression was examined in detail in HepG2 cells. Both PDTC and PEITC induced mafF, mafG and mafK expression, whereas tBHQ failed to markedly induce any of the three small Mafs. Where a response was observed, mafF was induced to the greatest extent compared with mafG and mafK, and this response was transcriptionally mediated. PDTC also induced small Maf expression in the other cell lines examined, with patterns of induction varying among cell lines. The differences in expression among the cell lines examined, coupled with the induction patterns observed, indicate that the three small maf genes are stress-responsive, but may be regulated via differing mechanisms. Furthermore, the fact that tBHQ, PDTC and PEITC induce EpRE activity, but that tBHQ fails to markedly induce any of the small Mafs, suggests that up-regulation of small Mafs is not an absolute requirement for EpRE-mediated gene expression.
Project description: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.
Project description:MafF-/-: MafG+/+: MafK-/- mice are viable, while MafF-/-: MafG-/-: MafK-/- mice are embryonic lethal. To get an insight into the cause of the lethality of small Maf triple knockout mice, transcriptome analysis was performed using whole embyos of MafF-/-: MafG-/-: MafK-/- at E10.5 and those of MafF-/-: MafG+/+: MafK-/- at E9.5 or E10.5. Because MafF-/-: MafG-/-: MafK-/- embryos exhibit growth retardation, the gene expression profile of MafF-/-: MafG-/-: MafK-/- embryos at E10.5 was compared with that of MafF-/-: MafG+/+: MafK-/- embyos at E9.5. The gene expression profile of MafF-/-: MafG+/+: MafK-/- embryos at E10.5 was also examined as an alternative control. Total RNA was prepared from pooled three embryos for each sample.
Project description:Although majority of the genes linked to early-onset cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. We report on small Maf transcription factors Mafg and Mafk as regulators of several non-crystallin human cataract-associated genes in fiber cells and establish their significance to this disease. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify Mafg and its co-regulators in the lens, and generated various null-allelic combinations of Mafg:Mafk mouse mutants for phenotypic and molecular analysis. By age 4 months, Mafg-/-:Mafk+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of Mafg-/-:Mafk+/- mouse lens reveals severely disorganized fiber cells, while microarray-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of Mafg-/-:Mafk+/- lens-DRGs with (1) binding motifs and genomic targets of small Mafs and their regulatory partners, (2) iSyTE lens expression data, and (3) interactions between DRGs in the String database, unravel a detailed small Maf regulatory network in the lens, several nodes of which are linked to cataract. This approach identifies 36 high-priority candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes oxidative stress and misregulation of sterol synthesis. These data identify Mafg and Mafk as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to human cataract.
Project description: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.
Project description:Members of the small Maf family (MafK, MafF, and MafG) are basic region leucine zipper (bZip) proteins that can function as transcriptional activators or repressors. The dimer compositions of their DNA binding forms determine whether the small Maf family proteins activate or repress transcription. Using a yeast two-hybrid screen with a GAL4-MafK fusion protein, we have identified two novel bZip transcription factors, Bach1 and Bach2, as heterodimerization partners of MafK. In addition to a Cap'n'collar-type bZip domain, these Bach proteins possess a BTB domain which is a protein interaction motif; Bach1 and Bach2 show significant similarity to each other in these regions but are otherwise divergent. Whereas expression of Bach1 appears ubiquitous, that of Bach2 is restricted to monocytes and neuronal cells. Bach proteins bind in vitro to NF-E2 binding sites, recognition elements for the hematopoietic transcription factor NF-E2, by forming heterodimers with MafK. Furthermore, a DNA binding complex that contained MafK as well as Bach2 or a protein related closely to Bach2 was found to be present in mouse brain cells. Bach1 and Bach2 function as transcription repressors in transfection assays using fibroblast cells, but they function as a transcriptional activator and repressor, respectively, in cultured erythroid cells. The results suggest that members of the Bach family play important roles in coordinating transcription activation and repression by MafK.
Project description:To elucidate the function of Nrf2-small Maf heterodimer, we constructed a tethered Nrf2-MafG (T-N2G) heterodimer using a flexible linker peptide and examined its function in the small Maf-deficient mouse embryonic fibroblast (MEF). Overall design: ChIP-seq was performed using anti-MafG antibody in the T-NG2-expressing MafF-/-::MafG-/-::MafK-/- (F0G0K0) MEFs, treated with 100µM Diethyl Maleate (DEM) or Vehcle for 4hr.
Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants
Project description:A group of cytoprotective genes is regulated by heterodimers composed of the cap'n'collar (CNC) family member Nrf2 and one of the small Maf (sMaf) proteins (MafF, MafG, or MafK) through the antioxidant response element (ARE, also referred to as the CNC-sMaf binding element [CsMBE]). Many lines of evidence support this model; however, a direct and specific evaluation of the Nrf2-sMaf heterodimer remains to be executed. To address this issue, we constructed a tethered Nrf2-MafG (T-N2G) heterodimer using a flexible linker peptide. We then introduced the T-N2G construct into cells lacking all three sMaf proteins to specifically evaluate the function of the tethered heterodimer without interference from other endogenous CNC-sMaf heterodimers or sMaf homodimers. In response to an Nrf2 activator, diethyl maleate, the T-N2G protein can widely activate the target genes of Nrf2 but not those of Nrf1, such as proteasome subunit genes. Genome-wide binding analysis showed that the T-N2G protein preferentially bound to the CsMBE motifs in the regulatory regions of the Nrf2 target genes. These results provide direct evidence that the Nrf2-MafG heterodimer acts as a transcriptional activator of Nrf2-dependent genes and show that this assay system will be a powerful tool to specifically examine the function of other CNC-sMaf heterodimers.
Project description:The small Maf proteins (sMafs) are basic region leucine zipper (bZIP)-type transcription factors. The basic region of the Maf family is unique among the bZIP factors, and it contributes to the distinct DNA-binding mode of this class of proteins. MafF, MafG and MafK are the three vertebrate sMafs, and no functional differences have been observed among them in terms of their bZIP structures. sMafs form homodimers by themselves, and they form heterodimers with cap 'n' collar (CNC) proteins (p45 NF-E2, Nrf1, Nrf2, and Nrf3) and also with Bach proteins (Bach1 and Bach2). Because CNC and Bach proteins cannot bind to DNA as monomers, sMafs are indispensable partners that are required by CNC and Bach proteins to exert their functions. sMafs lack the transcriptional activation domain; hence, their homodimers act as transcriptional repressors. In contrast, sMafs participate in transcriptional activation or repression depending on their heterodimeric partner molecules and context. Mouse genetic analyses have revealed that various biological pathways are under the regulation of CNC-sMaf heterodimers. In this review, we summarize the history and current progress of sMaf studies in relation to their partners.