Other transcriptional targets of Cap'n'collar homologs

Common signaling chains of various receptor families, despite some similarities, are able to provoke quite different cellular responses. This suggests that they are linked to different cascades and transcription factors, dependent on the context of the ligand binding moiety and the cell type. The ITAM (immunoreceptor tyrosine-based activation motif) containing gamma chain of the FcepsilonRI, FcgammaRI, FcgammaRIII and the T-cell receptor is one of these shared signaling molecules. In the context of the FcgammaRIII, the gamma chain activates the transcription factor Nrf1 or a closely related protein that specifically interacts with the extended kappa3 site in the TNFalpha promoter. A novel splice variant of Nrf1 with a 411 bp deletion of the serine-rich region, resulting in an overall structure reminiscent of the BTB and CNC homology (Bach) proteins, was isolated from the corresponding DC18 cells. In a gel shift analysis, this bacterially expressed splice variant binds to the TNFalpha promoter site after in vitro phosphorylation by casein kinase II (CKII). Cotransfection studies demonstrate that this splice variant mediates induced transcription at the TNFalpha promoter after stimulation/activation in a heterologous system (Prieschl, 1998).

Activation of cytochrome c (cyt c) transcription in electrically stimulated neonatal rat cardiac myocytes is preceded by transient expression of the activating protein-1 family of transcription factors (c-Fos, c-Jun, and JunB), as well as nuclear respiratory factor-1 (NRF-1). Mutations in either the NRF-1 or in the two cyclic AMP response elements on the cyt c promoter significantly reduce cyt c promoter activation produced either by electrical stimulation or by transfection of c-jun into nonpaced cardiac myocytes. Electrical stimulation of cardiac myocytes activates the c-Jun N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun were cotransfected. Physical association of NRF-1 protein with the NRF-1 enhancer element and of c-Jun with the cyclic AMP response element binding sites on the cyt c promoter was demonstrated by gel shift competition assays and by antibody super shifts. This is the first demonstration that induction of NRF-1 and c-Jun by pacing of cardiac myocytes directly mediates cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart (Xia, 1998).

The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence was investigated. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this role in vivo. To directly test these questions, the murine nrf2 gene was disrupted in vivo. While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) is markedly induced by a phenolic antioxidant in vivo in both wild type and heterozygous mutant mice, the induction is largely eliminated in the liver and intestine of homozygous nrf2-mutant mice. Nrf2 binds to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful model for the in vivo analysis of chemical carcinogenesis and resistance to anti-cancer drugs (Itoh, 1997).

Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) or pyrrolidine dithiocarbamate (PDTC) results in the up-regulation of the gamma-glutamylcysteine synthetase catalytic [GCS(h)] and regulatory [GCS(l)] subunit genes. Increased expression is associated with an increase in the binding of Nrf2 to electrophile response elements (EpRE) in the promoters of these genes. Nrf2 overexpression increases the activity of GCS(h) and GCS(l) promoter/reporter transgenes. Overexpression of an MafK dominant negative mutant decreases Nrf2 binding to GCS EpRE sequences, inhibits the inducible expression of GCS(h) and GCS(l) promoter/reporter transgenes, and reduces endogenous GCS gene induction. beta-NF and PDTC exposure also increases steady-state levels of MafG mRNA. In addition to Nrf2, small Maf and JunD proteins are detected in GCS(h)EpRE-protein complexes and, to a lesser extent, in GCS(l)EpRE-protein complexes. The Nrf2-associated expression of GCS promoter/reporter transgenes is inhibited by overexpression of MafG. Inhibition of protein synthesis by cycloheximide partially decreases inducibility by PDTC or beta-NF and results in significant increases in GCS mRNA at late time points, when GCS mRNA levels are normally declining. It is hypothesized that, in response to beta-NF and PDTC, the GCS subunit genes are transcriptionally up-regulated by Nrf2-basic leucine zipper complexes, containing either JunD or small Maf protein, depending on the particular GCS EpRE target sequence and the inducer. Following maximal induction, down-regulation of the two genes is mediated via a protein synthesis-dependent mechanism (Wild, 1999).

Oxidative stress-responsive transcription is regulated in part through cis-active sequences known as antioxidant response elements (ARE). Activation through the ARE involves members of the CNC-subfamily of basic leucine zipper proteins including Nrf1 and Nrf2. In particular, Nrf2 has been shown to coordinate induction of genes encoding antioxidant and phase 2 metabolizing enzymes in response to stimulation with electrophilic compounds and exposure to xenobiotics. The 65-kDa isoform of the Nrf1 gene functions as a repressor of Nrf2. Transient expression of p65Nrf1 suppressed Nrf2-mediated activation of ARE-dependent reporter genes in cells. Induction of endogenous ARE-genes is blocked in Hepa1c1c7 cells stably expressing p65Nrf1, leading to increased cell death. Consistent with these findings, electrophilic activation of ARE-gene expression is augmented by loss of p65Nrf1 function in Nrf1(-/-) fibroblasts, and the protective effects of oxidative preconditioning and ARE-gene expression are blocked in Nrf1(-/-) cells stably expressing p65Nrf1. Gel shift experiments demonstrated that p65Nrf1 binds the antioxidant response element as a heterodimer with small-Maf protein. Immunoprecipitation studies demonstrated that p65Nrf1 competes with Nrf2 for interaction with small-Maf protein and binding to the antioxidant response element in vivo. Together, these results demonstrate that p65Nrf1 has the potential to play an important role in modulating the response to oxidative stress by functioning as a transdominant repressor of Nrf2-mediated activation of ARE-dependent gene transcription (Wang, 2007).

Nrf1 is a member of the vertebrate Cap'n'Collar (CNC) transcription factor family that commonly contains a unique basic-leucine zipper domain. Among CNC family members, Nrf2 is known to regulate a battery of antioxidant and xenobiotic-metabolizing enzyme genes through the antioxidant response element (ARE). Although Nrf1 has also been shown to bind the ARE, it is unclear whether it plays a distinct role from Nrf2 in regulating genes with this element. To address this issue in vivo, mice were generated bearing a hepatocyte-specific disruption of the Nrf1 gene. Although Nrf2 knock-out mice did not exhibit liver damage when they were maintained in an unstressed condition, hepatocyte-specific deletion of Nrf1 caused liver damage resembling the human disease non-alcoholic steatohepatitis. Gene expression analysis revealed that the disruption of Nrf1 causes stress that activates a number of ARE-driven genes in an Nrf2-dependent manner, indicating that Nrf2 cannot compensate completely for loss of Nrf1 function in the liver. In contrast, expression of metallothionein-1 and -2 (MT1 and MT2) genes, each of which harbors at least one ARE in its regulatory region, was decreased in the Nrf1-null mutant mice. Whereas Nrf1 and Nrf2 bound the MT1 ARE with comparable affinity, Nrf1 preferentially activated the reporter gene expression through the MT1 ARE. This study has, thus, identified the first ARE-dependent gene that relies exclusively on Nrf1, suggesting that it plays a distinct functional role in regulating ARE-driven genes (Ohtsuji, 2008).

Cap'n'collar homologs, megakaryocytes and platelet production

Mechanisms of platelet production and release by mammalian megakaryocytes are poorly understood. Thrombocytopenic knockout mice were used to better understand these processes. Proplatelets are filamentous extensions of terminally differentiated megakaryocytes, thought to represent one mechanism of platelet release; however, these structures have largely been recognized in cultured cells and there has been no correlation between thrombocytopoiesis in vivo and proplatelet formation. Mice lacking transcription factor NF-E2 have a late arrest in megakaryocyte maturation, resulting in profound thrombocytopenia. In contrast to normal megakaryocytes, which generate abundant proplatelets, cells from these mice never produce proplatelets, even after prolonged stimulation with c-Mpl ligand. Similarly, megakaryocytes from thrombocytopenic mice with lineage-selective loss of transcription factor GATA-1 produce proplatelets very rarely. These findings establish a significant correlation between thrombocytopoiesis and proplatelet formation and suggest that the latter represents a physiologic mechanism for platelet release. Proplatelet formation by normal megakaryocytes and its absence in cells lacking NF-E2 are independent of interactions with adherent (stromal) cells. Similarly, thrombocytopenia in NF-E2(-/-) mice reflects intrinsic defects in the megakaryocyte lineage. These observations improve understanding of platelet production and validate the study of proplatelets to probe the underlying mechanisms (Lecine, 1998).

Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, an in vivo immunoselection assay was used: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). The TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2: (1) its promoter contains a functional NF-E2 binding site; (2) the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; (3) p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function (Deveaux, 1997).

The locus control region of the beta-globin gene is composed of four erythroid-specific hypersensitive sites. Hypersensitive site 2 is a powerful enhancer and contains a tandem repeat sequence for the transcription factors AP1 and NFE2 (activating protein 1 and nuclear factor erythroid 2, respectively). The human NRF2 (NFE2 related factor 2) has been isolated by bacterial expression screening using this core sequence as a probe. p45-NFE2, NRF1, and NRF2 belong to the CNC (cap 'n' collar) subfamily of the basic region-leucine zipper transcription factors, which exhibits strong homology at specific regions such as the CNC and the DNA binding and leucine zipper domains. Although the erythroid-specific p45-NFE2 has been implicated in globin gene regulation, p45-NFE2 null mice succumb to bleedings due to lack of platelets, yet those that survive exhibit only a mild anemia. To determine the function of NRF2, which is widely expressed in vivo, the genomic structure of the mouse NRF2 gene has been characterized, the Nrf2 gene has been disrupted by homologous recombination in mouse embryonic stem cells (ES cells), and NRF2-/- mice have been generated. Homozygous mutant mice develop normally, are not anemic, reach adulthood, and reproduce. These studies indicate that NRF2 is dispensable for mouse development (Chan, 1996).

Protein interactions of Cap'n'collar homologs: Interaction with Maf subfamily members

Mammalian globin gene expression is activated through NF-E2 elements recognized by basic-leucine zipper proteins of the AP-1 superfamily. The specificity of NF-E2 DNA binding is determined by several nucleotides adjacent to a core AP-1 motif, comprising a recognition site for transcription factors of the Maf subfamily. Earlier work proposed that p18(MafK) forms a heterodimer with hematopoietic-specific protein p45 NF-E2 to activate transcription through NF-E2 sites. However, there has been no direct evidence that p18(MafK) serves this function in vivo; in fact, mice lacking p18(MafK) have no mutant phenotype. A novel cDNA clone is described that encodes the human homolog of chicken MafG. Human MafG heterodimerizes with p45 NF-E2 and binds DNA with specificity identical to that of purified NF-E2 DNA binding activity. A tethered heterodimer of p45 and MafG is fully functional in supporting expression of alpha- and beta-globin, and in promoting erythroid differentiation in CB3, a p45-deficient mouse erythroleukemia cell line. These results indicate that human MafG can serve as a functional partner for p45 NF-E2, and suggest that the p45/MafG heterodimer plays a role in the regulation of erythropoiesis (Blank 1997).

The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the regulation of erythroid gene expression and platelet formation. To facilitate the characterization of NF-E2 functions in human cells, cDNAs were isolated encoding two members of the small Maf family: MafK and MafG. The human mafK and mafG genes encode proteins of 156 and 162 amino acid residues, respectively, whose deduced amino acid sequences show approximately 95% identity to their respective chicken counterparts. Expression of mafK mRNA is high in heart, skeletal muscle and placenta, whereas mafG mRNA is abundant in skeletal muscle and is moderately expressed in heart and brain. Both are expressed in all hematopoietic cell lines, including those of erythroid and megakaryocytic lineages. In electrophoretic gel mobility shift assays, binding to NF-E2 sites depends on formation of homodimers or heterodimers with p45 and p45-related CNC family proteins. The results suggest that the small Maf family proteins function in human cells through interaction with various basic-leucine zipper-type transcription factors (Toki, 1997).

A 1.6-kilobase pair full-length cDNA encoding a transcription factor homologous to the Maf family of proteins was isolated by screening a K562 cDNA library with the NFE2 tandem repeat probe derived from the globin locus control region. The protein, designated hMAF, contains a basic DNA binding domain and an extended leucine zipper but lacks any recognizable activation domain. Expressed in vitro, the hMAF protein is able to homodimerize in solution and band-shift the NFE2 tandem repeat probe. In addition to homodimers, hMAF can also form high affinity heterodimers with two members of the NFE2/CNC-bZip family (Nrf1 and Nrf2) but not with a third family member, p45-NFE2. Although hMAF/hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the same NFE2 site, they exert functionally opposite effects on the activity of a linked gamma-globin gene. In fact, whereas all hMAF/CNC-bZip heterodimers stimulate the activity of a gamma-promoter reporter construct in K562 cells, the association into homodimers that is induced by overexpressing hMAF inhibits the activity of the same construct. Thus variations in the expression of hMAF may account for the modulation in the activity of the genes that bear NFE2 recognition sites (Marini, 1997).

The widely expressed human transcription factor TCF11/LCR-F1/Nrf1 interacts with small Maf proteins and binds to a subclass of AP1-sites. Such sites are required for beta-globin 5' DNase I hypersensitive site 2 enhancer activity, erythroid porphobilinogen deaminase inducibility, hemin responsiveness by heme-oxygenase 1 and expression of the gene NAD(P)H:quinone oxidoreductase1. The optimal DNA-binding sequences for TCF11/LCR-F1/Nrf1 alone and as a heterodimer with MafG, identified by using binding-site selection, are reported. The heterodimer recognizes a 5'-TGCTgaGTCAT-3' binding-site that is identical to the established NF-E2-site, the antioxidant response element and the heme-responsive element, while the binding specificity of the homomer is less stringent. To investigate the activity of TCF11 through this selected site, both alone and in the presence of MafG, a transient transfection assay was used. TCF11 alone activates transcription while MafG alone acts as a repressor. When co-expressed, MafG interferes with TCF11 transactivation in a dose dependent manner. This indicates that MafG protein, which heterodimerizes efficiently with TCF11 in vitro (the heterodimer having a higher affinity for DNA than TCF11 alone), does not co-operate with TCF11 in transactivating transcription. It is proposed that since both these factors are widely expressed, they may act together to contribute to the negative regulation of this specific target site. Efficient positive regulation by TCF11 may require alternative partners with perhaps more restricted expression patterns (Johnsen, 1998).

The small Maf proteins, MafF, MafG, and MafK, possess a leucine zipper (Zip) domain that is required for homodimer or heterodimer complex formation with other bZip transcription factors. This study sought to determine the identity of the specific constituent that collaboratively interacts with Nrf2 to bind to the Maf recognition element in vivo. Studies in vitro suggested that Nrf2 forms heterodimers with small Maf proteins and then bind to Maf recognition elements, but the bona fide partner molecules supporting Nrf2 activity in vivo have not been definitively identified. Nrf2 activity is usually suppressed by a cytoplasmic repressor, Keap1, so disruption of the keap1 gene causes constitutive activation of Nrf2. Nrf2 hyperactivity results in hyperproliferation of keratinocytes in the esophagus and forestomach leading to perinatal lethality. However, simultaneous disruption of nrf2 rescues keap1-null mice from the lethality. This system was exploited to investigate whether small Mafs are required for Nrf2 function. keap1 and small maf compound mutant mice were generated and whether keratinocyte abnormalities persist in these animals was examined. The data show that loss of mafG and mafF in the keap1-null mice reversed the lethal keratinocyte dysfunction and rescues the keap1-null mutant mice from perinatal lethality. This rescue phenotype of mafG::mafF::keap1 triple compound mutant mice phenocopies that of the nrf2::keap1 compound mutant mice, indicating that the small Maf proteins MafG and MafF must functionally cooperate with Nrf2 in vivo (Motohashi, 2004).

Other protein interactions of Cap'n'collar homologs

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator that acts on a diverse set of nuclear genes required for mitochondrial respiratory function in mammalian cells. These genes encode respiratory proteins as well as components of the mitochondrial transcription, replication, and heme biosynthetic machinery. NRF-1 is shown to be a phosphoprotein in vivo. Phosphorylation occurs on serine residues within a concise NH2-terminal domain with the major sites of phosphate incorporation at serines 39, 44, 46, 47, and 52. The in vivo phosphorylation pattern can be approximated in vitro by phosphorylating recombinant NRF-1 with purified casein kinase II. Phosphate incorporation at the sites utilized in vivo results in a marked stimulation of DNA binding activity, which is not observed in mutated proteins lacking these sites. Pairwise expression of the wild-type protein with each of a series of truncated derivatives in transfected cells results in the formation of a dimer between wild-type and mutant forms demonstrating that a homodimer is the active binding species. Although NRF-1 can dimerize in the absence of DNA, phosphorylation does not enhance the formation of these dimers. These findings suggest that phosphorylation results in an intrinsic change in the NRF-1 dimer enhancing its ability to bind DNA (Gugneja, 1997).

Thyroid hormone (T3) and retinoic acid (RA) play important roles in erythropoiesis. The hematopoietic cell-specific bZip protein p45/NF-E2 interacts with T3 receptor (TR) and RA receptor (RAR) but not retinoid X receptor. The interaction is between the DNA-binding domain of the nuclear receptor and the leucine zipper region of p45/NF-E2 but is markedly enhanced by cognate ligand. Remarkably, ligand-dependent transactivation by TR and RAR is markedly potentiated by p45/NF-E2. This effect of p45/NF-E2 is prevented by maf-like protein p18, which functions positively as a heterodimer with p45/NF-E2 on DNA. Potentiation of hormone action by p45/NF-E2 requires its activation domain, which interacts strongly with the multifaceted coactivator cyclic AMP response element protein-binding protein (CBP). The region of CBP which interacts with p45/NF-E2 is the same interaction domain that mediates inhibition of hormone-stimulated transcription by AP1 transcription factors. Overexpression of the bZip interaction domain of CBP specifically abolishes the positive cross talk between TR and p45/NF-E2. Thus, positive cross talk between p45/NF-E2 and nuclear hormone receptors requires direct protein-protein interactions between these factors and with CBP, whose integration of positive signals from two transactivation domains provides a novel mechanism for potentiation of hormone action in hematopoietic cells (Cheng, 1997).

Tandem binding sites for the hematopoietic transcription factor NF-E2 in the beta-globin locus control region activate high-level beta-globin gene expression in transgenic mice. NF-E2 is a heterodimer consisting of a hematopoietic subunit p45 and a ubiquitous subunit p18. Human p45 contains a PPXY motif that binds WW domains. Murine NF-E2, which contains two PPXY motifs (PPXY-1 and -2) within its transactivation domain, differentially interacts with nine GST-WW domain fusion proteins. Quantitative analysis reveals high-affinity binding (KD = 5.7 nM) of p45 to a WW domain from a novel human ubiquitin ligase homolog (WWP1) expressed in hematopoietic tissues. The amino-terminal WW domain of WWP1 forms a multimeric complex with DNA-bound NF-E2. A WWP1 ligand peptide, isolated by phage display, and a peptide spanning PPXY-1 both inhibit p45 binding, whereas an SH3 domain-interacting peptide and a peptide spanning PPXY-2 do not. Mutation of PPXY-1, but not PPXY-2, inhibits the transactivation function of NF-E2, providing support for the hypothesis that WW domain interactions are important for NF-E2-mediated transactivation (Mosser, 1998).

NF-E2 is an erythroid-specific transcription factor required for expression of several erythroid-specific genes. By Far-Western blotting and yeast two-hybrid assay, p45, the large subunit of NF-E2 has been demonstrated to be capable of binding to a specific set of WW domain-containing proteins, including the ubiquitin ligase hRPF1. This binding is mediated through the interaction between the WW domains and a PY motif located within the amino-terminal region of p45. Interestingly, the carboxyl-terminal domain of mammalian RNA polymerase II binds a similar set of WW domains with which p45 interacts. These data suggest possible new pathways through which the processes of transcriptional regulation by NF-E2 could be regulated in erythroid and megakaryote cells (Gavva, 1997).

Mutation of Cap'n'collar homologs

Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. Loss of Nrf1 function in mice results in late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, mice were generated that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells (Leung, 2003).

Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), mice deficient in this transcription factor were generated. Targeted disruption was generated of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. No differences were observed in various blood parameters and chemistry values. Wild-type and Nrf3(-/-) mice were infected with acute lymphocytic choriomeningitis virus and no differences were found in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, mice deficient in multiple CNC factors were generated. Contrary expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. It is hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice (Derjuga, 2004).

Cap'n'collar homologs and stress response

Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcription factor but relatively little Nrf1. Regulation of intestinal antioxidant and detoxication enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed either a control diet or one supplemented with either synthetic cancer chemopreventive agents [butylated hydroxyanisole (BHA), ethoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol and kahweol palmitate, sulforaphane, coumarin (CMRN), or alpha-angelicalactone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and glutathione S-transferase (GST) enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with BHA or EQ; increases of about 2-fold were observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha-angelicalactone; and increases of 1.5-fold were measured with sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl synthetase [GCS(h)] was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary BHA or EQ: this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal antioxidant and detoxication proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction (McMahon, 2001).

Antioxidant-response element (ARE) and nuclear factor Nrf2-mediated expression and coordinated induction of genes encoding chemopreventive proteins, including NQO1, are critical mechanisms in chemoprotection. Recently, Nrf3, a new member of the Nrf family with substantial homology to Nrf2, was identified and cloned. This study investigated the role of Nrf3 in ARE-mediated gene expression and induction of NQO1 in response to antioxidants. Overexpression of Nrf3 in Hep-G2 cells leads to a concentration-dependent decrease in transfected and endogenous NQO1 gene expression and induction in response to antioxidant tert-butylhydroquinone (t-BHQ). Deletion mutation analysis revealed that Nrf3 repression of NQO1 gene expression requires heterodimerization and DNA binding domains but not transcriptional activation domain of Nrf3. Bandshift and supershift assays with in vitro transcribed and translated proteins and nuclear extracts from Hep-G2 cells treated with Me2SO and t-BHQ and immunoprecipitation assays demonstrated that Nrf3 associates with small Maf proteins to bind to the ARE. RNA interference specific to Nrf3 reduced intracellular Nrf3 leading to increased expression and induction of transfected and endogenous NQO1 gene expression in response to t-BHQ. These results combined suggest that Nrf3 is a negative regulator of ARE-mediated gene expression (Sankaranarayanan, 2004).

Nrf2 accumulates in nuclei upon exposure to oxidative stress, heterodimerizes with a small Maf protein, and activates the transcription of stress target genes through antioxidant response elements (AREs). Diethyl maleate (DEM), a well known activator of Nrf2, induces one of the small Maf genes, mafG. To elucidate the roles that MafG might play in the oxidative stress response, transcriptional regulation of the mouse mafG gene was examined. MafG utilizes three independent first exons that are each spliced to second and third coding exons. Among the small maf genes, mafG showed the strongest response to DEM, and of the three first exons, the highest-fold induction was seen with the proximal first exon (Ic). Importantly, one ARE (Ic-ARE) is conserved in the promoter flanking exon Ic of the human and mouse mafG genes. The Nrf2/MafG heterodimer binds the Ic-ARE and activates transcription, whereas DEM fails to activate mafG in nrf2-null mutant cells. Chromatin immunoprecipitation further revealed that both Nrf2 and small Maf proteins associate with the Ic-ARE in vivo. These results demonstrate that mafG is itself an ARE-dependent gene that is regulated by an Nrf2/small Maf heterodimer and suggest the presence of an autoregulatory feedback pathway for mafG transcriptional regulation (Katsuoka, 2004).

Degradation of Cap'n'collar homologs

Keap1 is a negative regulator of Nrf2, a bZIP transcription factor that mediates adaptation to oxidative stress. Previous studies have suggested this negative regulation is a consequence of Keap1 controlling the subcellular distribution of Nrf2. Keap1 also controls the total cellular level of Nrf2 protein. In the RL34 non-transformed rat liver cell line, Nrf2 was found to accumulate rapidly in response to oxidative stress caused by treatment with sulforaphane, and the accumulation resulted from inhibition of proteasomal-mediated degradation of the bZIP protein. By heterologously expressing in COS1 cells epitope-tagged Nrf2 and an Nrf2DeltaETGE mutant lacking the Keap1-binding site, in both the presence and absence of Keap1, it has been demonstrated that Nrf2 is subject to ubiquitination and proteasomal degradation independent of both Keap1 and the redox environment of the cell. In oxidatively stressed cells, this is the sole mechanism responsible for Nrf2 degradation. However, under homeostatic conditions Nrf2 is subject to a substantially more rapid mode of proteasomal degradation than it is in oxidatively stressed cells, and this rapid turnover of Nrf2 requires it to interact with Keap1. Within Nrf2, the N-terminal Neh2 domain is identified as the redox-sensitive degron. These data suggest that Keap1 negatively regulates Nrf2 by both enhancing its rate of proteasomal degradation and altering its subcellular distribution (McMahon, 2003).

The Nrf2 transcription factor is more rapidly turned over in cells grown under homeostatic conditions than in those experiencing oxidative stress. The variable turnover of Nrf2 is accomplished through the use of at least two degrons (discrete regions of primary sequence regulating degradation) and its redox-sensitive interaction with the Kelch-repeat protein Keap1. In homeostatic COS1 cells, the Neh2 degron confers on Nrf2 a half-life of less than 10 min. Analyses of deletion mutants of a Gal4(HA)mNeh2 fusion protein and full-length mNrf2 indicate that full redox-sensitive Neh2 destabilizing activity depends upon two separate sequences within this N-terminal domain. The DIDLID element (amino acids 17-32) is indispensable for Neh2 activity and appears necessary to recruit a ubiquitin ligase to the fusion protein. A second motif within Neh2, the ETGE tetrapeptide (amino acids 79-82), allows the redox-sensitive recruitment of Nrf2 to Keap1. This interaction, which occurs only in homeostatic cells, enhances the capacity of the Neh2 degron to direct degradation by functioning downstream of ubiquitination mediated by the DIDLID element. By contrast with the situation under homeostatic conditions, the Neh2 degron is neither necessary nor sufficient to account for the characteristic half-life of Nrf2 in oxidatively stressed cells. Instead, the previously uncharacterized, redox-insensitive Neh6 degron (amino acids 329-379) is essential to ensure that the transcription factor is still appropriately turned over in stressed cells, albeit with an increased half-life of 40 min. A model can now be proposed to explain how the turnover of this protein adapts in response to alterations in cellular redox state (McMahon, 2004).

Early developmental roles of Cap'n'collar homologs

Gastrulation in mammalian development is initiated at the posterior of the embryo at the border between embryonic epiblast (ectoderm), visceral endoderm, and extraembryonic ectoderm. At 6.5 days of development, a small patch of columnar epithelial cells at the posterior rim of the cup-shaped epiblast delaminates and moves into the region between the ectoderm and the endoderm. This structural change is propagated anteriorly and laterally to form a new structure designated the primitive streak. The streak contains a population of newly formed mesodermal cells that expands and migrates to produce a distinct third layer of tissue, the mesoderm. Previously, no transcription factor directly involved in mesoderm formation has been described. Brachyury (Drosophila homolog: T-related gene) is expressed in primitive mesoderm, however early mesoderm formation and gastrulation occur normally in Brachyury null mutants. Hepatocyte nuclear factors 4 (HNF4) (Drosophila homolog HNF4) and HNF3ß are also transcription factors expressed in the embryo at 6.5 days of development, but null mutations in these genes result in embryonic death later in development. HNF4 is required for the completion of gastrulation. The hnf4 gene is expressed in primitive endoderm. A null mutation in hnf3ß gene inhibits node and notochord formation. In this case, mesoderm is formed but is not correctly patterned. Mutations in the transcription factor genes Lim1, Otx2, and mammalian twist all affect development of specific subpopulations of mesoderm later in development (Farmer, 1997 and references).

LCR-F1 is a mammalian bZIP transcription factor containing a basic amino acid domain highly homologous to a domain in the Drosophila Cap'n'collar and Caenorhabditis elegans SKN-1 proteins. LCR-F1 contains a 30-amino-acid basic domain that is 70% homologous to CNC and 65% homologous to a domain in the C. elegans SKN-1 protein. SKN is involved in specifying mesodermal precursors that produce predominately pharynx and body wall muscle. LCR-F1 binds to AP1-like sequences in the human ß-globin locus control region and activates high-level expression of ß-globin genes. To assess the role of LCR-F1 in mammalian development, the mouse Lcrf1 gene was deleted in embryonic stem (ES) cells, and mice derived from these cells were mated to produce Lcrf1 null animals. Homozygous mutant embryos progress normally to the late egg cylinder stage at ~6.5 days of development, but development is arrested before 7.5 dpc. Lcrf1 mutant embryos failed to form a primitive streak and lack detectable mesoderm. These results demonstrate that LCR-F1 is essential for gastrulation in the mouse and suggest that this transcription factor controls expression of genes critical for the earliest events in mesoderm formation. Interestingly, Lcrf1 null ES cells injected into wild-type blastocysts contribute to all mesodermally derived tissues examined, including erythroid cells producing hemoglobin. These results demonstrate that the Lcrf1 mutation is not cell autonomous (mutant embryonic stem cells can be rescued by wild type cells) and suggest that LCR-F1 regulates expression of signaling molecules essential for gastrulation. The synthesis of normal hemoglobin levels in erythroid cells of chimeras derived from Lcrf1 null cells suggests that LCR-F1 is not essential for globin gene expression. LCR-F1 and the related bZIP transcription factors NF-E2 p45 and NRF2 must compensate for each other in globin gene regulation. One possible target for LCR-F1 regulation in early development is the nodal gene that encodes a TGF-ß-like factor secreted by ectodermal cells at the egg cylinder stage. The Bmp4 (Drosophila homolog DPP) gene is also a potential target. Alternatively, LCR-F1 may regulate a novel factor that functions alone or in conjunction with BMP4 and NODAL to induce mesoderm (Farmer, 1997).

NF-E2-related factor 3 (Nrf3) is a recently identified member of a family of transcription factors homologous to the Drosophila 'Cap 'n' collar' protein. The cnc gene is located immediately 3' to the Drosophila homeobox gene cluster and has been shown to regulate at least one of those genes, Deformed. Likewise, human and mouse CNC homologues are located immediately 3' to each of the four Hox complexes, although no genetic interactions have yet been demonstrated in vertebrates. Nrf3, adjacent to the Hox A cluster, is expressed during early development of the chicken embryo. Expression begins in the presumptive heart myocardium from the time of cardiac tube fusion through the looping process. Nrf3 transcripts then disappear from the heart and are next observed in the myotomal compartment of maturing somites, restricted to the medial portion along the rostrocaudal axis and fading after muscle precursors migrate. Central nervous system expression appears gradually and persists at low levels in ventricular neuroepithelial cells until at least embryonic day 6. Strong expression is observed in the early epiphysis, in the collecting ducts of the developing kidney and in individual cells of the yolk sac, underlying blood islands. This is the first description using in situ hybridization of the expression of a CNC family member and its dynamics through the course of early development (Etchevers, 2005).

Cap'n'collar homologs and retinal development

not really finished (nrf), a larval-lethal mutation in zebrafish generated by retroviral insertion, causes specific retinal defects. Analysis of mutant retinae reveals an extensive loss of photoreceptors and their precursors around the onset of visual function. These neurons undergo apoptosis during differentiation: all classes of photoreceptors are affected, suggesting an essential nrf function in the development of all types of photoreceptors. In the mutant, some photoreceptors escape cell death, are functional and, as judged by opsin expression, belong to at least three classes of cones and one class of rods. The protein encoded by nrf is a close homolog of human Nuclear Respiratory Factor 1 and avian Initiation Binding Repressor, transcriptional regulators binding the upstream consensus sequence RCGCRYGCGY. After 24 hours of development, prior to neuronal differentiation, nrf is expressed ubiquitously throughout the developing retina and central nervous system. At 48 hours, expression of nrf is detected in the ganglion cell layer, in the neurons of the inner nuclear layer, and in the optic nerve and optic tracts. At 72 hours nrf is no longer detectable by in situ hybridization. Mutants contain no detectable nrf mRNA and die within 2 weeks postfertilization, as larvae with reduced brain size. On the basis of its similarity with NRF-1 and IBR, nrf is likely involved in transcriptional regulation of multiple target genes, including those that encode mitochondrial proteins, growth factor receptors and other transcription factors. This demonstrates the power of insertional mutagenesis as a means for characterizing novel genes necessary for vertebrate retinal development (Becker, 1998).

Cap'n'collar homologs and cancer

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. Mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibit steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 show oxidative stress, and gene expression analysis shows decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer (Xu, 2005).

Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway

Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, a structural and functional analysis was performed on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, these mutations were modeled in Drosophila Lamin C, and the mutant lamins were expressed in muscle. The structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of the Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis (Dialynas, 2015).

return to Evolutionary Homologs part 1/2

cap'n'collar: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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