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Schering-Plough Research Institute, Kenilworth, New Jersey 07033
Received 30 June 2005/ Returned for modification 25 July 2005/ Accepted 25 September 2005
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ABSTRACT |
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INTRODUCTION |
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Nrf2 binds to antioxidant response elements (AREs) in the promoters of its target genes to activate transcription. The ARE has the core sequence 5'-TGACNNNGC-3' and responds to a wide variety of chemicals with potential to cause oxidative damage (27). The ARE and Nrf2 also drive the constitutive expression of many antioxidant and phase II drug-metabolizing enzymes presumably providing a level of protection required for normal cellular activities (7, 17, 27).
Based upon the homology of cross-species orthologues, Nrf2 has been divided into six domains, Neh1 to Neh6 (11) (Fig. 1A). Neh1 is located in the C-terminal half of the molecule and constitutes the basic DNA binding domain and the leucine zipper for dimerization. The Neh2 domain is located in the proximal N terminus and represents the region through which the repressor protein Keap1 recognizes and targets Nrf2 to a cullin 3 (cul3)-based E3 ubiquitin ligase for ubiquitylation and subsequent degradation by the 26S proteasome (5, 8, 11, 15, 37). Electrophiles disrupt the interaction between Keap1 and the Neh2 domain either by inducing phosphorylation of Nrf2 or by directly modifying cysteine residues in Keap1 (9, 33). Alternatively, it has also been reported that Keap1 may remain bound to the Neh2 domain during oxidative stress but cul3 is displaced from the complex (37). Either way, the result is that Nrf2 is stabilized, leading to an enhanced transcriptional response. Adjacent to Neh2 are the Neh4 and Neh5 regions, which were identified by deletion analysis as transcriptional activation domains (TADs). Although the overall mechanism by which Nrf2 transactivates its target genes is not well understood, it appears to be partly reliant on the abilities of the Neh4 and Neh5 domains to cooperatively recruit CREB binding protein (CBP) to ARE-regulated genes (13). Within the central part of Nrf2 lies the Neh6 domain, which has been shown to function as a degron, mediating the destabilization of the molecule only under conditions of oxidative stress (19).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid constructs.
Plasmids encoding full-length Nrf2 or Nrf2CT
16 tagged with the HA epitope at their N termini were generated by inserting the PCR-amplified rat cDNA into pHM6 (Roche Applied Science). To generate expression constructs containing HA epitope-tagged alanine substitution mutants of Nrf2, site-directed mutagenesis was performed using the QuikChange kit (Stratagene) with pHM6 containing the full-length rat Nrf2 cDNA as a template. For generation of 293-FlpIn TRex cell lines, Nrf2 or Nrf2CT16 tagged with the HA epitope was PCR amplified and cloned into pcDNA5/FRT/TO (Invitrogen). MafK and Keap1 expression plasmids, along with ARE-CAT and ARE-luciferase reporter constructs, were described previously (9, 21, 24, 27). Gal4-DNA binding domain Nrf2 fusion constructs were obtained by inserting the relevant PCR-amplified Nrf2 cDNA into pGBKT7 (BD Biosciences). The V5-tagged CHD6 expression construct was generated by amplifying a short fragment of the corresponding cDNA (548 to 1,153 bp) and cloning it into pCDNA3.1/V5-His (Invitrogen). All plasmid constructs were verified by DNA sequencing.
Cell culture.
Q293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, both of which were obtained from Invitrogen. 293/HA-Nrf2, 293/HA-Nrf2CT16, and 293/CAT were generated by cotransfecting pcDNA5/FRT/TO containing cDNAs encoding HA-Nrf2, HA-Nrf2CT16, or CAT with pOG44 (Invitrogen), which encodes Flp recombinase, into the parental FlpIn TREx 293 cell line (Invitrogen). The parental cells contain a single Flp recombinase target (FRT) site located at a transcriptionally active genomic locus to which a gene of interest can be targeted. Stable transfectants were selected and maintained in Dulbecco's modified Eagle's medium containing 10% tetracycline-reduced fetal bovine serum (BD Biosciences), 15 µg/ml blasticidin, and 150 µg/ml hygromycin B. Transfections were performed using Lipofectamine 2000 reagent (Invitrogen) as specified by the manufacturer.
Immunocytochemistry. Q293 cells were seeded on coverslips in six-well dishes and transfected as described above. Forty-eight hours posttransfection, the cells were fixed with formaldehyde (3.7%; 10 min), permeabilized with Triton X-100 (1%; 5 min), and then blocked with 0.1% (wt/vol) bovine serum albumin in phosphate-buffered saline for 20 min. The cells were subsequently incubated with anti-HA for 1 h and at the same time treated with RNase A. After being washed, the cells were incubated with Alexafluor 488-conjugated secondary antibodies (Invitrogen) in the presence of propidium iodide for 1 h. After being washed, coverslips were air dried and mounted using Prolong Gold antifade reagent (Invitrogen). Fluorescent images were obtained by confocal laser scanning microscopy (DM IRBE; Leica).
Immunoprecipitation and immunoblot analysis. Cells were cultured and transfected as described above. For immunoprecipitation, cells were lysed in immunoprecipitation (IP) buffer (1% NP-40, 150 mM NaCl, 50 mM Tris, pH 7.5) for 30 min on ice. Following clearing of lysates, 10% of the total volume was retained as input, and then the specified antibody was added and reaction mixtures were incubated at 4°C overnight with rotation. IPs were washed three times with IP buffer before complexes were eluted with sodium dodecyl sulfate (SDS) sample buffer. IPs using in vitro-translated proteins were conducted as described previously (9). For immunoblot analysis, cells were lysed directly in SDS sample buffer and resolved through 10% NuPAGE gels (Invitrogen). Western blotting was performed using standard procedures.
TaqMan analyses. Total RNA was isolated using Trizol reagent (Invitrogen) and then reverse transcribed using Superscript-RT (Invitrogen) and random hexamers (Promega). The relative levels of NQO1 and GCLM mRNAs were determined using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The following probe-primer sets were used: for NQO1, the forward primer was 5'-GCTGCCATGTATGACAAAGG-3', the reverse primer was 5'-CACTCTGAATTGGCCAGAGA-3', and the probe was 5'-[6-carboxyfluorescein (FAM)]-CCACTGGTGGCAGTGGCTCC-[6-carboxytetramethylrhoda mine (TAMRA)]-3'; for GCLM, the forward primer was 5'-GCGAGGAGCTTCATGATTG-3', the reverse primer was 5'-TGGAAACTCCCTGACCAAAT-3', and the probe was 5'-[FAM]-TGAATGGAGTTCCCAAATCAACCCA-[TAMRA]-3'; for CHD6, the forward primer was 5'-TCAAACAGCACTGCAACAAA-3', the reverse primer was 5'-TAGTCGATGTCAGGCAGAGG-3', and the probe was 5'-[FAM]-CATCCAGCTCCCTGGCAGGA-[TAMRA]-3'. The primer-probe set used for detection of Nrf2 has been described previously (22). A primer-probe set for 23hbp was obtained from Applied Biosystems and used as an internal control.
Gel shifts.
Gel shifts were performed essentially as described previously (20, 21) using a 43-base-pair, double-stranded oligonucleotide containing the ARE sequence of the rat NQO1 gene as a probe. The oligonucleotides were labeled at the 5' end using [
-32P]ATP and T4 polynucleotide kinase. In vitro-translated proteins were generated using the TNT-T7 coupled wheat germ extract system (Promega). In competition experiments, unlabeled DNA at a 10-, 20-, or 50-fold molar excess was preincubated with protein mixtures for 20 min at 25°C prior to the addition of the labeled probe. Reactions were fractionated using a 6% nondenaturing polyacrylamide gel in Tris-borate-EDTA.
Yeast two-hybrid screening.
A human liver Gal4 activation domain cDNA library (BD Biosciences) was screened with DBD-Nrf2 as specified by the manufacturer. Positive interactions were screened for on synthetic dropout (SD)-Leu-Trp-His-Ade-5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-
-Gal) plates, and cDNAs were isolated using the BD Biosciences Yeastmaker yeast plasmid isolation kit. In mating experiments, yeast bearing Gal4-activation domain and Gal4-DNA binding domain plasmids were grown overnight together in yeast extract-peptone-dextrose-adenine. Diploid yeasts were selected on SD-Leu-Trp plates and subsequently streaked onto SD-Leu-Trp-His-Ade-X--Gal plates to detect interacting proteins.
RNA interference (RNAi). HeLa cells were seeded in six-well plates at a density of 2.1 x 105 cells/well. After allowing the cells to attach for 2 h, double-stranded RNAs corresponding to CHD6, lamin A/C, or a nonspecific control (all from Dharmacon) were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) as specified by the manufacturer. After 48 h of incubation, the cells were treated with either 0.1% (vol/vol) dimethyl sulfoxide (DMSO) or 50 µM t-BHQ and incubated for a further 24 h prior to being harvested.
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RESULTS |
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16) (Fig. 1B) and tested the ability of the mutant protein to activate transcription. We reasoned that since Neh1 and Neh3 are contiguous, this small deletion at the proximal C terminus should cause minimum disruption to the dimerization and DNA binding activities of Nrf2. Human Q293 cells were cotransfected with increasing amounts of plasmids encoding either HA-tagged Nrf2CT16 or full-length Nrf2 and either the GSTA2 ARE-CAT or nqo1 ARE-luciferase reporter gene. As expected, wild-type Nrf2 increased reporter gene activity in a dose-dependent manner (Fig. 1C). Remarkably, even though the previously defined transactivation domains (Neh4 and Neh5) were still intact, Nrf2CT16 failed to activate transcription of either ARE-dependent reporter gene (Fig. 1C). The mutation had no effect on the expression levels of Nrf2 (Fig. 1D).
Because Nrf2CT16 fails to activate gene transcription, we investigated whether its expression might have a dominant-negative effect on wild-type Nrf2-driven gene expression. We cotransfected cells with expression plasmids for both proteins and determined ARE-dependent reporter gene activity. With a fixed amount of full-length Nrf2 plasmid present, increasing amounts of Nrf2CT16 vector in the transfection led to a dose-dependent decrease in reporter gene activity (Fig. 1E). This was true of both the GSTA2 and nqo1 reporter constructs. These results indicate that Nrf2CT16 can effectively compete with wild-type Nrf2 for binding to the ARE.
Nrf2CT16 heterodimerizes with MafK and binds the ARE in vitro.
To confirm that Nrf2CT16 retains its ability to bind to DNA, we first assessed whether Nrf2CT16 could dimerize with MafK. We cotransfected cells with plasmids for HA epitope-tagged Nrf2, Nrf2CT16, or Nrf2 with a deleted bZIP region (Nrf2bZIP) and V5-tagged MafK. Following harvesting, whole-cell lysates were incubated with immobilized V5 antibodies, and the IPs were analyzed by Western blotting. The results of this analysis show that full-length Nrf2 and Nrf2CT16 specifically associate with MafK, whereas Nrf2bZIP does not (Fig. 2A). Blotting of input samples with either HA or V5 antibodies revealed that all of the proteins were expressed in the expected cells and at similar levels (Fig. 2A). This experiment was repeated using in vitro-translated proteins with identical results (Fig. 2B).
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16 could bind DNA using gel shift assays. Incubation of in vitro-translated Nrf2, Nrf2CT16, or MafK alone with the radiolabeled ARE probe did not produce a detectable shifted complex. However, heterodimers of Nrf2-MafK or Nrf2CT16-MafK interacted specifically with the ARE, producing two shifted bands, as previously reported (21) (Fig. 2C). Importantly, no gross differences in band intensity could be observed between the wild-type Nrf2-MafK and Nrf2CT16-MafK complexes. To gain some insight into the affinity of the two complexes for the ARE, competition assays were conducted. When a 10-, 20-, or 50-fold molar excess of unlabeled probe was added to the binding reaction, a dose-dependent decrease in band intensity that was comparable between Nrf2-MafK and Nrf2CT16-MafK complexes was observed (Fig. 2D). Quantitative analysis of band intensities by densitometry showed that they were very similar across the range of competitor concentrations used (Fig. 2E). The Nrf2-MafK and Nrf2CT16-MafK complexes were not competed when a probe containing a mutated ARE was used (Fig. 2F). These findings indicate that Nrf2CT16 binds to the ARE with affinity similar to that of wild-type Nrf2.
Nrf2CT16 localizes to the nucleus.
The fact that Nrf2CT16 acts as a dominant-negative mutant implies that, like wild-type Nrf2, the protein is targeted to the nucleus. To confirm this possibility, the localizations of Nrf2CT16 and wild-type Nrf2 were examined by immunocytochemistry and confocal microscopy. Plasmids encoding either HA-tagged wild-type Nrf2 or Nrf2CT16 were transiently transfected into Q293 cells and, following fixation, their distribution was visualized using rat anti-HA primary and goat anti-rat immunoglobulin G Alexa 488 secondary antibodies. Strong nuclear fluorescence was observed in cells overexpressing either wild-type or mutant proteins, indicating that Nrf2 is a nuclear protein, consistent with our previous observation (23), and deleting the last 16 amino acids has no effect on its subcellular localization (Fig. 3).
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16 is unable to activate endogenous ARE-regulated genes.
The above data clearly show that although the DNA binding activity of Nrf2CT16 is not compromised, the protein is inactive in reporter gene assays. To put these results in a more physiologically relevant context, we investigated the ability of Nrf2CT16 to activate endogenous ARE-driven gene expression. We established cell lines in which plasmids encoding HA-Nrf2 or HA-Nrf2CT16, under the control of a tetracycline-sensitive promoter, were integrated into single identical genomic loci using FlpIn technology. A time course of Dox treatment showed that expression of HA-Nrf2 and HA-Nrf2CT16 was rapidly induced with identical kinetics, and the maximal expression levels (achieved after only 4 h) were the same for both proteins across the period examined (Fig. 4A). Next, we examined the endogenous expression of two well-characterized ARE-driven Nrf2 target genes (NQO1 and GCLM) in these cell lines, as well as in a control line (293/CAT) in which the chloramphenicol acetyltransferase (CAT) gene was integrated into the FRT site. Taqman analyses demonstrated that Dox treatment resulted in a rapid and sustained increase in NQO1 mRNA expression across the time course in 293/HA-Nrf2 cells (Fig. 4B). By contrast, no induction was observed in 293/HA-Nrf2CT16 or 293/CAT cells (Fig. 4B). Similarly, GCLM expression was also significantly increased by Dox treatment of 293/HA-Nrf2 cells, while no induction was observed in 293/HA-Nrf2CT16 or 293/CAT cells (Fig. 4B). Interestingly, the increase in GCLM mRNA in 293/HA-Nrf2 cells was not sustained across the time course and actually returned to basal levels at the 48-h time point. This is in contrast to the effects observed on NQO1 mRNA, where the level continued to rise throughout the time course. Thus, as yet uncharacterized mechanisms function to limit the ability of Nrf2 to continuously activate certain ARE-driven genes.
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16 to activate transcription is not solely a phenomenon observed for reporter genes in transient transfections, but that it is also transcriptionally silent on endogenous ARE-driven genes.
Nrf2CT16 is more stable than wild-type Nrf2 and is regulated normally by Keap1.
Although there were no gross differences in the steady-state levels of wild-type Nrf2 or Nrf2CT16, we also determined the half-lives (t1/2) of these proteins. Cells transiently expressing HA-tagged Nrf2 or Nrf2CT16 were treated with cycloheximide for 0, 15, 30, 45, 60, or 120 min before being harvested. Using immunoblot analysis, we found that the truncated protein appears to be more stable than wild-type Nrf2 (t1/2 of wild-type Nrf2 = 25 min; t1/2 of Nrf2CT16 = 45 min) (Fig. 5A and B). These findings suggest that the inability of Nrf2CT16 to activate transcription is intrinsically related to its transcriptional activity rather than its stability.
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16 can still be regulated by Keap1. Cotransfection of cells with plasmids encoding either wild-type Nrf2 or Nrf2CT16 with Keap1 led to a significant decrease in the steady-state level of the Nrf2 proteins (Fig. 5C), which was reflected in a decrease in their half-lives (data not shown). Treatment of cells with either t-BHQ or the proteosome inhibitor MG132 completely reversed the effects of Keap1; in the case of MG132, a ladder of higher-molecular-weight complexes was also observed, most probably representing ubiquitylated forms of Nrf2 and Nrf2CT16 (Fig. 5C).
Identification of residues critical to the activity of Neh3.
The fact that Nrf2CT16 binds DNA normally and acts as a dominant-negative mutant, effectively blocking the activity of wild-type Nrf2, implies that the final 16 amino acids of Nrf2 must have a defined role in activation of transcription. To clarify the region within the last 16 amino acids that is critical to its transcriptional activity, we constructed expression plasmids for full-length Nrf2 mutants in which sequential blocks of three residues (four in the case of M5) were mutated to alanine (M1 to M5) (Fig. 6A). The functional activities of these mutant Nrf2 proteins were examined by determining the effects of their overexpression on an ARE reporter gene. The results of these experiments indicate that the N-terminal part of this region appears to be the most critical, since the Nrf2 mutants M1 and M2 recapitulated the effects of the 16-amino-acid deletion (Fig. 6B). Mutants M3 and M5 showed partial loss of activity, although they still induced gene expression three- and fourfold, respectively, whereas M4 was as effective as wild-type Nrf2 (Fig. 6B). All mutants were expressed at levels similar to wild-type Nrf2 (Fig. 6C).
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CHD6 interacts with the Neh3 domain of Nrf2, and the VFLVPK motif is critical for this association.
Since our results indicate that the Neh3 domain is not required for DNA binding activity, we explored whether it may be involved in interacting with components of the transcriptional machinery. To assess this possibility, we used DBD-Nrf2, which contains the Gal4 DNA binding domain fused in frame to the Neh1 and -3 domains of Nrf2 (Fig. 7A), as bait in a yeast two-hybrid screen of a human liver cDNA library. We obtained multiple clones, many of which contain sequence encoding a protein identified as CHD6. CHD6 is a member of the chromo-ATPase/helicase DNA binding protein family and contains two tandem chromodomains and a DNA-helicase domain (28), making it a plausible candidate for an Neh3-interacting protein. To further assess this possibility, we created mutants of the DBD-Nrf2 construct in which the final 16 amino acids were deleted (DBD-CT16) or the M1, M2, and M4 mutations depicted in Fig. 6 were introduced (Fig. 7A). Subsequently yeast cells bearing the mutated plasmids were mated with yeast containing the Gal4-activation domain-CHD6 (AD-CHD6 [Fig. 7A]) construct. Strong growth was observed only for DBD-Nrf2/AD-CHD6 and DBD-M4/AD-CHD6 diploid yeast (Fig. 7B), which is consistent with the observation that the M4 mutant remains as transcriptionally active as wild-type Nrf2 (Fig. 6B). All other mutants failed to interact with AD-CHD6, which correlated perfectly with the loss of transcriptional activity associated with these deletions/substitutions (Fig. 7B and 6B). Thus, the loss of transcriptional activity associated with mutation of the VFLVPK motif correlates with loss of interaction with AD-CHD6.
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16, HA-Nrf2M1, HA-Nrf2M2, or HA-Nrf2M4. Following harvesting, lysates were incubated with immobilized HA antibodies, and the IPs were analyzed by Western blotting. The results of this experiment demonstrate that full-length Nrf2 and Nrf2M4 associate with CHD6, whereas Nrf2CT16, Nrf2M1, and Nrf2M2 interact much more weakly with the helicase (Fig. 8A), supporting the findings of our yeast two-hybrid experiment.
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These data indicate that the specific interaction between CHD6 and the Neh3 domain appears to be important for Nrf2 activity and suggest that Neh3 may be involved in recruiting accessory proteins or serve to bridge the transcription factor to the active transcription apparatus. The precise mechanisms whereby the interaction between Neh3 and CHD6 leads to gene transcription are not understood and remain to be explored.
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DISCUSSION |
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In this report, we present evidence demonstrating that the Neh3 domain at the C terminus of Nrf2 harbors a critical activity required for activation of transcription and may function as a TAD, along with the Neh4 and Neh5 domains. This conclusion is supported by our finding that deletion of the last 16 amino acids from Nrf2 results in a protein that is transcriptionally inactive. Although the truncated protein failed to activate ARE-dependent gene expression, its nuclear localization, as well as its ability to bind to the ARE with high affinity, are indistinguishable from those of wild-type Nrf2, suggesting that its inability to activate gene transcription is due to a defect in the transactivational activity. This is consistent with the observation that it can block wild-type Nrf2 from activating gene transcription and thus effectively act as a dominant-negative form of the transcription factor. Furthermore, although the truncated protein appears to be slightly more stable than wild-type Nrf2, it is efficiently targeted for degradation by Keap1 and is stabilized by either t-BHQ or MG132. These findings are consistent with the fact that Nrf2 is a modular protein comprising multiple functional domains that act independently of each other. These data provide strong evidence suggesting that the Neh3 domain may act as a TAD that is distinct and in addition to the TAD found in the Neh4-Neh5 domains; furthermore, it is absolutely required for Nrf2 to activate expression of its target genes.
Thus, the transcriptional activity of Nrf2 appears to be dependent on three separate domains that act in concert to recruit the full complement of proteins required to form an active transcriptional complex. This is supported by the fact that deletion of the TADs within the Neh4 and Neh5 domains also inactivates Nrf2, despite the presence of the Neh3 domain (13). We also noted that a Gal4-Neh3 fusion is unable to activate Gal4-dependent transcription in our yeast two-hybrid assay. The Neh3 domain and its interacting factors may not be capable of activating transcription in isolation but rather represent essential components of a larger multiprotein transcription complex that is formed on ARE-driven genes. These findings are reminiscent of p53, where deleting its N-terminal activation domain renders the protein unable to activate several of its target genes, even though the protein can still bind to the p68 coactivator protein (2, 4, 35).
Although the Neh4 and Neh5 domains have not been fully characterized, their activities are thought to be due in part to their cooperative binding of CBP (13). Using purified proteins, we have found that Nrf2CT16 interacts with p300 as competently as wild-type Nrf2 (data not shown). This finding suggests first that the structures of Neh4 and Neh5 are not perturbed by the deletion and secondly that Neh3 functions through a mechanism distinct from those of the other TADs. Furthermore, if CBP/p300 is still recruited to DNA by Nrf2CT16, then this would indicate that CBP/p300 alone is insufficient for transactivation of at least some Nrf2-dependent genes, supporting the hypothesis that the recruitment of multiple factors is required.
The activity of the Neh3 domain is reliant upon the VFLVPK motif, which is predominantly hydrophobic in nature and could be a site of interaction with a novel partner molecule. The critical F591 and L592 residues would most likely form essential interactions within such a protein complex. Notably, the VFLVPK motif is highly conserved between a number of CNC family members, including the drosophila CNC protein (Fig. 9), and likely represents a fundamental means of activating transcription that has been conserved throughout the evolution of the CNC-bZIP family. Sequences N terminal to the VFLVPK motif are also very highly conserved (Fig. 9), and this is consistent with our unpublished observations indicating that these regions are also critical for the activity of Nrf2.
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16. All of the available evidence demonstrates that Nrf2CT16 binds DNA normally; however, this could not be unequivocally confirmed in vivo. CHD6 is particularly interesting, since it is characterized by the presence of two tandem chromodomains and a DNA helicase domain, but its function is not well understood (28). However, the founding member of the CHD family, CHD1, is important for the expression of certain genes and associates with actively transcribed chromatin in both yeast and mammalian cells (29, 30, 31). CHD1 has been implicated in several processes, which may explain its importance in gene expression. First, CHD1 may be involved in transcription elongation, since it has been shown to associate with several factors involved in this process and can be localized to the coding regions of certain genes (29). Secondly, CHD1 can alter nucleosome structure to produce regions of DNase I hypersensitivity in a test substrate, possibly providing access to DNA for additional DNA-interacting factors (31). Finally, one of the chromodomains of CHD1 specifically interacts with methylated lysine 4 in histone H3, a modification that is associated with transcriptional activity, and by doing so recruits enzymes with histone acetyltransferase activity to chromatin (25). Importantly, the activity of CHD1 is reliant upon both its helicase domain and chromodomains, both of which are highly conserved in CHD6 (14, 25, 29). Therefore, recruitment of CHD6 to Nrf2-dependent genes by the Neh3 domain has the potential to nucleate multiple activities associated with transcriptional activation. These data suggest that the specific interaction between CHD6 and the Neh3 domain may be important for Nrf2 activity. The precise role of CHD6 in ARE-driven gene expression remains to be explored.
In summary, as a modular protein, Nrf2 possesses a number of distinct functional domains the concerted actions of which are critical to the maintenance of cellular redox homeostasis. We have established that the Neh3 domain harbors a transactivation-like activity and functions in parallel with the Neh4 and Neh5 domains to activate transcription of Nrf2 target genes. The function of the Neh3 domain is reliant upon the VFLVPK motif, which is conserved across species and among other members of the CNC-bZIP family. The Neh3 domain may function as a protein-protein interaction module to facilitate the recruitment of putative coactivator proteins, such as CHD6, to Nrf2-dependent genes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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