cap'n'collar
SKN-1, a C. elegans Cap'n'collar homolog involved in establishment of cellular and embryonic polarity The autonomous or cell-intrinsic developmental properties of early embryonic blastomeres in
nematodes are thought to result from the action of maternally provided determinants. After the first
cleavage of the C. elegans embryo, only the posterior blastomere, P1, has a cell-intrinsic ability to
produce pharyngeal cells. The product of the maternal gene skn-1 is required for P1 to produce
pharyngeal cells. Skn-1 protein is nuclear localized and P1 appears to
accumulate markedly higher levels of Skn-1 protein than its sister, the AB blastomere. The distribution of Skn-1 protein was examined in embryos from mothers with maternal-effect mutations in
the genes mex-1, par-1, and pie-1. These results suggest that mex-1(+) and par-1(+) activities are
required for the unequal distribution of the Skn-1 protein and that pie-1(+) activity may function to
regulate the activity of Skn-1 protein in the descendants of the posterior blastomere P1 (Bowerman, 1993).
After fertilization in C. elegans, activities encoded by the maternally expressed par genes appear to
establish cellular and embryonic polarity. Loss-of-function mutations in the par genes disrupt
anterior-posterior (a-p) asymmetries in early embryos and result in highly abnormal patterns of cell
fate. Little is known about how the early asymmetry defects are related to the cell fate patterning
defects in par mutant embryos, or about how the par gene products affect the localization and activities
of developmental regulators known to specify the cell fate patterns made by individual blastomeres.
Examples of such regulators of blastomere identity include the maternal proteins MEX-3 and GLP-1,
expressed in the anterior at high levels, and SKN-1 and PAL-1, expressed in the posterior at high levels in early
embryos. To better define par gene functions, the expression patterns of MEX-3, PAL-1
and SKN-1 were analyzed, and mex-3, pal-1, skn-1 and glp-1 activities were analyzed in par mutant embryos. Mutational inactivation of each par gene results in a unique phenotype, but in no case is a complete loss of a-p asymmetry observed. It is concluded that no single par gene is required for all a-p
asymmetry and it is suggested that, in some cases, the par genes act independently of one another to
control cell fate patterning and polarity (Bowerman, 1997).
The SKN-1 transcription factor specifies early embryonic cell fates in Caenorhabditis elegans. SKN-1
binds DNA at high affinity as a monomer, by means of a basic region like those of basic-leucine zipper
(bZIP) proteins, which bind DNA only as dimers. How the SKN-1 DNA-binding
domain (the Skn domain) promotes stable binding of a basic region monomer to DNA was investigated. A flexible arm
at the Skn domain amino terminus binds in the minor groove, but a support segment adjacent to the
carboxy-terminal basic region can independently stabilize basic region-DNA binding. Off DNA, the
basic region and arm are unfolded and, surprisingly, the support segment forms a molten globule of four
alpha-helices. On binding DNA, the Skn domain adopts a tertiary structure in which the basic region
helix extends directly from a support segment alpha-helix, which is required for binding. The remainder
of the support segment anchors this uninterrupted helix on DNA, but leaves the basic region exposed in
the major groove. This is similar to the way in which the bZIP basic region extends from the leucine zipper,
indicating that positioning and cooperative stability provided by helix extension are conserved
mechanisms that promote binding of basic regions to DNA (Carroll, 1997).
Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early
in the development of Caenorhabditis elegans. This transcription factor contains a basic region, but it
binds to DNA as a monomer. Because other transcription factors containing basic regions bind as
dimers, this finding implies that Skn represents a new DNA recognition motif. It has been proposed
that the basic region helix of Skn is stabilized for binding by tertiary contacts to other parts of the
protein. This proposal was tested by carrying out circular dichroism (CD) and NMR experiments
on the Skn domain and five truncated proteins. The basic region of Skn is
shown to be unstructured in solution and does not contact other parts of the protein; like other basic region peptides,
it folds into a helix only on binding specifically to DNA. However, there is a stably folded helical
module in the Skn domain, and one of the helices in this module terminates immediately before the start
of the basic region. This pre-organized helix contains a surface rich in basic amino acids, and it is proposed
that this helix contacts the DNA distal to the basic region proper, providing an extra long
helical recognition surface that helps to stabilize monomeric binding. Homology between the Skn
domain and several basic-region leucine zipper (bZIP) domains raises the possibility that the affinity
and perhaps the specificity of DNA binding by bZIP proteins can be modulated by incorporating a
stably folded helical segment that contacts the DNA just below the basic region proper (Pal, 1997).
Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early in the development of Caenorhabditis elegans. It has been reported that the DNA-binding domain is a molten globule and that the structure cannot be defined because there are no long-range nuclear Overhauser effects (NOEs). Working with short Skn domain fragments and using 13C-labeled proteins, 28 long-range NOEs have been identified that establish a tertiary fold for the Skn domain. The internal region of the Skn domain consists of three stable helices and one conformationally labile helix organized into a nascent helix-turn-helix-turn-helix-turn-helix motif. The N and C termini of the Skn domain are unstructured and emerge from the same end of the folded domain. This structure is consistent with biochemical data on binding of the Skn domain to DNA that show that the N and C termini bind in the adjacent minor and major grooves from the same face of the DNA helix. The NMR solution structure of the Skn domain should be useful for developing a complete understanding of the DNA recognition event, including any conformational changes that take place upon binding (Lo, 1998).
In Caenorhabditis elegans, the predicted transcription factor SKN-1 is required
for embryonic endodermal and mesodermal specification and for maintaining
differentiated intestinal cells post-embryonically. The SKN-1 DNA-binding region
is related to the Cap'n'Collar (CNC) family of basic leucine zipper proteins,
but uniquely, SKN-1 binds DNA as a monomer. CNC proteins are absent in C.
elegans, however; and their involvement in the endoderm and mesoderm suggests
some functional parallels to SKN-1. Using a cell culture assay, it has been shown that
SKN-1 induces transcription and contains three potent activation domains. The
functional core of one domain is a short motif, the DIDLID element, which is
highly conserved in a subgroup of vertebrate CNC proteins. The DIDLID element is
important for SKN-1-driven transcription, suggesting a likely significance in
other CNC proteins. SKN-1 binds to and activates transcription through the
p300/cAMP-responsive element-binding protein-binding protein (CBP) coactivator,
supporting the genetic prediction that SKN-1 recruits the C. elegans p300/CBP
ortholog, CBP-1. The DIDLID element appears to act independently of p300/CBP,
however, suggesting a distinct conserved target. The evolutionarily preservation
of the DIDLID transcriptional element supports the model that SKN-1 and some CNC
proteins interact with analogous cofactors and may have preserved some similar
functions despite having divergent DNA-binding domains (Walker, 2000).
The evolutionarily conserved p38 mitogen-activated protein kinase (MAPK) cascade is an integral part of the response to a variety of environmental stresses. The C. elegans PMK-1 p38 MAPK pathway regulates the oxidative stress response via the CNC transcription factor SKN-1. In response to oxidative stress, PMK-1 phosphorylates SKN-1, leading to its accumulation in intestine nuclei, where SKN-1 activates transcription of gcs-1, a phase II detoxification enzyme gene. These results delineate the C. elegans p38 MAPK signaling pathway leading to the nucleus that responds to oxidative stress (Inoue, 2005).
Oxidative stress contributes to the etiology of various degenerative diseases such as ischemia and the process of aging. In vertebrates, a major mechanism of oxidative stress defense is orchestrated by the two NF-E2-related factors Nrf1 and Nrf2, which belong to the Cap-N-Collar (CNC) family of transcription factors. Nrf proteins induce expression of a battery of phase II detoxification enzymes. Nrf proteins accumulate in cell nuclei in response to oxidative stress. In the nematode Caenorhabditis elegans, the SKN-1 protein is required for oxidative stress resistance. SKN-1 is distantly related to the Nrf proteins and induces phase II detoxification gene transcription. Oxidative stress induces SKN-1 to accumulate in intestinal nuclei. This oxidative stress response thus appears to be widely conserved (Inoue, 2005).
TORC2 signaling antagonizes SKN-1 to induce C. elegans mesendodermal embryonic development
The evolutionarily conserved target of rapamycin (TOR) kinase controls fundamental metabolic processes to support cell and tissue growth. TOR functions within the context of two distinct complexes, TORC1 and TORC2. TORC2, with its specific component Rictor, has been recently implicated in aging and regulation of growth and metabolism. This study identified rict-1/Rictor (homolog of Drosophila Rictor) as a regulator of embryonic development in C. elegans. The transcription factor skn-1 establishes development of the mesendoderm in embryos, and is required for cellular homeostasis and longevity in adults. Loss of maternal skn-1 function leads to mis-specification of the mesendodermal precursor and failure to form intestine and pharynx. Genetic inactivation of rict-1 suppressed skn-1-associated lethality by restoring mesendodermal specification in skn-1 deficient embryos. Inactivation of other TORC2 but not TORC1 components also partially rescues skn-1 embryonic lethality. The SGK-1 kinase (homolog of the vertebate serum- and glucocorticoid-inducible kinase SGK) mediates these functions downstream of rict-1/TORC2, as a sgk-1 gain-of-function mutant suppresses the rict-1 mutant phenotype. These data indicate that TORC2 and SGK-1 antagonize SKN-1 during embryonic development (Ruf, 2013).
Reactive oxygen species (ROS) play important signaling roles in metazoans, but also cause significant molecular damage. Animals tightly control ROS levels using sophisticated defense mechanisms, yet the transcriptional pathways that induce ROS defense remain incompletely understood. In the nematode Caenorhabditis elegans, the transcription factor SKN-1 is considered a master regulator for detoxification and oxidative stress responses. This study shows that MDT-15, a subunit of the conserved Mediator complex, is also required for oxidative stress responses in nematodes. Specifically, mdt-15 is required to express SKN-1 targets upon chemical and genetic increase of SKN-1 activity. mdt-15 is also required to express genes in SKN-1-dependent and -independent fashions downstream of insulin/IGF-1 signaling and for the longevity of daf-2/insulin receptor mutants. At the molecular level, MDT-15 binds SKN-1 through a region distinct from the classical transcription factor binding KIX-domain. Moreover, mdt-15 is essential for the transcriptional response to and survival on the organic peroxide tert-butyl-hydroperoxide (tBOOH), a largely SKN-1-independent response. The MDT-15 interacting Nuclear Hormone Receptor, NHR-64, is specifically required for tBOOH but not arsenite resistance, but NHR-64 is dispensable for the transcriptional response to tBOOH. Hence NHR-64 and MDT-15's mode of action remain elusive. Lastly, the role of MDT-15 in oxidative stress defense is functionally separable from its function in fatty acid metabolism, as exogenous polyunsaturated fatty acid complementation rescues developmental, but not stress sensitivity phenotypes of mdt-15 worms. These findings reveal novel conserved players in the oxidative stress response and suggest a broad cytoprotective role for MDT-15 (Goh, 2913).
Dietary restriction (DR) has been shown to prolong longevity across diverse taxa, yet the mechanistic relationship between DR and longevity remains unclear. MicroRNAs (miRNAs) control aging-related functions such as metabolism and lifespan through regulation of genes in insulin signaling, mitochondrial respiration, and protein homeostasis. A network analysis was conducted of aging-associated miRNAs connected to transcription factors PHA-4/FOXA and SKN-1/Nrf (homologs of Drosophila Forkhead and Cap'n'collar respectively), which are both necessary for DR-induced lifespan extension in Caenorhabditis elegans. This network analysis has revealed extensive regulatory interactions between PHA-4, SKN-1, and miRNAs and points to two aging-associated miRNAs, miR-71 and miR-228, as key nodes of this network. This study shows that miR-71 and miR-228 are critical for the response to DR in C. elegans. DR induces the expression of miR-71 and miR-228, and the regulation of these miRNAs depends on PHA-4 and SKN-1. In turn, PHA-4 and SKN-1 were shown to be negatively regulated by miR-228, whereas miR-71 represses PHA-4. This study has discovered new links in an important pathway connecting DR to aging. By interacting with PHA-4 and SKN-1, miRNAs transduce the effect of dietary-restriction-mediated lifespan extension in C. elegans. Given the conservation of miRNAs, PHA-4, and SKN-1 across phylogeny, these interactions are likely to be conserved in more-complex species (Smith-Vikos, 2014).
The monophyly of Mandibulata (the division of arthropods uniting pancrustaceans and myriapods) is consistent with several morphological characters, such as the presence of sensory appendages called antennae and the eponymous biting appendage, the mandible. Functional studies have demonstrated that the patterning of the mandible requires the activity of the Hox gene Deformed and the transcription factor cap-n-collar (cnc) in at least two holometabolous insects: the fruit fly Drosophila melanogaster and the beetle Tribolium castaneum. Expression patterns of cnc from two non-holometabolous insects and a millipede have suggested conservation of the labral and mandibular domains within Mandibulata. However, the activity of cnc is unknown in crustaceans and chelicerates, precluding understanding of a complete scenario for the evolution of patterning of this appendage within arthropods. To redress these lacunae, this study investigated the gene expression of the ortholog of cnc in Parhyale hawaiensis, a malacostracan crustacean, and two chelicerates: the harvestman Phalangium opilio, and the scorpion Centruroides sculpturatus. In the crustacean P. hawaiensis, the segmental expression of Ph-cnc is the same as that reported previously in hexapods and myriapods, with two distinct head domains in the labrum and the mandibular segment. In contrast, Po-cnc and Cs-cnc expression is not enriched in the labrum of either chelicerate, but instead is expressed at comparable levels in all appendages. In further contrast to mandibulate orthologs, the expression domain of Po-cnc posterior to the labrum is not confined within the expression domain of Po-Dfd. Thus, expression data from two chelicerate outgroup taxa suggest that the signature two-domain head expression pattern of cnc evolved at the base of Mandibulata. The observation of the archetypal labral and mandibular segment domains in a crustacean exemplar supports the synapomorphic nature of mandibulate cnc expression. The broader expression of Po-cnc with respect to Po-Dfd in chelicerates further suggests that the regulation of cnc by Dfd was also acquired at the base of Mandibulata. To test this hypothesis, future studies examining panarthropod cnc evolution should investigate expression of the cnc ortholog in arthropod outgroups, such as Onychophora and Tardigrada (Sharma, 2014).
Cap'n'collar homologs regulation erythropoesis and the globin locus The NF-E2-binding sites or Maf recognition elements (MAREs) are essential cis-acting elements in the regulatory regions of erythroid-specific genes recognized by the erythroid transcription factor NF-E2, composed of p45 and MafK. Recently, two p45-related factors Nrf1 and Nrf2 were isolated, and they are now collectively grouped as the Cap'n'collar (CNC) family. CNC factors bind to MARE through heterodimer formation with small Maf proteins. A novel CNC factor, Nrf3, encoding a predicted 73-kDa protein with a basic region-leucine zipper domain highly homologous to those of other CNC proteins has been identifed and characterized. In vitro and in vivo analyses show that Nrf3 can heterodimerize with MafK and that this complex binds to the MARE in the chicken beta-globin enhancer and can activate transcription. Nrf3 mRNA is highly expressed in human placenta and B cell and monocyte lineage. Chromosomal localization of human Nrf3 is 7p14-15, which lies near the hoxA gene locus. Since the genetic loci of p45, nrf1, and nrf2 have been mapped close to those of hoxC, hoxB, and hoxD, respectively, the present study strongly argues for the idea that a single ancestral gene for the CNC family members may have been localized near the ancestral Hox cluster and have diverged to give rise to four closely related CNC factors through chromosome duplication (Kobayashi, 1999).
NF-E2 binding sites, located in distant regulatory sequences, may be important for high level alpha- and
beta-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little
or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part,
to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2-related factors,
Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for
Nrf-2, the Nrf-2 gene was disrupted by homologous recombination. Nrf-2-deficient mice have no
detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of
NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both
NF-E2 and Nrf-2 expressed normal levels of alpha- and beta-globin. Mature mice with combined
deficiency of NF-E2 and Nrf-2 do not exhibit a defect in erythroid maturation beyond that seen with
loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2-deficient mice is not the
result of compensation by Nrf-2 (Martin, 1998).
The CNC-basic leucine zipper (CNC-bZIP) family is a subfamily of bZIP proteins identified from
independent searches for factors that bind the AP-1-like cis-elements in the beta-globin locus control
region. Three members (p45-Nf-e2, Nrf-1 and Nrf-2) have been identified in mammals. Expression of
p45-Nf-e2 is largely restricted to hematopoietic cells while Nrf-1 and Nrf-2 are expressed in a wide
range of tissues. To determine the function of Nrf-1, a targeted disruption of the Nrf-1 gene was carried
out. Homozygous Nrf-1 mutant mice are anemic due to a non-cell autonomous defect in definitive
erythropoiesis: they die in utero (Chan, 1998).
Much of the understanding of the process by which enhancers activate transcription has been gained
from transient-transfection studies in which the DNA is not assembled with histones and other
chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural
chromatin context in vivo, a minichromosome was constructed containing the human epsilon-globin gene
and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are
maintained at stable copy number in human erythroid cells. Expression of the minichromosomal
epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the
chromosomal gene. The chromatin structure of the epsilon-globin gene was determined in both the
active and inactive states. The transcriptionally inactive locus is covered by an array of positioned
nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase
I-hypersensitive site 2 (HS2) that actively transcribe the epsilon-globin gene, the nucleosome at the
promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All
or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter
and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2
structure itself, depends on the presence of the NF-E2 binding motif in HS2. The nucleosome at the
promoter that is altered upon activation, is positioned over the transcriptional elements of the
epsilon-globin gene (i.e., the TATA, CCAAT, and CACCC elements) and the GATA-1 site at -165.
The simple availability of erythroid transcription factors that recognize these motifs is insufficient to
allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin
structure. These observations are consistent with the idea that one role of the beta-globin LCR is to
maintain promoters free of nucleosomes. The restricted structural change observed upon
transcriptional activation may indicate that the LCR need only make a specific contact with the
proximal gene promoter to activate transcription. It is concluded that NF-E2 plays an essential role in the remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5'
hypersensitive site 2 of the beta-globin locus control region (Gong, 1996).
The erythroid transcription factor NF-E2 is an obligate heterodimer composed of two different subunits
(p45 and p18), each containing a basic region-leucine zipper DNA binding domain. NF-E2 plays a critical
role in erythroid differentiation as an enhancer-binding protein for expression of the beta-globin gene.
Dimethyl sulfoxide treatment of wild-type murine erythroleukemia cells (but not a
mutant clone of dimethyl sulfoxide-resistant cells) significantly increases NF-E2 activity, which involves
both up-regulation of DNA binding and transactivation activities. Both activities are reduced
markedly by treatment of cells with 2-aminopurine but not by genistein. Activation of the
Ras-Raf-MAP kinase signaling cascade significantly increases NF-E2 activity, but this is suppressed
when MafK is overexpressed. Domain analysis reveals an activation domain in the NH2-terminal
region of p45 and a suppression domain in the basic region-leucine zipper of MafK. These findings
indicate that induction of NF-E2 activity is essential for erythroid differentiation of murine
erythroleukemia cells and that serine/threonine phosphorylation may be involved in this process. These data also suggest that a MafK homodimer can suppress transcription, not only by competition for the
DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active
transcription repressor (Nagai, 1998).
Regulated expression of genes in the beta-globin cluster depends on sequences located between 5
and 20 kb upstream of the epsilon gene, known as the locus control region (LCR). beta-Globin
expression in murine erythroleukemia (MEL) cells depends on NF-E2, a transcription factor that
binds to enhancer sequences in the LCR. To gain insight into the mechanism of globin gene activation
by NF-E2, an NF-E2 null MEL cell line was used to map regions of NF-E2 required for beta-globin
expression. Within the transactivation domain, two discrete proline-rich regions are required for
rescue of beta-globin expression. The first is located at the N-terminus of NF-E2, while the second
was located N-terminal to the cap 'n collar (CNC) domain. Other proline-rich sequences are
dispensable, indicating that proline content per se does not determine NF-E2 activity. Mutations within
the conserved CNC domain markedly diminish rescue of beta-globin expression. This domain is
required, in addition to the basic leucine zipper domain, for DNA binding activity. The requirement for
discrete proline-rich sequences within the transactivation domain suggests that globin gene expression
in MEL cells depends on specific interactions between NF-E2 and downstream effector molecules (Bean, 1997).
The interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the
human beta-globin locus control region and the globin gene promoters was used as a paradigm to examine the
mechanisms governing promoter/enhancer interactions in this locus. Enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA
box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription
factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2,
p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation
domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein,
expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support
enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails
to reactivate expression of the endogenous beta- or alpha-globin loci in this cell line. Protein-protein
interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the
transcription initiation complex: TATA binding protein associated factor TAFII130. These findings
suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to
the individual promoters (Amrolia, 1997).
Stress response elements, which mediate induction of the mouse heme oxygenase-1 (HO-1) gene by several agents, resemble the binding site for the activator protein-1 (Jun/Fos), Maf, and Cap'n'Collar/basic leucine zipper (CNC-bZIP) families of
proteins. In L929 fibroblasts, significant activation of an HO-1 enhancer-reporter fusion gene was observed only with the CNC-bZIP class of proteins with Nrf2 exhibiting the highest level of trans-activation, between 25- and 30-fold. To further examine the role of this factor in HO-1 gene regulation, a dominant-negative mutant, Nrf2M, was generated and conditionally
expressed in L929 cells. The mutant protein was detected in cytoplasmic and nuclear fractions but does not affect cell growth.
Under conditions of Nrf2M overexpression, HO-1 mRNA accumulation in response to heme, cadmium, zinc, arsenite, and
tert-butylhydroquinone is 85%-95% inhibited. In contrast, overexpression of a dominant-negative mutant of c-Jun
decreases L929 cell growth but does not inhibit HO-1 gene activation. Nrf2 does not homodimerize, but CNC-bZIP-small Maf
protein heterodimers and Nrf2-Jun protein complexes are proposed to function as trans-activators. Co-expression of Jun
proteins or p18, however, has no significant effect or else inhibit Nrf2-mediated trans-activation. Taken together, these results implicate Nrf2 in the induction of the HO-1 gene but suggest that the Nrf2 partner in this function is a factor other than p18 or Jun proteins (Alam, 1999).
Nrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, a yeast two-hybrid screening assay was performed to identify any Nrf1-interacting proteins. In this study, a cDNA encoding residues 126-475 of MCRS2 were isolated from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. This study demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems. To find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354-447 of Nrf1 as well as the residues 314-475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2. This study has shown that MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation (Wu, 2009).
Expression of antioxidant and phase 2 xenobiotic metabolizing enzyme genes is regulated through cis-acting sequences known as antioxidant response elements. Transcriptional activation through the antioxidant response elements involves members of the CNC (Cap 'n' Collar) family of basic leucine zipper proteins including Nrf1 and Nrf2. Nrf2 activity is regulated by Keap1-mediated compartmentalization in the cell. Given the structural similarities between Nrf1 and Nrf2, attempts were made to investigate whether Nrf1 activity is regulated similarly to Nrf2. Nrf1 also resides normally in the cytoplasm of cells. Cytoplasmic localization however, is independent of Keap1. Colocalization analysis using green fluorescent protein-tagged Nrf1 and subcellular fractionation of endogenous Nrf1 and fusion proteins indicate that Nrf1 is primarily a membrane-bound protein localized in the endoplasmic reticulum. Membrane targeting is mediated by the N terminus of the Nrf1 protein that contains a predicted transmembrane domain, and deletion of this domain resulted in a predominantly nuclear localization of Nrf1 that significantly increased the activation of reporter gene expression. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, caused an accumulation of a smaller form of Nrf1 that correlated with detection of Nrf1 in the nucleus by biochemical fractionation and immunofluorescent analysis. These results suggest that Nrf1 is normally targeted to the endoplasmic reticulum membrane and that endoplasmic reticulum stress may play a role in modulating Nrf1 function as a transcriptional activator (Wang, 2006).
Other transcriptional targets of Cap'n'collar homologs Continued: Evolutionary Homologs part 2/2
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