cap'n'collar

EVOLUTIONARY HOMOLOGS (part 1/2)

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).

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).

Other transcriptional targets of Cap'n'collar homologs

Continued: Evolutionary Homologs part 2/2

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