Transcriptional Regulation

cnc is maintained independently of the activity of homeotic genes. cnc is activated in labral primordia by the maternal genes bicoid and torso (through the torso pathway), as tailless expression retracts from the anterior pole. Activation in mandibular primordia requires zygotic gap gene products, including activation by Buttonhead, and repression by Orthodenticle and Snail (Mohler 1993).

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterized Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity (Janody, 2000).

The results indicate that early anterior expression of a labral determinant, cnc, and of two foregut determinants, wg and hh, is repressed when zygotic expression of hb is allowed to persist at the anterior pole of the Drosophila blastoderm embryo. Expression of cnc, wg and hh is under the positive regulation of bcd and torso but no zygotic gene has yet been implicated in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and that torso-induced anterior repression of hb is necessary for their positive control by torso. To determine whether the positive control of cnc, wg and hh by torso could be the result of a double negative control involving hb, expression of these genes was analysed in hb zygotic mutant embryos derived from torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb at the pole, expression of these genes should be recovered in hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is not recovered in hb minus embryos derived from torso females whereas it is normal in hb minus embryos. This indicates that, although necessary, the anterior repression of hb is not sufficient to mediate Torso positive control on cnc, wg and hh early anterior expression (Janody, 2000).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The embryonic head phenotype of col1 hemizygous mutant embryos indicates a loss of skeletal structures derived from the intercalary, and possibly mandibular, segments without transformation toward another segment identity. To investigate this segmentation phenotype in more detail, col expression was compared with that of the segment polarity genes hh and wg. At the blastoderm stage, the posterior limit of col expression is parasegmental (PS0/PS1), since it precisely abuts the mandibular stripe of hh-expressing cells. Whether its anterior limit is also parasegmental cannot be answered at this stage because the expression of segment polarity genes in pre-gnathal segments is not yet established at this stage. Examination of early stage 11 embryos shows that col expression overlaps the intercalary hh stripe and abuts the intercalary Wg spot, indicating a parasegmental anterior border for col expression. At this stage however, col expression has been lost from the posterior part of PS0, since it does not overlap mandibular Wg expression. The cnc gene, which codes for a b-ZIP transcription factor, has been postulated to act as a segment identity gene in the mandibular segment. Consistent with col being expressed in PS0, col and cnc expression only partly overlap, in the region corresponding to the anterior mandibular segment. Together, these data indicate a parasegmental register of col expression at the blastoderm stage, which is subsequently restricted to anterior PS0 (Crozatier, 1999b).

A determination was made of whether col mutations affect the expression of wg and En, which mark the anterior and posterior compartments of each segment, respectively. In col1 hemizygous embryos, both the intercalary stripe of En and the spot of wg expression are missing. Since col expression does not overlap the intercalary Wg spot, the loss of this spot in col mutant embryos suggested that col does not regulate wg expression directly but possibly by an hh-dependent mechanism. It has indeed been found that in col mutant embryos, the intercalary stripe of hh is also absent, or much reduced. Together, these results show that col controls hh, en and wg expression in the intercalary segment and is required for establishing the PS(-1)/PS0 parasegmental border. The head skeleton structures ventral arm (VA) and lateral-gräten (LG), which are, respectively, either missing or reduced in col mutant embryos, are also affected in two other head mutants: crocodile (croc), which codes for a forkhead-domain protein, and cnc. These structures are also affected in embryos mutant for the homeotic genes Dfd and lab, which are expressed, respectively, in the mandibular and maxillary segments, and in the intercalary segment. col expression was examined in embryos mutant for croc, cnc, Dfd or lab. In none of these embryos was there a change in col transcription. Conversely, no changes could be detected for croc, Dfd or Lb expression in col1 hemizygous embryos, indicating that expression of each of these three genes is independent of col. In contrast, col is required for cnc transcription in the posterior intercalary segment at stage 9-10. Because this region is anterior to the region of overlap between col and cnc expression at the blastoderm stage, it is concluded that this region corresponds to a secondary site of cnc expression initiated at stage 9, under control of col activity. In cnc mutant embryos, intercalary hh expression is normal, indicating that hh and cnc are regulated by col, independent of one another (Crozatier, 1999b).

Because knot encodes a transcription factor, it was of interest to determine whether kn is required for the proper regulation of segment-specific homeotic genes. Notably, kn mutant embryos (zygotic + maternal) show an altered expression of cnc in the early embryo. At early gastrulation, the mandibular stripe of cnc expression is approximately one cell narrower than normal. In stage 10, cnc expression is normally found throughout the anterior compartment of the mandibular segment. However, in kn mutant embryos at stage 10, mandibular cnc expression is restricted to the posterior portion of the anterior compartment of the mandibular segment (which later gives rise to the mandibular lobe) and absent from the anterior-most portion. In wild-type stage 12 embryos cnc is found in both the hypopharyngeal and mandibular lobes. In kn mutant embryos at stage 12, mandibular cnc expression is restricted to the mandibular lobes, while the hypopharyngeal lobes fail to form. Unfortunately, because the cells that normally form the hypopharyngeal lobe are no longer marked with cnc in the kn mutant embryos, it is not possible to determine their new, alternative fate. This early alteration of the homeotic gene cnc expression, prior to the manifestation of the hypopharyngeal lobe, indicates that kn functions in establishing the cell fate of hypopharyngeal lobe. In contrast, no alteration in the expression of the two HOX genes, lab and Dfd, expressed in flanking ectodermal regions was observed. In addition, no altered expression was found in the mandibular region of kn mutant embryos of kn itself prior to stage 11, when mandibular-specific expression of kn ceases and kn becomes expressed in a segmentally reiterated pattern (Seecoomar, 2000).

Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster

Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. This study shows that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. These data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc (Nagy, 2013).

Earlier genetic studies have established that Myc is required for proper expression of hundreds of housekeeping genes and is therefore essential for cell growth and proliferation. Myc is a typical example of a nuclear oncogene: a transcription factor that drives tumor progression if its expression is deregulated in mammalian cells. Its mechanisms of promoting cell growth are likely different in many ways from that of cytoplasmic oncogenes such as kinases encoded by PI3K and AKT genes, also frequently activated in various cancers. Overexpression of these drives cell growth in Drosophila as well, but Myc also increases the nuclear:cytoplasmic ratio in hypertrophic cells, unlike activation of PI3K/AKT signaling. PI3K and AKT suppress basal and starvation-induced autophagy, while their inactivation strongly upregulates this process. In contrast, this study shows that both basal and starvation-induced autophagy requires Myc, and that overexpression of Myc increases UPR, leading to PERK-dependent induction of autophagy, and presumably to accumulation of cytoplasmic p62 that activates antioxidant responses. Autophagy deficiency predisposes to cancer at least in part through accumulation of the selective autophagy cargo p62, resulting in activation of antioxidant responses and tumor formation. These analyses show that both of these cytoprotective pathways can be activated simultaneously, and are required in parallel to sustain Myc-induced overgrowth in Drosophila cells (Nagy, 2013).

Autophagy and antioxidant responses have been considered to act as tumor suppressor pathways in normal cells and during early stages of tumorigenesis, while activation of these processes may also confer advantages for cancer cells. Lack of proper vasculature in solid tumors causes hypoxia and nutrient limitation. These stresses in the tumor microenvironment have been suggested to elevate UPR and autophagy to promote survival of cancer cells. This study demonstrates that genetic alterations similar to those observed in cancer cells (that is, deregulated expression of Myc) can also activate the UPR, autophagy and antioxidant pathways in a cell-autonomous manner in Drosophila. These processes are likely also activated as a consequence of deregulated Myc expression in human cancer cells based on a number of recent reports, similar to the findings in Drosophila presented in this study. First, chloroquine treatment that impairs all lysosomal degradation pathways is sufficient to reduce tumor volume in Myc-dependent lymphoma models. Second, ER stress and autophagy induced by transient Myc expression increase survival of cultured cells, and PERK-dependent autophagy is necessary for tumor formation in a mouse model. Data suggest that UPR-mediated autophagy and antioxidant responses may also be necessary to sustain the increased cellular growth rate driven by deregulated expression of Myc (Nagy, 2013).

Myc has proven difficult to target by drugs. Myc-driven cancer cell growth could also be selectively prevented by blocking cellular processes that are required in cancer cells but dispensable in normal cells, known as the largely unexplored non-oncogene addiction pathways. Previous genetic studies establish that autophagy is dispensable for the growth and development of mice, although knockout animals die soon after birth due to neonatal starvation after cessation of placental nutrition. Tissue-specific Atg knockout mice survive and the animals are viable, with potential adverse effects only observed in aging animals. Genetic deficiencies linked to p62 are also implicated in certain diseases, but knockout mice grow and develop normally and are viable. Similarly, Nrf2 knockout mice are viable and adults exhibit no gross abnormalities, while these animals are hypersensitive to oxidants. Mice lacking PERK also develop normally and are viable. All these knockout studies demonstrate that these genes are largely dispensable for normal growth and development of mice, and that progressive development of certain diseases is only observed later during the life of these mutant animals. There are currently no data regarding the effects of transient inhibition of these processes, with the exception of the non-specific lysosomal degradation inhibitor chloroquine, originally approved for the treatment of malaria, which is already used in the clinic for certain types of cancer (Nagy, 2013).

Based on these knockout mouse data, UPR, autophagy and antioxidant responses may be considered as potential non-oncogene addiction pathways: strictly required for Myc-dependent overgrowth (this study) and tumor formation, but dispensable for the growth and viability of normal cells, both in Drosophila and mammals. One can speculate that the transient inactivation of these pathways will have even more subtle effects than those observed in knockout mice, but this needs experimental testing. While it is difficult to extrapolate data obtained in Drosophila (or even mouse) studies to human patients, it is tempting to speculate that specific drugs targeting UPR, autophagy and antioxidant responses may prove effective against Myc-dependent human cancers, perhaps without causing adverse side-effects such as current, less specific therapeutic approaches. Notably, widely used anticancer chemotherapy treatments are known to greatly increase the risk that cancer survivors will develop secondary malignancies. Moreover, the autophagy and antioxidant pathways appear to be required in parallel during Myc-induced overgrowth in Drosophila cells. If a similar genetic relationship exists in Myc-dependent human cancer cells, then increased efficacy may be predicted for the combined block of key enzymes acting in these processes (Nagy, 2013).

Elucidation of the genetic alterations behind increased UPR, autophagy and antioxidant responses observed in many established human cancer cells may allow specific targeting of these pathways, and potentially have a tremendous benefit for personalized therapies. In addition to non-specific autophagy inhibitors such as chloroquine, new and more specific inhibitors of selected Atg proteins are being developed. Given the dual roles of autophagy during cancer initiation and progression, a major question is how to identify patients who would likely benefit from taking these drugs. For example, no single test can reliably estimate autophagy levels in clinical samples, as increases in autophagosome generation or decreases in autophagosome maturation and autolysosome breakdown both result in accumulation of autophagic structures. Based on this study's data and recent mammalian reports, elevated Myc levels may even turn out to be useful as a biomarker before therapeutic application of inhibitors for key autophagy, UPR or antioxidant proteins in cancer patients (Nagy, 2013).

Targets of Activity

After stage 11, cnc and forkhead are required for the continued expression of dorsal pharyngeal domains of wingless and hedgehog (Mohler 1995).

The genetic function of the homeotic gene Deformed (< I>Dfd) is required in the cnc mutant background to produce ectopic mouth hooks, and it has been proposed that Dfd and cnc function in combination to specify mandibular identity. One of the downstream genes that is activated by Dfd in maxillary cells is Distal-less (Dll). Dll is required for the formation of the larval appendage primordia and the distal regions of adult appendages. In the maxillary segment, Dll is expressed in two patches of cells: a dorsal patch that gives rise to the maxillary sense organ and a ventral patch that consists of the primordia for the maxillary cirri. The dorsal maxillary domain of Dll expression is largely independent of Dfd function, while the ventral maxillary patch of Dll is activated by Dfd through a 3' enhancer (O’Hara, 1993). In cnc2E16 mutant embryos, Dll is ectopically expressed in ventral mandibular cells, suggesting that cncB, one of the transcripts coded for by cnc, represses Dll transcription in mandibular cells. In hs-cncB embryos 30 minutes after heat shock, when Dfd protein abundance is normal, Dll expression is repressed in the ventral maxillary segment but other domains of Dll expression in the head and thorax are relatively unaffected, indicating that CncB selectively represses the Dfd-dependent portion of the Dll expression pattern. In hs-cncC and hs-cncA embryos, the ventral maxillary expression of Dll is not selectively repressed. Reporter gene expression from the 3' enhancer follows the expression of Dll as the enhancer is ectopically activated in the ventral mandibular region in cnc mutants and repressed in hs-cncB embryos (McGinnis, 1998).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Ruscha, 2000).

The early phase reflects a requirement for gap gene function for normal expression of pb to occur during later stages. Specifically, btd, gt, and hb have been identified as being required for proper gnathal expression of pb. The function of the head gap gene btd has been shown to be required only during early stages of embryogenesis. The expression patterns of gt and hb are such that they are no longer expressed in the labial segment at the time when pb expression begins. This is taken as a strong indication that the gap genes influence pb indirectly. Consistent with this hypothesis, no gt or hb binding sites could be detected in the regulatory elements of the pb reporter. In the case of hb, the role that various trans-acting factors might play in mediating loss of pb expression in the labial segment was investigated. Expression of Scr, the positive regulator of pb in the labial segment, is not eliminated. Further, repression of pb is not attributable to expansion of tsh expression. One possibility is that another negative regulator is being expressed such that Scr can no longer activate pb. Given the negative regulatory interactions that occur between the gap genes, it is possible that one of the other gap genes might be misexpressed and downregulate pb. However, it may be misexpression of cnc or some other gene that has yet to be identified. Alternatively, the 'hit-and-run' hypothesis, proposed to explain the long-term repression of Ultrabithorax (Ubx) by hb, may describe how transient expression of the gap genes is required very early in development to permit later expression of pb. In this hypothesis, heritable changes in chromatin structure, mediated by the PcG genes, are invoked to explain how hb regulates Ubx long after hb expression has ceased. In the case of pb regulation, one or more of these gap genes may be required to alter chromatin structure in and around the pb locus, thereby allowing the various trans-acting factors access to the pb cis-acting regulatory elements (Rusch, 2000).

During the middle phase, the initial expression pattern of pb is set by a variety of trans-acting factors. Focus was placed on the identification of those factors that determine the ectodermal pattern of pb expression along the A/P axis of the embryo. The Hox genes Dfd and Scr act as positive regulators of pb and Dfd can bind to pb regulatory elements in vitro. It is thought likely that Scr also regulates pb directly based on the similarity with which mutations in Dfd and Scr affect expression of pb and the pb reporter. In addition to the Hox genes, the region-specific homeotics cnc and tsh have been identified as negative regulators of pb and serve to restrict pb expression to the gnathos. It is not clear whether these genes regulate pb directly, though in the case of tsh the sequence TGGAAAAGT has been identified in the 500-bp regulatory fragment used in the pb reporter; this sequence is very similar to the identified tsh binding site. While this regulatory paradigm does not completely describe the regulation of the endogenous gene, based on the presence of pb residual expression, it is sufficient to explain the behavior of the 500-bp pb reporter. This mechanism of regulation places pb downstream of the Hox genes and is the first instance in Drosophila where one Hox gene is positively and directly regulated by another, a distinction previously accorded only to vertebrate Hox genes. Studies by others have suggested that wg may be mediating the nonautonomous residual expression of pb that is uncovered by mutations in Dfd and/or Scr. With the exception that wg has the strongest effect on pb expression of the segment polarity genes tested, the results shed little light on the mechanism that underlies this phenomenon. However, non-cell autonomous signaling has been implicated to explain regulation of ectodermal pb function by mesodermal expression of Scr; perhaps the residual expression in the embryo is an example of this pathway. Further experiments, including identification of an enhancer that mediates this residual expression, are needed (Rusch, 2000).

Beyond antioxidant genes in the ancient NRF2 regulatory network

NRF2, a basic leucine zipper transcription factor encoded by the gene NFE2L2, is a master regulator of the transcriptional response to oxidative stress. NRF2 (Drosophila homolog Cap-n-collar) is structurally and functionally conserved from insects to humans, and it heterodimerizes with the small MAF transcription factors to bind a consensus DNA sequence (the antioxidant response element, or ARE) and regulate gene expression. This study used genome-wide chromatin immunoprecipitation (ChIP-seq) and gene expression data to identify direct NRF2 target genes in Drosophila and humans. These data have allowed construction of the deeply conserved ancient NRF2 regulatory network - target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways, metabolism, and a number of less expected genes. Enhancer reporter assays and electrophoretic mobility shift assays were used to confirm NRF2-mediated regulation of ARE (antioxidant response element) activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that and NRF2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear NRF2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the NRF2-ARE pathway, and indicate that NRF2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis have been central to this pathway since its inception (Lacher, 2015).

Protein Interactions

cnc and Deformed act in overlapping sets of structures. Nevertheless, cnc expression is independent of Deformed (Mohler 1995).

The cnc locus encodes three transcript and protein isoforms. The cncB transcript is expressed in an embryonic pattern that includes the labral, intercalary and mandibular segments, while cncA and cncC are expressed ubiquitously. CncB suppresses the segmental identity function of the Hox gene Deformed (Dfd) in the mandibular segment of Drosophila embryos. Evidence has been provided that the CncB-mediated suppression of Dfd requires the Drosophila homolog of the mammalian small Maf proteins, Maf-S, and that the suppression occurs even in the presence of high amounts of Dfd protein. Interestingly, the CncB/Maf-S suppressive effect can be partially reversed by overexpression of Homothorax (Hth), suggesting that Hth and Extradenticle proteins antagonize the effects of CncB/Maf-S on Dfd function in the mandibular segment. In embryos, multimers of simple CncB/Maf-S heterodimer sites are transcriptionally activated in response to CncB, and in tissue culture cells the amino-terminal domain of CncB acts as a strong transcriptional activation domain. There are no good matches to CncB/Maf binding consensus sites in the known elements that are activated in response to Dfd and repressed in a CncB-dependent fashion. This suggests that some of the suppressive effect of CncB/Maf-S proteins on Dfd protein function might be exerted indirectly, while some may be exerted by direct binding to as yet uncharacterized Dfd response elements. Ectopic CncB is sufficient to transform ventral epidermis in the trunk into repetitive arrays of ventral pharynx. The functions of CncB are compared to those of its vertebrate and invertebrate homologs, p45 NF-E2, Nrf and Skn-1 proteins, and it is suggested that the pharynx selector function of CncB is highly conserved on some branches of the evolutionary tree (Veraksa, 2000).

Mammalian homologs of Cnc (p45 NF-E2 and Nrf proteins) have been shown to bind DNA as obligate heterodimers with small Maf proteins (MafK/p18, MafF and MafG). An apparent Drosophila ortholog of the mammalian small Mafs was identified in a yeast two-hybrid screen for genes encoding peptides capable of interacting with the common b-Zip domain of Cnc proteins. BLAST searches with a corresponding cDNA sequence detect only one small maf gene, corresponding to CG9954, in the near complete Drosophila genome. The gene has been called Drosophila maf-S (S for Small), and the protein Maf-S. In the basic region that contacts DNA, Maf-S shares a domain of perfect identity with mammalian small Maf proteins, and partial identity in the leucine zipper and other protein regions. In situ hybridizations show that maf-S RNA is maternally deposited in the Drosophila egg, and the transcripts are present in all or nearly all embryonic cells throughout development, making the Maf-S protein available for potential interactions with all Cnc isoforms. In addition, there is an enrichment in maf-S transcripts in certain areas of the embryo, against the ubiquitous expression background. At stage 8, slightly higher levels are detected around cephalic furrow and in the mesoderm. At stage 11 and beyond, there is an enrichment in maf-S transcripts in the anterior and posterior midgut, in the nervous system, as well as in the involuting intercalary segment that will give rise to ventral pharynx (Veraksa, 2000).

Double stranded RNA interference was used in an attempt to test the phenotypic effects of loss of maf-S function. After injection of maf-S dsRNA into the head region of precellular blastoderm embryos, only 1.5% of embryos hatched as first instar larvae. The cuticles of injected embryos show head defects that are remarkably similar to those found in cncB mutants: duplications of mouth hooks, shortened lateralgraten, missing or deformed median tooth and truncated ventral pharynx. Immunostaining of maf-S dsRNA-injected embryos with Cnc-specific antibody reveals that the levels of CncB protein are not significantly different from wild type. Results of the dsRNA interference experiment suggest that Maf-S is an obligate functional partner of CncB. Without the small Maf subunit, CncB is unable to promote the development of intercalary, mandibular and labral head structures and to repress the function of Dfd. The maf-S gene, which maps to chromosomal segment 57A, is uncovered by Df(2R)exu2. Homozygotes for this deletion, which lack the zygotic dose of maf-S and other nearby genes, exhibit mild head defects that include disruptions of the pattern of ventral pharyngeal transverse ribs (T-ribs). The weakness of this phenotype relative to the RNAi result may be due to the maternal contribution from maf-S, which is unaffected in the zygotic deletion mutants (Veraksa, 2000).

Despite a high degree of primary sequence similarity between the DNA binding domains of CncB and NF-E2, the optimal recognition site for the Cnc isoforms in association with Drosophila Maf might be different. CncA and CncB optimal binding sites were sought by selecting specific sequences from a pool of degenerate oligonucleotides by Cnc/Maf-S heterodimers. Flag-tagged full-length CncA or CncB proteins were cotranslated in vitro with Maf-S and bound to an oligonucleotide containing a core of 14 ambiguous positions. All selected sites start from the same position (T), suggesting that this and nearby nucleotides influence the selection process. The sequence selected in this experiment by Cnc/Maf-S heterodimers (TGCTGAGTCAT) is identical to the preferred site selected by TCF11/MafG, and is very similar to the consensus binding site derived for p45 NF-E2/p18 MafK from gel shift competition experiments. The TGCTGAG part of the site corresponds to the Maf-bound sequences in the sites for mammalian CNC family proteins, and also to the T-MARE (TRE[phorbol-12-O-tetradecanoate-13-acetate (TPA)-responsive element]-type Maf recognition element) consensus half-site for large Maf homodimers. The GTCAT half-site is contacted by Cnc homologs, and also is a perfect match to the binding site for the C. elegans Skn-1 protein that binds DNA as a monomer. Monomers of CncA or CncB do not appreciably bind to the Cnc/Maf consensus. Maf-S does not bind as a homodimer either to this sequence or to the T-MARE consensus. This property of the Drosophila small Maf protein is different from mammalian small Mafs, which form stable homodimer complexes on T-MARE-like sites. When CncA or CncB are cotranslated with Maf-S, abundant and stable complexes form on Cnc/Maf binding sites, suggesting that DNA binding is achieved only by Cnc/Maf heterodimers and that such binding is highly cooperative (Veraksa, 2000).

Previous genetic and molecular data have established that CncB has several functions in the developing Drosophila embryo: it is required for the formation of labral and intercalary structures such as median tooth and pharynx, and it is required to inhibit Dfd function in the mandibular segment. A summary model has been proposed in which these functions are mediated in part by the transcriptional activator properties of CncB, and are achieved through specific DNA binding with Maf-S to an 11-bp CncB/Maf-S heterodimer binding site. In the intercalary and anterior mandibular segments of the embryo, heterodimerization of CncB with Maf can activate some downstream target genes, and the heterodimers are required to develop ventral pharyngeal structures (T-ribs and ventral arms). Furthermore, CncB, in the context of the ventral epidermis of other head and trunk segments, is sufficient to promote pharynx identity, and thus cncB functions as a pharynx selector gene (Veraksa, 2000).

The function of CncB in suppressing the maxillary-promoting function of Dfd is carried out in the mandibular cells. Early in development CncB and Dfd are coexpressed throughout the mandibular segment, but by stage 13 Dfd expression retracts and becomes localized to a row of cells in the posterior of the segment. In these cells, CncB and Dfd are both required to specify lateralgraten and the base of the mouth hook. Although Dfd function is required for the normal structures that derive from the posterior mandibular segment, the maxillary-promoting function of Dfd is inhibited in these cells. In the anterior mandibular segment, CncB represses both the function and expression of Dfd. Since Dfd protein activates its own transcription, inhibition of Dfd function inevitably results in silencing the endogenous transcription of the Dfd gene. Persistent expression of Dfd in the posterior mandibular cells is likely to be mediated by regulatory regions that are different from the autoactivation enhancers and are insensitive to CncB-mediated repression. Based on the present data, it is suggested that CncB-mediated repression of Dfd function is at least in part indirect, for several reasons. (1) There are no close matches to the consensus CncB/Maf-S binding site in known Dfd response elements, despite the fact that these elements can be repressed by ectopic expression of CncB, e.g. modules C, E, and F from the 2.7 kb Dfd epidermal autoregulatory enhancer. (2) The CncB-specific amino terminal region, crucial for the suppression of Dfd function, contains a strong transcriptional activation domain. (3) A CncA fusion protein with a simple heterologous transcriptional activation domain from VP16 can also partially repress Dfd-dependent maxillary structures upon overexpression. (4) Chimeric Dfd response elements containing Cnc/Maf binding sites adjacent to Dfd binding sites are not repressed by overexpression of CncB, as might be expected if the action of CncB on Dfd were direct. Instead, the endogenous Dfd-dependent maxillary expression of 4CE is enhanced in HS-CncB embryos when compared to non-heat-shocked controls. Suppression of Dfd function by CncB may therefore be achieved by transcriptional activation of an intermediate gene encoding a repressor or corepressor that would interfere with the function of Dfd. Another possibility is that CncB might compete for a common cofactor or coactivator (such as Exd, Hth or CBP), or it might activate a specific Dfd modifying enzyme such as a kinase or phosphatase. It is still possible that the suppressive effect that CncB protein exerts on Dfd function may involve CncB mediated transcriptional repression on as yet uncharacterized response elements. A binding interaction between Dfd and CncB proteins has been observed in GST pull-down assays, but the biological significance of this result is as yet unclear (Veraksa, 2000).

Repression of Dfd function by CncB is analogous to the phenomenon of phenotypic suppression (also known as posterior prevalence), which denotes the ability of more posterior Hox proteins to suppress the function of more anterior Hox proteins in the same cells. 'Anterior prevalence' of CncB over Dfd provides additional morphological diversity in the mandibular and maxillary segments. The indirect way by which CncB acts on Dfd maxillary function has a precedent in Hox phenotypic suppression. The inhibitory effects of posterior Hox proteins on Antennapedia function can apparently be exerted via an indirect pathway involving phosphorylation of Antp protein by casein kinase II. Dfd protein is also phosphorylated in vitro by CKII, but whether this phosphorylation is relevant to the suppression by CncB is unknown (Veraksa, 2000).



cnc functions as a required homeotic segment-specific selector gene, continually expressed in labral and mandibular parasegments [Image] of the head. Earliest expression is in the late blastoderm, found in the anterior portion of the mandibular segment (parasegment 0), just anterior to the cephalic furrow, and in the clypeolabral lobe (parasegment -4). labial and spalt, two other homeotic genes affecting the head, are independent of cnc. cnc represses maxillary development in the mandibular segment. cnc defines the dorsal pharyngeal region and the posterior lateral and ventral portion of the pharynx (Mohler, 1995).

cap'n'collar expression in the anterior compartment of the mandibular segment has two roles, depending on whether or not Deformed is expressed. Where Deformed is not expressed, cnc promotes hypopharyngeal development of the mandibular segment. Where Deformed is expressed, cnc promotes mandibular development in combination with Deformed (Mohler, 1995).

Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila

Keap1/Nrf2 signaling defends organisms against the detrimental effects of oxidative stress and has been suggested to abate its consequences, including aging-associated diseases like neurodegeneration, chronic inflammation, and cancer. Nrf2 is a prominent target for drug discovery, and Nrf2-activating agents are in clinical trials for cancer chemoprevention. However, aberrant activation of Nrf2 by keap1 somatic mutations may contribute to carcinogenesis and promote resistance to chemotherapy. To evaluate potential functions of Keap1 and Nrf2 for organismal homeostasis, the pathway was characterized in Drosophila. Keap1/Nrf2 signaling in the fruit fly is shown to be activated by oxidants, induces antioxidant and detoxification responses, and confers increased tolerance to oxidative stress. Importantly, keap1 loss-of-function mutations extend the lifespan of Drosophila males, supporting a role for Nrf2 signaling in the regulation of longevity. Interestingly, cancer chemopreventive drugs potently stimulate Drosophila Nrf2 activity, suggesting the fruit fly as an experimental system to identify and characterize such agents (Sykiotis, 2008).

Three RNA isoforms are transcribed off the Drosophila cap'n'collar locus, designated cncA, cncB, and cncC. These isoforms encode three different proteins, which share their C-terminal regions and thus comprise the same DNA-binding domain. However, the three Cnc translation products differ at their N-termini: CncC encompasses CncB, which in turn encompasses CncA. The CncB isoform is required together with a small Maf subunit for the development of embryonic head structures (Veraksa, 2000). The unique N terminus of CncC predicts a different role for this isoform: it shows similarity to the Neh2 domain of Nrf2, which contains the Keap1-binding ETGE motif and an upstream hydrophobic region. Based on this distinctive homology, it has been suggested that CncC might be the Drosophila counterpart of Nrf2 (Kobayashi, 2002). As a first step to addressing this idea and the potential role of CncC in antioxidant responses, its expression was examined in Drosophila. In larvae, cncC mRNA is most abundantly expressed in the alimentary canal, and it shows a strong, distinctive staining pattern. This is reminiscent of the broad expression of mammalian Nrf2 in the digestive tract, which, like the skin and airways, is a major frontier where the organism comes into direct contact with its environment. cncC mRNA expression was also detected in the Malpighian tubules (which are detoxification organs) and in the salivary glands, but not in imaginal discs or in the fat body. cncC mRNA is also present in adult female and male flies as determined by RT-PCR, consistent with a role in the homeostasis of the mature organism (Sykiotis, 2008).

The Drosophila genome also harbors a likely homolog of vertebrate keap1 genes. This gene and its product have not yet been functionally characterized. Multiple sequence alignments show that the predicted Drosophila Keap1 (dKeap1) protein has striking similarity with its mammalian and zebrafish homologs. Similar to cncC, dkeap1 mRNA is expressed in the alimentary canal and in the Malpighian tubules of third instar larvae and adult flies, and it shows a similar distribution as cncC but with a weaker, more diffuse staining pattern. keap1 mRNA is also detected in the salivary glands, the brain, and the ring gland, but not in imaginal discs or in the fat body. These findings demonstrate that the candidate Drosophila homologs of Nrf2 and Keap1 are expressed in the fly's digestive tract, which represents the first line of defense to ingested environmental stressors, and in the Malpighian tubules, which are major sites of detoxification. Expression of keap1 and cncC in the adult gut and Malpighian tubules was verified by RT-PCR on dissected adult tissues. Consistent with the conservation of the Keap1-binding motif in CncC, the physical association between Drosophila Keap1 and CncC has been suggested by a genome-wide yeast two-hybrid experiment. This interaction predicts that Keap1 should act as a negative regulator of CncC in vivo (Sykiotis, 2008).

This study shows that the Drosophila CncC and Keap1 proteins are genuine Nrf2 pathway components, regulate antioxidant response element (ARE)-mediated transcription and detoxification gene expression, and are crucial for the organism's defense against oxidative stress. gstD1 is a prototypical oxidative stress response gene; it encodes a well-known detoxification enzyme, and its transcription unit is preceded by a consensus ARE sequence. Keap1/CncC signaling activates the gstD-GFP transgene indicating that reporter activity is regulated by one or more functional ARE sequences in the cloned enhancer. Indeed, the gstD enhancer harbors a 9 bp sequence (TGACcggGC) that perfectly matches the consensus core ARE sequence (TGAYnnnGC). Moreover, this 9 bp core element lies at the center of a 20 bp sequence (TCAgcATGACcggGCAaaaa), which also conforms perfectly to the extended ARE consensus sequence (TMAnnRTGAYnnnGCRwwww) (Sykiotis, 2008).

These findings should accelerate genetic approaches to the functional characterization of the Nrf2 pathway aimed at the comprehensive elucidation of its roles in health and disease. In this respect, the lifespan extension of keap1 heterozygous male flies is an example of new insight derived from Drosophila. Very recently, the Nrf2-related SKN-1 protein was found to be required for longevity induced by dietary restriction in C. elegans (Bishop, 2007). This finding supports the concept that Cnc family members have an evolutionarily conserved function in lifespan regulation. The current results suggest that this role extends beyond calorie restriction and establish a function for Keap1 (which is not conserved in worms) in this context. It was previously reported that the transcriptional activity of Nrf2 decreases in aging mice (Suh, 2004). Based on these findings, it will be very interesting to investigate the role of Keap1/Nrf2 in the aging of vertebrates (Sykiotis, 2008).

The effect of keap1 heterozygosity on stress resistance and longevity shows a marked sexual dimorphism, with significant, albeit modest, effects discernible only in males. Although the basis for this sex difference is presently unknown, sexually dimorphic genetic effects on Drosophila lifespan are not uncommonly reported. It is important to note that the sexual dimorphism in the longevity of keap1 heterozygotes is reflected in their Paraquat sensitivity. In males, removal of one gene copy of keap1 increases Paraquat resistance, whereas females do not show this effect. While these findings imply that females react differently to keap1 heterozygosity, such a difference is not manifested at the level of gstD1 expression, which is significantly elevated in keap1 heterozygotes of both sexes. These data suggest that although keap1 heterozygosity increases stress response gene levels in both sexes, this translates into a benefit for Paraquat tolerance and longevity only in male flies. Importantly, when CncC is overexpressed, Paraquat resistance is enhanced in both males and females. Taken together, the data suggest that females can also benefit from increased CncC activity, but keap1 heterozygosity is not sufficient to yield stress protection and extend lifespan in females. Wild-type female flies generally live longer and are more stress resistant than males, and this effect is also seen in the current study. The extended lifespan of male keap1 heterozygotes is very similar to that of female flies. Given the modest, albeit significant, benefits observed in males, it is proposed that keap1 heterozygosity is not sufficient to increase stress tolerance and extend lifespan beyond certain biological limits (Sykiotis, 2008).

The involvement of Nrf2 in both cancer prevention and lifespan regulation adds to the intricate links between cancer and aging. In addition to its role in cancer prevention, the antioxidant and cell-protective properties of Nrf2 make it an important target for drug discovery in the prevention and treatment of oxidative stress- and aging-related diseases, including neurodegenerative disorders. Many Drosophila models of neurodegeneration are available, rendering the fruit fly an excellent system for the preclinical testing of this strategy. Thus, the characterization of Keap1/Nrf2 signaling in Drosophila facilitates studies that will examine the preventive and/or therapeutic effects of Nrf2-activating cancer chemopreventive agents in neurodegenerative diseases. Studies utilizing oltipraz, in particular, are especially appealing, since it is a relatively safe agent that has been used for the treatment of schistosomiasis (a parasitic disease) and has been tested in human clinical trials for cancer chemoprevention (Sykiotis, 2008).

Integration of UPRER and oxidative stress signaling in the control of intestinal stem cell proliferation

The Unfolded Protein Response of the endoplasmic reticulum (UPRER: see Drosophila Inositol-requiring enzyme-1) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. This study shows that the UPRER is a critical regulator of intestinal stem cell (ISC) quiescence in Drosophila melanogaster. ISCs were found to require activation of the UPRER for regenerative responses, but a tissue-wide increase in ER stress was found to trigger ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. The results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium (Wang, 2014 PubMed).

Effects of Mutation or Deletion

Embryonic lethal mutants lack mandibular and labral head structures (Mohler, 1995). cnc mutant larvae possess head skeletons missing the labrum and dorsal pouch (labral structures), the transverse-ribs and ventral arms (hypopharyngeal structures) and the lateralgräte (mandibular structure). cnc mutants are also missing the posterior pharyngeal wall (labral structure), as well as having some duplicate maxillary structures. Such duplication is due to the fact that CNC represses maxillary development in the mandibular segment (Mohler, 1995).

Proteins produced by the homeotic genes of the Hox family assign different identities to cells on the anterior/posterior axis. Relatively little is known about the signaling pathways that modulate their activities or the factors with which they interact to assign specific segmental identities. To identify genes that might encode such functions, a screen was carried out for second site mutations that reduce the viability of animals carrying hypomorphic mutant alleles of the Drosophila homeotic locus, Deformed. Genes mapping to six complementation groups on the third chromosome were isolated as modifiers of Deformed function. Products of two of these genes, sallimus and moira , have been previously proposed as homeotic activators since they suppress the dominant adult phenotype of Polycomb mutants. Mutations in hedgehog, which encodes secreted signaling proteins, were also isolated as Deformed loss-of-function enhancers. hedgehog mutant alleles also suppress the Polycomb phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with Polycomb, indicating that the screen identified genes that are not general homeotic activators. Two of these genes, cap 'n' collar and defaced, have defects in embryonic head development that are similar to defects seen in loss of function Deformed mutants (Harding, 1995).

In Drosophila, dorsoventral polarity is established by the asymmetric positioning of the oocyte nucleus. In egg chambers mutant for cap 'n' collar, the oocyte nucleus migrates correctly from a posterior to an anterior-dorsal position, where it remains during stage 9 of oogenesis. However, at the end of stage 9, the nucleus leaves its anterior position and migrates towards the posterior pole. The mislocalization of the nucleus is accompanied by changes in the microtubule network and a failure to maintain Bicoid and Oskar mRNAs at the anterior and posterior poles, respectively. Gurken mRNA associates with the oocyte nucleus in cap 'n' collar mutants and initially the local secretion of Gurken protein activates the Drosophila EGF receptor in the overlying dorsal follicle cells. However, despite the presence of spatially correct Grk signaling during stage 9, eggs laid by cap 'n' collar females lack dorsoventral polarity. cap 'n' collar mutants, therefore, allow for the study of the influence of Grk signal duration on DV patterning in the follicular epithelium (Guichet, 2001).

cnc is a complex locus coding for three protein isoforms (CncA, CncB, CncC) which share a basic-leucine zipper domain at the carboxy terminus. While CncA and CncC are expressed ubiquitously, CncB is expressed specifically in the head region of early embryos where it is required for the repression of deformed function and the formation of intercalary and labral structures. Double-stranded RNA interference experiments have shown that CncA and CncC are dispensible for embryonic development. The two P-insertions used in this study affect all three isoforms. CncB is not expressed during oogenesis, thus the mutant phenotypes observed are due to a lack of either CncA, CncC, or both isoforms. Judging from their structure, both proteins probably function as transcription factors, as has been demonstrated for CncB and the Cnc homologs of vertebrates and other invertebrates. At present, no genes are known to be regulated by Cnc proteins during oogenesis. However, the cnc phenotype reveals two new aspects as to how DV polarity is established during oogenesis. (1) The initial asymmetric movement of the oocyte nucleus has to be followed by a separate process of stable anchoring of the nucleus at the anterior cortex. (2) An early pulse of asymmetric Egf signaling is insufficient to induce stable DV follicle cell patterning, indeed Egf receptor activation by Gurken has to persist until stage 10A to establish the DV axis of the Drosophila egg (Guichet, 2001).

In cnc mutant egg chambers, nuclear movement occurs normally. The nucleus remains cortically localized even after its posterior displacement. Since interference with components of the dynactin complex leads to the dissociation of the nucleus from the cortex, it is believed that the dynactin complex is not affected by the loss of cnc function. However, the polarization of the microtubule network is aberrant in stage 10A cnc oocytes. Higher numbers of microtubules accumulate in the posterior region of the oocyte at the expense of the anterior cortical ring, which dominates the microtubule network of wild-type stage-9 to -10A egg chambers. This second microtubule reorganization could either be the cause of or result from the late displacement of the nucleus. In the first case, cnc would be required for a process that stabilizes and maintains the microtubule polarity after stage 8. Prolonged signaling from posterior follicle cells might be necessary to suppress the reestablishment of microtubule organizing centers (MTOCs) at the posterior pole. The reception of such a signal or its transmission to the cytoplasm might be impaired in the absence of cnc function. In this model, the reassembly of MTOCs in posterior regions would lead to the redistribution of free tubulin and consequently weaken anterior MTOCs. The nucleus would subsequently migrate towards these ectopic posterior MTOCs. BCD mRNA also would become mislocalized since it is known to move, like the nucleus, towards the minus ends of microtubules, i.e., towards the MTOCs (Guichet, 2001).

In the other scenario, cnc would be required specifically for oocyte nucleus anchoring at the anterior cortex. Anterior anchoring might be necessary since there is a massive influx of cytoplasm from the nurse cells to the anterior pole of the oocyte during egg chamber growth. If the nucleus is not properly anchored, these transport processes might dislodge the nucleus from the anterior pole. Why would this mispositioning of the nucleus lead to the reorganization of the microtubule network? Such microtubule reorganizations have not been described in other mutant backgrounds where the nucleus does not reach the anterior cortex, such as grk, cni, mago, and DLis-1. It has been shown that the nucleus gets encaged by microtubules when it arrives at the anterior pole in wild-type oocytes, indicating that the anteriorly localized nucleus acquires a microtubule-nucleating activity. This activity might remain associated with the mispositioned nucleus in cnc egg chambers and might subsequently cause the increased microtubule density in the posterior half of the cnc oocytes (Guichet, 2001).

In both scenarios, the mislocalization of OSK mRNA remains somehow enigmatic. OSK should not localize to the same region to which BCD is transported. However, normal OSK transport from the anterior to the posterior might just be blocked by the mispositioned nucleus and its associated microtubules. Thus OSK might be trapped in the vicinity of the ectopic nucleus on its way to the posterior pole (Guichet, 2001).

Although at present it is impossible to distinguish between these two explanations for the cnc phenotype, the observed correlation between ectopic microtubules and nuclear position supports the second scenario. It is therefore proposed that two processes of oocyte nucleus anchoring can be distinguished: the general anchoring to the oocyte cortex and the spatially restricted anchoring to the anterior surface of the oocyte. The first process involves the dynein/dynactin complex, which controls cortical anchoring and nuclear movement. This process is likely to be unaffected in cnc. The second process is required after migration to tether the nucleus to the anterior surface of the oocyte. It keeps the nucleus in place during the growth of the oocyte after stage 9 of oogenesis. This process might be affected in cnc mutant egg chambers. As a transcription factor, Cnc might control the expression of proteins, which function as components of the anterior anchor (Guichet, 2001).


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

date revised: 15 August 2015

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