Gene name - cap-n-collar
Cytological map position - 94E 3,4
Function - transcription factor
Keyword(s) - head gap gene
Symbol - cnc
Genetic map position - 3-81.2
Classification - basic leucine zipper
Cellular location - nuclear
|Recent literature||Karim, M.R., Taniguchi, H. and Kobayashi, A. (2015). Constitutive activation of Drosophila CncC transcription factor reduces lipid formation in the fat body. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26049108
Accumulating evidence indicates that the vertebrate stress-response transcription factors Nrf1 and Nrf2 are involved in hepatic lipid metabolism. To elucidate the precise roles of Nrfs in this process, this study analyzed the physiological role of CncC in lipid metabolism as a Drosophila model for vertebrate Nrf1 and Nrf2. It was examined whether CncC activity was repressed under physiological conditions through a species-conserved NHB (N-terminal homology box 1) domain, similar to that observed for Nrf1. Deletion of the NHB1 domain (CncCΔN) led to CncC-mediated rough-eye phenotypes and the induced expression of the CncC target gene gstD1 both in vivo and in vitro. Thus, the affect of CncCΔN overexpression on the formation of the fat body, which is the major lipid storage organ, was explored. Intriguingly, CncCΔN caused a significant reduction in lipid droplet size and triglyceride (TG) levels in the fat body compared to wild type. It was found that CncCΔN induced a number of genes related to innate immunity that might have an effect on the regulation of cellular lipid storage. This study provides new insights into the regulatory mechanism of CncC and its role in lipid homeostasis.
|Tamada, M. and Zallen, J. A. (2015). Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling. Dev Cell 35: 151-161. PubMed ID: 26506305
Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. This study identified a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle-positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation.
|Li, X., Chatterjee, N., Spirohn, K., Boutros, M. and Bohmann, D. (2016). Cdk12 is a gene-selective RNA polymerase II kinase that regulates a subset of the transcriptome, including Nrf2 target genes. Sci Rep 6: 21455. PubMed ID: 26911346
The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. This study carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. The study suggests that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to "stress-defence mode" when challenged by external stress.
|Chatterjee, N., Tian, M., Spirohn, K., Boutros, M. and Bohmann, D. (2016). Keap1-independent Regulation of Nrf2 activity by protein acetylation and a BET bromodomain protein. PLoS Genet 12: e1006072. PubMed ID: 27233051
Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. This study identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation.
|Brock, A. R., Seto, M. and Smith-Bolton, R. K. (2017). Cap-n-collar promotes tissue regeneration by regulating ROS and JNK signaling in the Drosophila wing imaginal disc. Genetics [Epub ahead of print]. PubMed ID: 28512185
Regeneration is a complex process that requires an organism to recognize and repair tissue damage, as well as grow and pattern new tissue. This study describes a genetic screen to identify novel regulators of regeneration. The Drosophila melanogaster larval wing primordium was ablated by inducing apoptosis in a spatially and temporally controlled manner, and the tissue was allowed to regenerate and repattern. To identify genes that regulate regeneration, a dominant modifier screen was carried out by assessing the amount and quality of regeneration in adult wings heterozygous for isogenic deficiencies. Thirty-one regions on the right arm of the third chromosome were identified that modify the regenerative response. Interestingly, several distinct phenotypes were observed: mutants that regenerated poorly, mutants that regenerated faster or better than wild type, and mutants that regenerated imperfectly and had patterning defects. One deficiency region was mapped to cap-n-collar (cnc), the Drosophila Nrf2 ortholog, which is required for regeneration. Cnc regulates reactive oxygen species levels in the regenerating epithelium, and affects JNK signaling, growth, debris localization, and pupariation timing. This study presents the results of the screen and proposes a model wherein Cnc regulates regeneration by maintaining an optimal level of reactive oxygen species to promote JNK signaling.
|Pomatto, L. C., Wong, S., Carney, C., Shen, B., Tower, J. and Davies, K. J. (2017). The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster. Aging (Albany NY). PubMed ID: 28373600
Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C (Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H2O2) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female flies. Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H2O2, and blocking adaptive increases in proteasome expression also prevented full adaptation. This adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H2O2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keap1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.
|Tan, S. W. S., Lee, Q. Y., Wong, B. S. E., Cai, Y. and Baeg, G. H. (2017). Redox homeostasis plays important roles in the maintenance of the Drosophila testis germline stem cells. Stem Cell Reports [Epub ahead of print]. PubMed ID: 28669604
Oxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. This study utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in activity of Nrf2 and its cytoplasmic inhibitor Keap1 decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.
|Bhide, S., Trujillo, A. S., O'Connor, M. T., Young, G. H., Cryderman, D. E., Chandran, S., Nikravesh, M., Wallrath, L. L. and Melkani, G. C. (2018). Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan. Aging Cell: e12747. PubMed ID: 29575479
Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. This study modeled human disease-causing mutations in the Drosophila Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Simultaneous over-expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.
|Carlson, J., Swisse, T., Smith, C. and Deng, H. (2019). Regulation of position effect variegation at pericentric heterochromatin by Drosophila Keap1-Nrf2 xenobiotic response factors. Genesis: e23290. PubMed ID: 30888733
The Keap1-Nrf2 signaling pathway plays a central role in the regulation of transcriptional responses to oxidative species and xenobiotic stimuli. The complete range of molecular mechanisms and biological functions of Keap1 and Nrf2 remain to be fully elucidated. To determine the potential roles of Keap1 and Nrf2 in chromatin architecture, the effects of their Drosophila homologs (dKeap1 and CncC) were examined on position effect variegation (PEV), which is a transcriptional reporter for heterochromatin formation and spreading. Loss of function mutations in cncC, dKeap1, and cncC/dKeap1 double mutants all suppressed the variegation of w(m4) and Sb(V) PEV alleles, indicating that reduction of CncC or dKeap1 causes a decrease of heterochromatic silencing at pericentric region. Depletion of CncC or dKeap1 in embryos reduced the level of the H3K9me2 heterochromatin marker, but had no effect on the transcription of the genes encoding Su(var)3-9 and HP1. These results support a potential role of dKeap1 and CncC in the establishment and/or maintenance of pericentric heterochromatin. This study provides preliminary evidence for a novel epigenetic function of Keap1-Nrf2 oxidative/xenobiotic response factors in chromatin remodeling.
|Reedy, A. R., Luo, L., Neish, A. S. and Jones, R. M. (2019). Commensal microbiota induced redox signaling activates proliferative signals in the intestinal stem cell microenvironment. Development. PubMed ID: 30658986
A distinct taxon of the Drosophila microbiota, Lactobacillus plantarum, is capable of stimulating the generation of reactive oxygen species (ROS) within cells, and inducing epithelial cell proliferation. This study shows microbial-induced ROS generation within Drosophila larval stem cell compartments exhibits a distinct spatial distribution. Lactobacilli-induced ROS is strictly excluded from defined midgut compartments that harbor adult midgut progenitor (AMP) cells, forming a functional "ROS sheltered zone" (RSZ). The RSZ is undiscernible in germ-free larvae, but forms following mono-colonization with L. plantarum. L. plantarum is a strong activator of the ROS-sensitive CncC/Nrf2 signaling pathway within enterocytes. Enterocyte-specific activation of CncC stimulated the proliferation of AMPs, demonstrating that pro-proliferative signals are transduced from enterocytes to AMPs. Mechanistically, this study shows that the cytokine Upd2 is expressed in the gut following L. plantarum colonization in a CncC dependent fashion, and may function in lactobacilli-induced AMP proliferation and intestinal tissue growth and development.
Even a cursory look at the Drosophila head [Images] reveals an incredible elaboration of numerous structures: discrete, diverse and unique. What produces these complex yet well organized and differentiated results? Early in head differentiation the anterior posterior axis is marked by a subdivision into seven segments. The element primarily responsible for this subdivision is the action of gap genes, expressed in well defined anterior-posterior positions. Among the gap genes, buttonhead regulates cnc activation, and subsequently cnc regulates genes responsible for labral and mandibular development, specifically in the dorsal portion of the labral segment and the posterior lateral and ventral portion of the mandibular segment. cnc also functions in conjunction with Deformed, a homeotic gene expressed in the head.
In the trunk, segment polarity genes are activated by pair-rule genes, but this is not the case in the anterior of the embryo. Here gap genes activate segment polarity genes. The segment polarity genes hedgehog and wingless are two important targets of cnc and forkhead, expressed in the anterior and posterior gut anlagen. cnc is expressed in the labral region of the foregut, fated to give rise to the dorsal pharynx and fkh is expressed in the adjacent esophagus. fkh is responsible for the maintenance but not the initiation of wg synthesis in the invaginating esophageal primordium. cnc is responsible for the maintenance of wg in the dorsal pharyngeal domain of wingless expression. Expression of hedgehog is similarly affected in cnc and fkh mutants. It is not known whether the actions of cnc and fkh on hh and wg are direct or indirect (Mohler, 1995).
Deletion mutants of cnc coding sequences indicate that cnc functions are required for the normal development of both labral and mandibular structures (Mohler, 1995). In place of the missing mandibular structures, some maxillary structures - mouth hooks and cirri - are ectopically produced (Harding, 1995; Mohler, 1995). The genetic function of the homeotic gene Deformed (Dfd) is required in the cnc mutant background to produce ectopic mouth hooks, and Mohler (1995) have proposed that Dfd and cnc function in combination to specify mandibular identity. A protein isoform (CncB) from the Drosophila cap n collar locus has been characterized that selectively represses cis-regulatory elements that are activated by the Hox protein Deformed. Analysis of the cnc gene reveals the presence of three isoforms: cncA, cncB, and cncC. The expression patterns of the three transcript isoforms were analyzed using exon-specific probes both on wild-type and EMS-induced cnc mutants. In wild-type embryos, a cncB probe detects cytoplasmic transcripts limited to the mandibular and labral segments from cellular blastoderm to the end of embryogenesis. The cncB transcripts are expressed throughout both anterior and posterior regions of the mandibular lobes. In contrast, a cncA-specific probe detects a ubiquitous distribution of presumably maternal RNA at syncytial and early cellular blastoderm stages. After cellular blastoderm, cncA transcripts are not detectable until stage 14, when the level of ubiquitous cytoplasmic transcript increases and remains high for the remainder of embryogenesis. cncC-specific probes also detect a ubiquitous distribution of mRNA in syncytial stage embryos and a low level ubiquitous expression pattern in embryos after stage 14 (McGinnis, 1998).
Based on the above results, the labral and mandibular stripes of transcription that were detected by Mohler, (1991) using a probe including the cnc common exons (A2 and A3), correspond primarily to cncB transcripts. Since cncB is the transcript isoform that is expressed throughout the entire mandibular segment during mid-embyronic stages, cncB is likely to encode the principal function that modulates Dfd function in the mandibular segment (Harding, 1995). To further test this hypothesis, an assay was carried out to see whether cncB transcript or protein abundance is altered in embryos homozygous for the cnc2E16 and cncC7, mutations known to reveal interaction with Dfd. The pattern of zygotic RNA expression detected with a cncB probe is unaltered in the EMS-induced cnc mutant embryos. The signal due to cncA and cncC transcripts is also unchanged in these mutants. However, the use of polyclonal antiserum raised against the common domain of the cnc isoforms (anti-Cnc) indicates that CncB protein expression is strikingly reduced in both cnc2E16 and cncC7 mutant embryos. In wild-type embryos, the anti-Cnc antiserum exhibits a low-level ubiquitous staining in syncytial embryos, presumably due to maternally deposited CncA and CncC isoforms. From cellular blastoderm (stage 5) until stage 14, the staining detected by the anti-Cnc antiserum is localized in the nuclei of mandibular and labral cells. Although the anti-Cnc antiserum used in these experiments cross-reacts with all three Cnc proteins, only cncB RNA expression is localized in mandibular and hypopharyngeal regions from stages 6 through 14. cnc2E16 mutants (and cncC7 mutants) accumulate much lower levels of Cnc antigen in both mandibular and labral cells of stage 11 embryos. These results provide further evidence that the cnc2E16 and cncC7 mutations result in a loss of cncB function, and is consistent with the idea that CncB protein is required to prevent the maxillary-promoting function of Dfd from being active in mandibular cells (McGinnis, 1998).
In another test of the functions of the Cnc protein isoforms, each of the cncA, cncB and cncC open reading frames were placed under the control of the heat-shock promoter in P-element vectors and transgenic fly strains were generated carrying these constructs. Using the Cnc common-region antiserum to stain heat-shocked embryos, it appears that all three isoforms are produced at similar levels, localized in nuclei and possess similar stabilities after ectopic expression. However, their morphogenetic and regulatory effects are quite dissimilar. Heat-shock-induced ectopic expression of CncA during embryogenesis has no effect on embryonic morphology. Nearly all of the hs-cncA embryos hatch and proceed through larval development, and many eclose as viable adults. In contrast, ectopic expression of CncB at mid-stages (4-10 hours) of embryonic development is lethal. When ectopic expression is induced at 6 to 8 hours after egg lay, a defective embryonic head phenotype, which resembles the mutant phenotype of strong Dfd hypomorphs is produced. These hs-cncB embryos develop with rudimentary mouth hooks, H-piece and cirri. In addition, the anterior portion of the lateralgräten are truncated. All of these structures are components of the head skeleton that are absent or abnormal in Dfd mutant embryos. The head defects seen in the hs-cncB embryos also include an absent or abnormal dorsal bridge, a structure that is usually unaffected in Dfd mutant embryos. Many other head structures that develop in a Dfd-independent manner, such as the antennal sense organ, vertical plates and T-ribs develop normally in the hs-cncB embryos. The hs-cncB head defects are produced at high penetrance (>95%) by heat shocks in mid-embryogenesis (4-10 hours). In 10%-70% of these embryos, depending on the stage of heat shock, abdominal denticles near the ventral midline are replaced with naked cuticle. Ectopic induction of hs-cncC at 6-8 hours of development also results in highly penetrant defects in head development that include the loss of maxillary mouth hooks and cirri as well as head involution defects that are more profound than those induced by hs-cncB. In addition to the morphological defects described for CncB, ectopic CncC induces the formation of an abnormal head sclerite that develops as an extension of the normal lateralgräten. The position and appearance of this extra fragment of head skeleton suggests that it might correspond to ectopic production of lateralgräten or longitudinal arms of the H-piece (McGinnis, 1998).
Since CncB encodes a function that is required and sufficient to antagonize the maxillary-promoting effects of the Hox gene Dfd, it is reasonable to ask if CncB protein acts upstream to repress Dfd transcription, or in parallel to inhibit Dfd protein function? It is possible for CncB to do both, since Dfd protein function is required to establish an autoactivation circuit that provides persistent Dfd transcription in maxillary and mandibular cells. In wild-type embryos at stage 9, both Dfd and CncB proteins are expressed throughout the entire mandibular segment. By stage 11, Dfd protein is present at lower levels in the anterior, when compared to posterior mandibular nuclei, while CncB protein persists at relatively high levels throughout the segment. Finally, at stage 13, Dfd protein expression is no longer detected in anterior mandibular nuclei, although it is still abundant in posterior nuclei. cnc is required for this progressive repression of Dfd expression in the anterior mandibular segment, since cnc null mutants as well as the EMS-induced mutants show inappropriate persistence of Dfd transcripts and protein after stage 11 in anterior mandibular cells. All of these data suggest that CncB is not capable of repressing Dfd expression before stage 11. But after this stage, CncB represses the maintenance phase of Dfd transcription in mandibular cells, perhaps by repressing the autoactivation circuit that is normally established during stages 9 and 10 (Zeng et al., 1994). CncB is found to be sufficient to repress Dfd transcription outside the mandibular segment. When CncB is ectopically expressed in embryos, Dfd transcript levels in the maxillary segment are reduced, especially in the anterior region of the segment. Only the CncB isoform is capable of repressing Dfd transcription. Neither the ectopic expression of CncA nor CncC have an effect on the abundance or pattern of Dfd transcripts in the maxillary epidermis. Since the phenotypic effect of hs-cncC in epidermal cells strongly resembles that of hs-cncB, this indicates that the effect of Cnc gene products on maxillary epidermal development may not require repression of Dfd transcription per se. However, various experiments show that the maxillary-promoting function of Dfd protein is reduced in the presence of CncB; this could either be due to CncB-mediated repression of the Dfd autoactivation circuit in ectopic positions or to CncB repression of downstream target elements of Dfd protein, or to both of these effects. It is concluded that CncB provides a mechanism to modulate the specificity of Hox morphogenetic outcomes, which results in an increase in the segmental diversity in the Drosophila head. (McGinnis, 1998).
In Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question of which gene activities fulfill the role of the second order regulators still remains to be solved. This study provides first molecular evidence that the Helix-Loop-Helix-COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, a direct physical association has been identified between Collier and the basic-leucine-zipper transcription factor Cap'n'collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages (Ntini, 2011b).
In the context of an analysis to identify cis-regulatory elements controlling expression of segment polarity genes in the embryonic head, an intercalary-specific cis-regulatory element of hh—ic-CRE—was isolated within the upstream 6.43 kb region (Ntini, 2011a). The ~ 1 kb enhancer fragment (− 4085 to − 3077 bp) mediates reporter expression in the hh expressing cells of the posterior part of the intercalary segment, when combined with the endogenous hh promoter (− 120 to + 99 bp;). Further functional dissection of this element showed that the 450 bp ?1mF5 subfragment (− 3914 to − 3465 bp) mediates the intercalary-specific expression with slightly delayed onset, while the 335 bp F5_R4 subfragment (− 3799 to − 3465 bp) constitutes the minimum sequence required for the intercalary expression, but mediates an additional spotty metameric pattern in the trunk (Ntini, 2011). Because a high degree of phylogenetic conservation in non-coding DNA sequence implicates a functional role in vivo, such as recognition and DNA-binding by sequence-specific transcription factors, the sequence of the ic-CRE was subjected to phylogenetic conservation analysis within the genome of twelve Drosophila species, and different in silico analyses were performed to detect putative transcription factor binding sites. The minimum 335 bp ic-CRE consists of six highly conserved sequence blocks. A series of complete block deletions designed in the context of the minimum ic-CRE in combination with the endogenous hh promoter resulted in non-functional elements. This could be either because individual binding motifs were disrupted or inter-motif distances crucial for transcription factor binding and operation were disturbed. A point mutagenesis screen was conducted in the context of the 450 bp ic-CRE to extract crucial cis-regulatory information in respect to the conserved in silico identified transcription factor binding sites (Ntini, 2011b).
The ic-CRE responds to the homeotic transformation of the mandibular into an intercalary segment resulting from ectopic ems expression by a duplication of its expression pattern. However, despite this and the fact that the Hox gene labial is active in the intercalary segment, disrupting the homeodomain binding sites in conserved sequence blocks III or IV by point mutations did not abolish the ic-CRE mediated reporter expression. In contrast, disrupting a putative binding site for the fork head transcription factor Sloppy paired 1 (Slp1) in block IV eliminated the ic-CRE-mediated reporter expression. This is consistent with the reduced reporter expression in an RNAi-mediated knock-down of slp1, which is a proposed head gap-like and pair rule segmentation gene (Ntini, 2011b).
Another in silico prediction was found in conserved block II at position − 3771 to − 3755 bp that scores the binding matrix of the mammalian COE factor Olf1. Disrupting this site by point-mutation resulted in the complete abolishment of the ic-CRE mediated reporter expression, indicating that the site is absolutely required for the function of the 450 bp ic-CRE. Olf1 is the mammalian COE homolog of Collier and the endogenous hh expression in the intercalary segment is abolished in a col loss-of-function mutant (col1. Likewise, the ic-CRE-mediated expression pattern is abolished in col1 or col knock-down. In addition, the DNA-binding domain of Collier displays a high degree of primary sequence identity (86%) to the mammalian homolog. High degree of primary sequence identity in the DNA-binding domain, shared among the members of the COE family allows for a similar DNA-binding specificity: both Collier and the Xenopus homologs recognize the mammalian DNA target sequences in vitro. Therefore, the Olf1 prediction identified in silico within the ic-CRE is regarded as a putative Collier binding site and referred to as a Collier recognition site (Ntini, 2011b).
Apart from this functionally required Collier recognition site at − 3773 to − 3751 bp, scanning in silico the 6.43 kb upstream hh enhancer using MatInspector with a similarity cut-off of 1, 0.8 (core, matrix) identifies one more Olf1 prediction within the ic-CRE at position − 3967 to − 3945 bp. The 6.43 kb upstream enhancer of hh was also submitted to rVISTA using the nucleotide positions 3–19 of the binding matrix of Olf1. When setting the highest possible similarity cut-off 0.95, 0.85 (core, matrix), so that at least one prediction is generated, then only the functionally required Collier recognition site CAATTCCCCAATGGCAT (at − 3771 to − 3755) within the ic-CRE is detected. Lowering the matrix similarity threshold by 0.05, using cut-off 0.95, 0.8, generates three additional predictions. These are two distant sites, GAGACACTTGGGATGAG at − 3963 to − 3947 and CACACCACGGGGAAGCG at − 2872 to − 2856, and one promoter-proximal site CACTTCCCTTGCGCATA at − 212 to − 196. The first distant site is within the ic-CRE, 190 bp upstream of the functionally required Collier recognition site, and is also predicted by the MatInspector. Interestingly, in contrast to the functionally required Collier recognition site within the ic-CRE, none of the other predicted sites are phylogenetically conserved among the twelve Drosophila species. Considering the displayed short-range homotypic clustering (within 200 bp), it is, however, possible that the weaker predictions may contribute to the transcriptional outcome of the ic-CRE, even though they might be recognized with minor affinity by Collier in vivo (Ntini, 2011b).
In order to verify that the in silico identified and functionally required Collier recognition site within the ic-CRE is indeed occupied by Collier in vivo, chromatin immunoprecipitations (ChIP) from Drosophila embryonic nuclear extracts were performed with an antibody against Collier. In the anti-Col ChIPs, the functionally required Collier binding site within the ic-CRE was specifically enriched in comparison to mock ChIPs, which indicates that the site is indeed occupied by Collier in vivo (Ntini, 2011b).
In the case of the mammalian COE homolog of Collier, it was previously deciphered that the mouse transcription factor EBF contains two distinct and functionally independent transcription activation domains, the second one within the C-terminal region. Although Drosophila Collier has been genetically implicated as an activator of downstream segment polarity gene expression, its transcriptional activation potential had not yet been analyzed. In Drosophila two Collier isoforms are expressed from the col gene locus. The cDNAs encoding Collier A (also termed Col2) and Collier B (Col1) differ from each other by 465 bp due to alternative splicing. The two protein isoforms share the same first 528 N-terminal amino acids and differ in the C-terminal 29 amino acids for Collier A and 47 amino acids for Collier B. No specific expression pattern of collier A could be detected by double in situ hybridization using an RNA probe specific for collier B and a probe that hybridizes with both transcripts (Ntini, 2011b).
Therefore the transcriptional activation potential of each of the two Collier isoforms was examined by reporter assays in Drosophila S2 R+ cell transfections. In the reporter construct the functionally required and in vivo occupied Collier site was cloned in a single copy upstream of the endogenous hh promoter (− 120 to + 99 bp) driving luciferase gene expression. Both Collier isoforms activate luciferase expression when independently co-transfected with the reporter construct, indicating that both isoforms possess transcriptional activation potential. A truncated form of ColA lacking the last 23 C-terminal amino acids (ColA 1–534) displays a significantly reduced activation potential (~ 84% decrease), which indicates that a transcriptional activation domain must reside within either C-terminal region of both isoforms. Disrupting the Collier recognition site by point mutations decreased the mediated reporter activation by ~ 48% in the case of Collier A and ~ 44% in the case of Collier B. Taking into consideration that disrupting the Collier binding site in the context of the ic-CRE resulted in a complete abolishment of the mediated reporter expression in vivo, and that the same mutation does not support Collier DNA-binding in vitro, it is assumed that part of the reporter activation assessed in cell transfection may be achieved by Collier transactivating via unknown system-provided DNA-binding activities on the regulatory sequences of the reporter plasmid. Moreover, Collier carries a perfect SUMOylation motif within the N-terminus, predicted with the highest threshold value. The protein sequence TSLKEEP at amino acid position 44-50 matches the SUMOylation motif. Additional members of the COE transcription factor family contain also a SUMOylation motif at this conserved position. Apart from antagonizing ubiquitin-mediated degradation, sumoylation has been implicated in modifying transcriptional activation/repression potential of transcription factors. Mutant versions of Collier A and Collier B where the K within the SUMOylation motif is mutated towards R (ColA RK and ColB RK) display reduced activation potential, implying a possible role for sumoylation in regulation of Collier transcriptional activity (Ntini, 2011b).
Data is presented consistent with the cap-n-collar isoform CncB performing as a sequestering factor or inhibitor of Collier DNA-binding to its cognate site found within the ic-CRE. Furthermore, fluorescent immunostaining revealed that only a small fraction of the expressed Collier protein is nuclear localized in vivo. Conversely, CncB protein greatly accumulates in the nuclei. Prediction of nuclear localization signals (NLS) in silico generates no results for Collier, while CncB contains an NLS within the bZIP domain (aa 617–680). Interestingly, Collier carries a perfect SUMOylation motif in the very N-terminus, predicted with the highest threshold value. Apart from antagonizing ubiquitin-mediated degradation and modifying transcriptional activation/repression potential of transcription factors, sumoylation has also been implicated in protein nucleo-cytoplasmic translocation. Alternatively, in the absence of a nuclear localization signal, Collier import in the nucleus may be realized by heterodimerization with a protein that carries an NLS. This would increase the probability that Collier is recruited into combinatorial control mechanisms, which has already been implicated in muscle specification. Furthermore, nuclear accumulation of CncB, in converse to a relatively low concentration of nuclear Collier protein, indicated by the fluorescent immunostainings, may facilitate the sequestering function of CncB to antagonize and overcome the DNA-binding activity of Collier on the ic-CRE in the cells of the anterior most part of the mandibular segment during the establishment of procephalic hh expression, and at later stages in the hh expressing cells of the intercalary lobes (Ntini, 2011b).
In this respect it is interesting to note that despite the intrinsic transcriptional activation properties of the Cnc homologs, CncB acts to suppress both the expression and the homeotic selector (maxillary structures promoting) function of Deformed (Dfd) in the mandibular segment. In particular, CncB represses the maintenance phase of Dfd transcription in the mandibular cells, most probably by interfering with the positive regulatory function of Deformed within the Dfd autoactivation circuit. Overexpression of CncB partially represses Dfd-responsive transcriptional target elements in vivo. Interestingly, interaction between CncB and Dfd proteins has been reported. Perhaps the negative regulation of Dfd expression and function caused by CncB results from CncB interfering with Dfd binding to its cognate target cis-regulatory elements in vivo, as a consequence of a direct physical interaction at protein level with a sequestering effect similar to the interaction with Collier reported in this study (Ntini, 2011b).
The isolation of an intercalary-specific cis-regulatory element from the hh upstream region supports a unique mode for anterior head segment-specific transcriptional control of segment polarity gene expression. Thus, not only cross-regulatory interactions among segment polarity genes during the maintenance phase, but also the initial establishment of procephalic segment polarity gene expression seems to be unique for each of the anterior head segments. The previously proposed mode of second order regulation in anterior head patterning, resulting in activation of hh in the posterior part of the intercalary segment, is mediated by the HLH-COE factor Collier evidently via direct DNA binding. The reported physical interaction between Collier and CncB is likely to attenuate the activating function of Collier in the hh expressing cells of the posterior part of the intercalary segment at a later developmental stage, and it might also be involved in eliminating the potential of target activation by Collier in the anterior most part of the mandibular segment where the two factors are co-expressed (Ntini, 2011b).
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).
Three EMS-induced mutant alleles of cnc (cnc2E16, cncC7 and cncC14) in a screen for mutations that interact with the Hox gene Dfd (Harding, 1995). Embryos homozygous for these EMS-induced alleles have ectopic duplications of maxillary mouth hooks and cirri, but retain normal labral structures and some normal mandibular structures, e.g. the lateralgräten and median tooth. This contrasts with the phenotype of deletion mutants of cnc, which lack all mandibular and labral derivatives. The difference between the phenotypes of the EMS-induced alleles when compared to the deletion alleles prompted a consideration of the possibility that multiple functions are encoded in the cnc locus. Previous studies detected one transcript isoform at cnc, but the current molecular analyses of the locus indicates that three transcript and protein isoforms are produced from the cnc gene. A probe homologous to the region that encodes the b-ZIP region of cnc detects three different sizes of polyadenylated RNAs on embryonic northern blots. These will be referred to as the cncA, cncB and cncC transcripts. The 3.3 kb cncA transcript is present in 0-2 hour embryos, presumably from maternal stores and is also abundantly expressed in 12-24 hour embryos. The 5.4 kb cncB transcript is absent from 0-2 hour embryos, but present at all other embryonic stages. The 6.6 kb cncC transcript is present in 0-2 hour embryos, is barely detected in 2-12 hour embryos and is detected at relatively higher levels in 12-24 hour embryos. The sequence of all of the coding exons and exon/intron boundaries for all isoforms on the cnc2E16 and cncC7 mutant chromosomes was determined in an attempt to find the molecular lesion responsible for the decreased amount of CncB protein in the mutant embryos. However, no nucleotide substitutions were detected when the coding and splice site sequences were compared with parental chromosome sequence. Though the location of the mutations that alter CncB protein expression are not yet known, they could plausibly reside in translational regulatory sequences for cncB (McGinnis, 1998).
To identify cDNAs corresponding to the cncA, cncB and cncC transcripts, 212 cDNA clones from libraries covering all stages of Drosophila embryonic development were isolated and characterized. The first class of cDNAs corresponds to the cncA transcript. This is the same class characterized by Mohler, 1991, and is distinguished by the incorporation of exon A1. Exons A2 and A3, which encode the CNC and b-ZIP domains, are present in cncA and the other two isoforms of cnc. A probe containing exon A1 sequences specifically hybridizes the 3.3 kb cncA transcript on northern blots. The cncA open reading frame begins with an ATG codon near the 5' end of exon A2 and is predicted to encode a 533 amino acid protein (Mohler, 1991). A second class of cDNAs from the locus corresponds to cncB transcripts. Such cDNAs lack sequences from exon A1, but contain five additional exons (B1-B5) spliced onto the 5' end of exon A2. Each of these additional exons is found upstream of exon A1. A probe containing the B1-B4 exons detects the 5.4 kb cncB transcript and the 6.6 kb cncC transcript on Northern blots. The total extent of the cncB transcription unit is approximately 17 kb. Since exon A2 sequences contain no stop codons upstream of the initiating ATG for the CncA codons, the open reading frame in cncB transcripts includes the entirety of the CncA protein, as well as an additional 272 codons from exons B3, B4, B5 and A2. The predicted 805 amino acid CncB protein thus is distinguished from CncA by a 272 amino acid region that includes His-Pro repeats, Ala-repeats, a Pro-repeat and Val-Gly repeats, but the region exhibits no extended sequence similarity to other proteins in database searches, other than the CNC/b-ZIP domain that it shares with CncA. The third class of cDNAs from the locus corresponds to cncC transcripts. These cDNAs have identical sequences as the cncB cDNAs, except that exon B1 is absent, and five additional exons (C1-C5) are spliced onto the 5' end of exon B2. Each of these five additional exons are found upstream of the B1 exon. A probe containing the C1-C4 exons detects the 6.6 kb cncC transcript on Northern blots. Since exon B2 and the 5' end of exon B3 contain no stop codons upstream of the initiating ATG for the CncB codons, the ATG-initiated open reading frame in cncC transcripts includes the entirety of the CncB protein, as well as an additional 491 codons that derive from the C3, C4, C5, B2 and B3 exons. The extent of the entire cncC transcription unit is approximately 39 kb. The 491 amino acid CncC-specific domain at the N terminus of the predicted 1296 residue CncC protein includes regions that are rich in Ser and Thr residues, other regions with abundant concentrations of Glu and Asp residues, but exhibits no extended sequence similarity to other proteins in database searches. Interestingly, the fuzzy onions gene, which encodes a testis protein required for mitochondrial fusion in Drosophila spermatids (Hales, 1997), is encoded in the sequence interval between the C5 and B1 exons (McGinnis, 1998).
Bases in 5' UTR - 94
Bases in 3' UTR - 1028
Amino Acids - 533
The leucine zipper of CNC is longer than bZIP proteins and contains six heptad repeats. The function of the leucine zipper is considered as a protein interaction domain. The leucine zipper of CNC is divergent from the typical sequence (Mohler, 1991).
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