cubitus interruptus : Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - cubitus interruptus

Synonyms - ci dominant

Cytological map position - 102A3

Function - putative transcription factor

Keywords - segment polarity, hedgehog pathway, oncogene

Symbol - ci

FlyBase ID:FBgn0004859

Genetic map position - 4-0.0

Classification - zinc finger

Cellular location - cytoplasmic and nuclear



NCBI link: Entrez Gene
ci orthologs: Biolitmine

Recent literature
Shi, Q., Li, S., Li, S., Jiang, A., Chen, Y. and Jiang, J. (2014). Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator.Proc Natl Acad Sci U S A 111: E5651-5660. PubMed ID: 25512501
Summary:
Hedgehog (Hh) signaling governs many developmental processes by regulating the balance between the repressor (CiR/GliR) and activator (CiA/GliA) forms of Cubitus interruptus (Ci)/glioma-associated oncogene homolog (Gli) transcription factors. Although much is known about how CiR/GliR is controlled, the regulation of CiA/GliA remains poorly understood. This study, carried out in larval wing discs demonstrates that Casein kinase 1 (CK1) sustains Hh signaling downstream of Costal2 and Suppressor of fused (Sufu) by protecting CiA) from premature degradation. Hh stimulates Ci phosphorylation by CK1 at multiple Ser/Thr-rich degrons to inhibit its recognition by the Hh-induced MATH and BTB domain containing protein (HIB), a substrate receptor for the Cullin 3 family of E3 ubiquitin ligases. In Hh-receiving cells, reduction of CK1 activity accelerated HIB-mediated degradation of CiA, leading to premature loss of pathway activity. Evidence that GliA is regulated by CK1 in a similar fashion and that CK1 acts downstream of Sufu to promote Sonic hedgehog signaling. Taken together, this study not only reveals an unanticipated and conserved mechanism by which phosphorylation of Ci/Gli positively regulates Hh signaling but also provides the first evidence that substrate recognition by the Cullin 3 family of E3 ubiquitin ligases is negatively regulated by a kinase.

Zhou, Z., Yao, X., Li, S., Xiong, Y., Dong, X., Zhao, Y., Jiang, J. and Zhang, Q. (2015). Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling. Dev Cell 34: 58-72. PubMed ID: 26120032
Summary:
Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. This study shows that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-Cul3 E3 ligases. Furthermore, Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, it was shown that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. These findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers.

Li, C., Kan, L., Chen, Y., Zheng, X., Li, W., Zhang, W., Cao, L., Lin, X., Ji, S., Huang, S., Zhang, G., Liu, X., Tao, Y., Wu, S. and Chen, D. (2015). Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation. Cell Res [Epub ahead of print]. PubMed ID: 26403189
Summary:
Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. This study identified that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.

Gurdziel, K., Lorberbaum, D. S., Udager, A. M., Song, J. Y., Richards, N., Parker, D. S., Johnson, L. A., Allen, B. L., Barolo, S. and Gumucio, D. L. (2015). Identification and validation of novel Hedgehog-responsive enhancers predicted by computational analysis of Ci/Gli binding site density. PLoS One 10: e0145225. PubMed ID: 26710299
Summary:
The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) contain more than one Ci/Gli site, prompting this study to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. A computational algorithm was developed to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) gene. It is concluded that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci.

Nguyen, D., Fayol, O., Buisine, N., Lecorre, P. and Uguen, P. (2016). Functional interaction between HEXIM and Hedgehog signaling during Drosophila wing development. PLoS One 11: e0155438. PubMed ID: 27176767
Summary:
Studying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcriptional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. This study examines the function of HEXIM during Drosophila development. It was found that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knockdown initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knockdown mutant phenotype does not result from the apoptotic ablation of imaginal disc but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, ci was shown to be a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism. 

Han, Y., Shi, Q. and Jiang, J. (2015). Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction. Proc Natl Acad Sci U S A 112: 6383-6388. PubMed ID: 25941387
Summary:
The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. This study shows that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. It was also found that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an 'exposed' location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, this study identifies a previously unidentified and conserved Sufu-binding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.
Garcia Garcia, E., Little, J. C. and Kalderon, D. (2017). The exon junction complex and Srp54 contribute to Drosophila Hedgehog signaling via ci RNA splicing. Genetics [Epub ahead of print]. PubMed ID: 28637711
Summary:
Hedgehog (Hh) regulates the Cubitus interruptus (Ci) transcription factor in Drosophila melanogaster by activating full-length Ci-155 and blocking processing to Ci-75 repressor. However, the interplay between regulation of Ci-155 levels and activity, as well as processing-independent mechanisms that affect Ci-155 levels have not been explored extensively. This study has identified Mago Nashi (Mago) and Y14 core Exon Junction Complex (EJC) proteins, as well as the Srp54 splicing factor as modifiers of Hh pathway activity under sensitized conditions. Mago inhibition reduced Hh pathway activity by altering the splicing pattern of ci to reduce Ci-155 levels. Srp54 inhibition also affected pathway activity by reducing ci RNA levels but additionally altered Ci-155 levels and activity independently of ci splicing. Further tests using ci transgenes and ci mutations confirmed evidence from studying the effects of Mago and Srp54 that relatively small changes in the level of Ci-155 primary translation product alter Hh pathway activity under a variety of sensitized conditions. ci transgenes lacking intron sequences or the presumed translation initiation codon were used for an alternatively spliced ci RNA to provide further evidence that Mago acts principally by modulating the levels of the major ci RNA encoding Ci-155, and to show that ci introns are necessary to support production of sufficient Ci-155 for robust Hh signaling and may also be important mediators of regulatory inputs.
Pan, C., Xiong, Y., Lv, X., Xia, Y., Zhang, S., Chen, H., Fan, J., Wu, W., Liu, F., Wu, H., Zhou, Z., Zhang, L. and Zhao, Y. (2017). UbcD1 regulates Hedgehog signaling by directly modulating Ci ubiquitination and processing. EMBO Rep. PubMed ID: 28887318
Summary:
The Hh pathway controls many morphogenetic processes in metazoans and plays important roles in numerous pathologies and in cancer. Hh signaling is mediated by the activity of the Gli/Ci family of transcription factors. Several studies in Drosophila have shown that ubiquitination by the ubiquitin E3 ligases Slimb and Rdx(Hib) plays a crucial role in controlling Ci stability dependent on the levels of Hh signals. If Hh levels are low, Slimb adds K11- and K48-linked poly-ubiquitin chains on Ci resulting in partial degradation. Ubiquitin E2 enzymes are pivotal in determining the topologies of ubiquitin chains. However, which E2 enzymes participate in the selective ubiquitination-degradation of Ci remains elusive. This study finds that the E2 enzyme UbcD1 negatively regulates Hh signaling activity in Drosophila wing discs. Genetic and biochemical analyses in wing discs and in cultured cells reveal that UbcD1 directly controls Ci stability. Interestingly, UbcD1 is found to be selectively involved in Slimb-mediated Ci degradation. Finally, it was shown that the homologs of UbcD1 play a conserved role in modulating Hh signaling in vertebrates.
Cambon, M. and Sanchez, O. (2019). Analysis of the transcriptional logic governing differential spatial expression in Hh target genes. PLoS One 14(1): e0209349. PubMed ID: 30615641
Summary:
This work provides theoretical tools to analyse the transcriptional effects of certain biochemical mechanisms (i.e. affinity and cooperativity) that have been proposed in previous literature to explain the proper spatial expression of Hedgehog target genes involved in Drosophila development. Specifically this study has focused on the expression of decapentaplegic, wingless, stripe and patched. The transcription of these genes is believed to be controlled by enhancer modules able to interpret opposing gradients of the activator and repressor forms of the transcription factor Cubitus interruptus (Ci). This study is based on a thermodynamic approach, which provides expression rates for these genes. These expression rates are controlled by transcription factors which are competing and cooperating for common binding sites. Mathematical representations have been made of the different expression rates which depend on multiple factors and variables. The expressions obtained with the model have been refined to produce simpler equivalent formulae which allow for their mathematical analysis. Thanks to this, the correlation between the different interactions involved in transcription and the biological features observed at tissue level can be evaluated. These mathematical models can be applied to other morphogenes to help understand the complex transcriptional logic of opposing activator and repressor gradients.
Sharma, S. K., Ghosh, S., Geetha, A. R., Mandal, S. and Mandal, L. (2019).. Cell adhesion-mediated actomyosin assembly regulates the activity of Cubitus interruptus for hematopoietic progenitor maintenance in Drosophila. Genetics. PubMed ID: 31138608
Summary:
The actomyosin network is involved in crucial cellular processes including morphogenesis, cell adhesion, apoptosis, proliferation, differentiation and collective cell migration in Drosophila, C. elegans and mammals. This study demonstrates that Drosophila larval blood stem-like progenitors require actomyosin activity for their maintenance. Genetic loss of actomyosin network from the progenitors caused a decline in their number. Likewise, the progenitor population increased upon sustained actomyosin activation via phosphorylation by Rho-associated kinase. This study shows that actomyosin positively regulates larval blood progenitors by controlling the maintenance factor Cubitus interruptus (Ci). Overexpression of the maintenance signal via a constitutively activated construct (ci.HA) failed to sustain Ci-155 in the absence of actomyosin components like Zipper (zip) and Squash (sqh), thus favoring protein kinase A (PKA)-independent regulation of Ci activity. Furthermore, this study demonstrates that a change in cortical actomyosin assembly mediated by DE-cadherin modulates Ci activity, thereby determining progenitor status. Thus, loss of cell adhesion and downstream actomyosin activity results in desensitization of the progenitors to Hh signaling, leading to their differentiation. These data reveal how cell adhesion and actomyosin network cooperate to influence patterning, morphogenesis, and maintenance of the hematopoietic stem-like progenitor pool in the developing Drosophila hematopoietic organ.
Li, Y., Sun, X., Gao, D., Ding, Y., Liu, J., Chen, J., Luo, J., Zhang, J., Liu, Q. and Zhou, Z. (2020). Dual functions of Rack1 in regulating Hedgehog pathway. Cell Death Differ. PubMed ID: 32467643
Summary:
Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. This study shows that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci-Rack1-Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci-Rack1-Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, this study finds the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. These findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.
Zhou, M., Han, Y., Wang, B., Cho, Y. S. and Jiang, J. (2022). Dose-dependent phosphorylation and activation of Hh pathway transcription factors. Life Sci Alliance 5(11). PubMed ID: 36271509
Summary:
Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. This study shows that graded Hh in Drosophila induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. This study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition.
Gao, Y., Shan, Z., Jian, C., Wang, Y., Yao, X., Li, S., Ti, X., Zhao, G., Liu, C. and Zhang, Q. (2023). HIB/SPOP inhibits Ci/Gli-mediated tumorigenesis by modulating the RNA Polymerase II components stabilities. iScience 26(8): 107334. PubMed ID: 37554435
Summary:
Hedgehog (Hh) signaling mediated by transcription factor Ci/Gli plays a vital role in embryonic development and adult tissue homeostasis in invertebrates and vertebrates, whose dysregulation leads to many human disorders, including cancer. However, till now, cofactors of Ci/Gli which can affect tumorigenesis are not well known. Through genetic screen, this study found overexpression of active Ci alone is not sufficient to generate tumor-like eye phenotype in Drosophila, however, its overexpression combined with knockdown of hib causes a striking tumor-like big eye phenotype. Mechanistically, HIB/SPOP inhibits Ci/Gli-mediated tumorigenesis by modulating the RNA polymerase II (RNAPII) components Rpb3/Rpb7 stabilities in E3 ligase dependent manner. In addition, Ci/Gli can promote HIB/SPOP-mediated Rpb7/Rpb3 degradation. Taken together, these results indicate Ci/Gli needs to hook up with suitable RNAPII together to achieve the tumor-like eye phenotype and HIB/SPOP plays dual roles through controlling Ci/Gli and Rpb3/Rpb7 protein stabilities to temper Ci/Gli/RNAPII-mediated tumorigenesis.
BIOLOGICAL OVERVIEW

cubitus interruptus is a segment polarity gene. Mutations in segment polarity genes cause a disruption in pattern formation in each segment of the fly. This is a serious problem because the differentiation between anterior and posterior compartments of a segment are crucial to appropriate differentiation for each appendage. In humans, for example, segment polarity makes possible the correct differentiation between the thumb and the little finger.

The Drosophila melanogaster wing imaginal disc is subdivided into an anterior (A) and a posterior (P) compartment. P cells heritably express the selector gene engrailed (en) which directs these cells to secrete the short-range signaling molecule Hedgehog (Hh) and at the same time makes P cells refractory to the Hh signal. In contrast, A cells do not express En and, as a consequence, can receive and respond to Hh. The response to Hh requires Smoothened (Smo), a seven-pass transmembrane protein, and the transcription factor Cubitus interruptus (Ci), the Drosophila Gli homolog. Ci is expressed exclusively in A cells, where it can exist in two forms. A repressor form of Ci (Ci[rep]) is generated in A cells that do not receive the Hh signal, and an activator form of Ci (Ci[act]) is generated in A cells that receive the Hh signal. Both forms of Ci control the transcription of the decapentaplegic (dpp) gene, which, as a consequence, is expressed only in a thin strip of A cells along the A/P boundary. dpp encodes a member of the transforming growth factor ß (TGFß) superfamily which induces the expression of target genes in a concentration dependent manner in both compartments. The stable and precise positioning of the Dpp morphogen source is crucial for growth and patterning of the entire wing. It is critically dependent on the continuous segregation of En-expressing (Hh-secreting) and non-En-expressing cells into distinct but apposing P and A compartments, respectively (Dahmann, 2000 and references therein).

A functional approach has been taken to define the minimal regions of both the CI protein and the cis-acting regulatory regions of the gene patched, sufficient to mediate its transcriptional activation. The zinc finger domain of CI alone, when fused to the herpes simplex activation domain, can activate transcription of patched in imaginal discs, indicating that the specificity of CI activity is determined by its putative DNA binding domain. In addition, CI can serve as a transcriptional activator in a yeast synthetic promoter. Finally, a 758-bp patched upstream regulatory element, that directs robust expression along the anteroposterior compartment boundary, contains three consensus CI zinc finger binding sites, which when mutated completely abolish expression. All told, circumstantial evidence is leaning to the conclusion that CI acts as a transcription factor to regulate Hedgehog target genes (Alexandre, 1996).

Although Cubitus interruptus is thought to have roles as a transcription factor repressing hh expression and activating target genes, it localizes in the cytoplasm of anterior compartment cells. How can a cytoplasmic protein act as a transcription factor? Different forms of Ci protein are found in wing imaginal disc cells. Whereas sequences bearing the N-terminal domain and the central zinc finger domains are located uniformly throughout the anterior compartment, the C-terminal is found at the A/P compartment border and is found less prominently in the rest of the anterior compartment. Antibody to the N-terminal domain recognizes proteins with approximate molecular weights of 155 and 75 kDa. The size of the larger species is consistent with the predicted open reading frame of ci cDNA, while the 75 kDa species is a novel Ci product and is designated Ci75. Ci75 is produced from the full-length protein by proteolysis. The C-terminal domain tethers Ci in the cytoplasm. Residues between 703 and 850 fulfil this function. In some anterior cells, Ci is cleaved to generate a form that lacks the tethering domain. This form translocates to the nucleus where it represses hh and other target genes, including patched. In fact, DNA-binding activity can be detected in Ci fragments containing the zinc finger region (Aza-Blanc, 1997).

Hh is believed to inhibit proteolysis of Ci. Three results supply evidence for this: antibody to the N-terminal domain of Ci stains nuclei of anterior domain cells at the compartment border at a diminished level; Ci75 disapppears from CI producing cells treated with Hh-conditioned medium, and in wing discs overexpressing Hh, levels of Ci75 and of nuclear staining decrease. It is suggested that this inhibition leads to the observed patterns of expression of key target genes at the compartment border. Because Hh inhibits proteolysis of Ci, it has not been explained why cells anterior to the A-P border, exposed to high Hh levels, do not express Hh. Perhaps sufficient levels of Ci75 remain in these cells to repress hh or hh is repressed by another, as yet unknown, factor in these border cells (Aza-Blanc, 1997).

Since ectopic expression of full-length Ci in the posterior compartment induces patched, but ectopic activation of Ci75 does not, an activator function appears to be associated with expression of full-length Ci. Indeed, the C-terminal region of Ci might contain a transcriptional activation domain. The simplest model is that ptc is directly induced by an activated form of the full-length Ci protein. These experiments do not explain the expression of dpp at the A-P border (Aza-Blanc, 1997).

Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Evidence is provided for the existence of a distinct activator form of Ci, which does not arise by mere prevention of Ci proteolysis, but rather depends on a separate regulatory step subject to Hh control. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. Thus, limb development is organized by the integration of different transcriptional outputs of Hh signaling (Methot, 1999).

Although ci was identified more than 60 years ago, analysis of its role has been severely hindered by two obstacles. First, the ci gene is located on the fourth chromosome, imposing major technical difficulties in studying its role during development. Second, no bona fide null allele has been available. These difficulties have been overcome by identifying a true null allele and generating genomic transgenes that carry all or only some of the activities of the ci+ locus inserted at other chromosomal locations, enabling the generation of mutant cell clones by Flp-mediated mitotic recombination (Methot, 1999 and references).

A candidate null allele of ci is ci94. This mutation was generated by the imprecise excision of a P element located in the promoter region of the ci gene. Homozygous ci94 mutant embryos die and display a segment polarity defect with a deficit of naked cuticle. The molecular lesion of ci94 was determined by sequence analysis and found to contain a 5 kb deletion. This deletion removes the promoter and the first exon of ci and consequently the sites for transcriptional and translational initiation. Thus, ci94 represents a bona fide null allele of ci (Methot, 1999).

A candidate allele of ci that may code for a repressor form of ci is ciCell-2 (here referred to as ciCell). This allele gives rise to a protein that is smaller than wild-type Ci and accumulates to high levels throughout the anterior compartment of wing imaginal discs. ciCell differs from wild-type ci by an 8 bp deletion that is expected to result in a truncation of the Ci protein product at amino acid 975. Expression of a transgene encoding CiCell protein represses transcription of Ci target genes in the wing imaginal disc. These findings and the dominant phenotype associated with ciCell suggest that the mutant protein acts as a constitutive inhibitor of Hh target gene expression (Methot, 1999).

Homozygous ci94 animals are rescued to adulthood by one copy of a ci+ transgene that contains 16 kb of the ci locus. Rescued flies are healthy, fertile, and exhibit no obvious phenotypes. In contrast, ciCell homozygous animals cannot be rescued by one copy of the genomic construct, but survive to late pupal stages with rare adult escapers when two copies are present. This result is consistent with the interpretation that CiCell functions in a dominant-negative manner. Indeed, a single ciCell allele (in ciCell /ci94 animals) is rescued with one copy of the ci+ transgene. Wings of such animals exhibit an extreme form of the dominant ciCell phenotype, that is, a fusion of the longitudinal veins L3 and L4. This phenotype is absent in ciCell /ci94 animals rescued by two copies of the ci+ transgene (Methot, 1999).

Clones of cells homozygous mutant for ci94 (and thus completely lacking any Ci product) were generated by Flp-mediated mitotic recombination using the ci+ transgene. ci- clones located in the the anterior (A) compartment of the wing imaginal disc compartment ectopically express a hh-lacZ reporter gene, albeit at levels lower than those of endogenous hh-lacZ expression in posterior (P) compartment cells. By contrast, clones of A compartment cells mutant for ciCell (ciCell /ci94 clones) do not express the hh-lacZ gene. These results show that Ci is normally required in A compartment cells to repress hh transcription and that physiological levels of CiCell (from a single copy of ciCell) are sufficient to supply this function (Methot, 1999).

It has been proposed that Ci repressor activity is negatively regulated by Hh signaling. To analyze the influence of Hh signaling on Ci repressor activity in vivo, the P compartment of the wing imaginal disc was used as an assay system. Although P cells normally do not express Ci, they express hh, providing them simultaneously with Hh ligand and with a reporter gene to assay the repressor activity of Ci (hh itself). The ability of P cells to transduce the Hh signal was controlled by manipulating the function of smoothened (smo), which encodes the transducing component of the Hh receptor complex. P cells homozygous mutant for smo express hh at wild-type levels. However, smo mutant P cells of discs that express Ci ubiquitously from a transgene fail to express hh, indicating that Ci functions as a potent repressor of hh expression in the absence of the Hh signal transduction. Since the hh-lacZ gene is expressed in neighboring smo+ P cells that express the same ci transgene, it appears that reception of the Hh signal in these cells prevents Ci from repressing hh transcription. Thus, the ability of Ci to function as a transcriptional repressor is regulated by Hh. By contrast, CiCell represses hh transcription in P cells regardless of the presence of smo activity. It is concluded that CiCell escapes Hh regulation and acts as a constitutive repressor (Methot, 1999).

Anterior compartment cells along the AP compartment boundary express high levels of ptc in response to Hh signaling, in contrast to cells in the remainder of the A compartment, which express only low levels of ptc. During a late stage of wing imaginal disc development, these 'boundary' A compartment cells also express en. The upregulation of these target genes could result from the reduction of Ci repressor activity in response to Hh signaling. Clones of A compartment cells homozygous for the ci94 mutation do not appear to express high levels of ptc or to express en, regardless of where they arise, except for large clones in the notum that are associated with weak upregulation of a ptc-lacZ gene. Strikingly, clones located very close to the A/P compartment boundary abolished Ptc and En expression. Since elimination of Ci leads to a loss, rather than a gain, of ptc and en expression, it is concluded that Hh signaling normally upregulates the expression of these genes by creating an activating form of Ci rather than eliminating the expression or activity of a repressing form (Methot, 1999).

To test whether the transcriptional activator function of Ci is created in response to Hh signaling, P compartment cells were again used as an assay system. Clones of smo mutant cells in the P compartment of wing discs that ubiquitously express ci do not express ptc-lacZ, indicating that Ci is unable to induce ptc-lacZ expression in the absence of Hh input. By contrast, neighboring P cells that are smo+, and hence able to receive the Hh signal, readily express ptc-lacZ. The equivalent behavior has previously been described for A compartment cells that fail to upregulate ptc in the absence of smo function. Thus, the activator activity of Ci is not constitutive, but rather depends on the reception of the Hh signal (Methot, 1999).

Full-length Ci (Ci-155) undergoes proteolytic cleavage to give rise to the N-terminal fragment Ci-75. Hh signaling blocks the repressor activity of Ci. Thus, Hh signaling might generate the activating form of Ci merely by stabilizing the intact protein that has an inherent activating function. To test this hypothesis, a form of Ci was created that is not cleaved in vivo. The Ci cleavage site has been mapped to a region encompassing amino acids 650 to 700. In an initial attempt to remove the cleavage site, sequences encoding amino acids 612 to 712 of the Ci protein were deleted. However, Western blot analysis of this mutant protein (CiC) reveals that it can still be proteolyzed to Ci-75. Sequences encoding amino acids 611 to 760 were then deleted. This mutant protein, referred to as CiU, does not yield appreciable amounts of Ci-75 and therefore appears to be resistant to proteolysis (Methot, 1999).

Ci, CiC, and CiU were expressed ubiquitously in wing discs in which smo mutant clones were also induced. hh-lacZ expression is abolished in P compartment clones of smo- cells expressing Ci. The same result is obtained with CiC. By contrast, CiU expression does not abolish hh-lacZ expression in smo- P cells, indicating that CiU is unable to provide repressor function even in the absence of Hh signaling. This result indicates that cleavage of Ci-155 to Ci-75 is a necessary step in the formation of Ci repressor (Methot, 1999).

Since CiU cannot undergo proteolysis to Ci-75, it was then asked whether it functions as a constitutively active form of Ci. Ubiquitous expression in wing discs reveals that CiU, like CiC and Ci, readily activates ptc-lacZ in P compartment cells, but not in anterior A cells, nor in smo mutant P compartment cells. Thus, CiU functions as an activator form of Ci, but it does so only in Hh-receiving cells, indicating that the activating function of the CiU protein depends on Hh signal transduction. It is concluded that the prevention of proteolysis to Ci-75 is not sufficient to cause Ci to function as an activator and that activator function requires an additional Hh-dependent step (Methot, 1999).

Evidence has been provided that Ci exhibits two activities in its control of target gene expression: an activator activity (named Ci[act]) and a repressor activity (Ci[rep]), both of which are regulated by Hh. These results also indicate that for some genes, Ci acts only as an activator of transcription (ptc, en), and for others, exclusively as an inhibitor (hh). The most critical target gene of Hh in its control of wing development is dpp. To determine which of Ci's activities control dpp expression, the expression of dpp-lacZ reporter genes was examined in wing discs carrying ci mutant clones. ci94 mutant cells located in the A compartment invariably express dpp-lacZ. However, the level of expression is less than that observed in wild-type cells located along the AP boundary. About 30% of the clones also cause nonautonomous expression of dpp-lacZ in neighboring cells at higher levels than inside the mutant clones. To determine whether dpp-lacZ expression observed both within and surrounding clones of ci94 mutant cells is due to ectopic Hh expressed by the mutant cells, ci94;hh- double mutant clones were generated. All clones analyzed exhibit autonomous dpp-lacZ expression at levels lower than the endogenous dpp-lacZ stripe. Nonautonomous dpp-lacZ expression is never observed. These results show that dpp expression within ci- clones is Hh independent, but nonautonomous dpp expression around the ci mutant clones is Hh dependent. Thus, one function of Ci in the absence of Hh signaling is to repress a low, latent transcription of dpp in A cells. Consistent with this interpretation, it was found that a single copy of the ciCell allele (in ciCell /ci94 clones) completely suppressed ectopic dpp expression in the A compartment as well as normal dpp expression in cells near the AP boundary. However, the observation that the levels of dpp expression in ci null clones are significantly lower than those in cells receiving the Hh signal indicates that Ci is also required to activate high levels of dpp transcription. Lack of Ci is not sufficient to induce maximal dpp expression. This can best be seen in ci94 clones located within the dpp-lacZ stripe along the A/P boundary where the low dpp expression levels of mutant cells contrast with those of their neighboring wild-type cells. Thus, the results indicate that dpp transcription in the A compartment is regulated by a combination of repressor and activator activities of Ci (Methot, 1999).

One key finding is that Hh controls at least two aspects of Ci function. Apart from negatively regulating the generation of a repressor form of Ci, Hh signaling tightly controls the formation of an activator form of Ci. The simplest model to account for both steps of Hh regulation would be that the formation of Ci[act] is a direct consequence of preventing the formation of Ci[rep] and vice versa. However, the mere prevention of Ci-75 formation does not render the full-length Ci protein active by default. This argument is based on the observation that an uncleavable form of Ci, CiU, lacks activator activity in the absence of Hh input. Thus, the results suggest the existence of an additional step of Hh regulation that converts Ci into a transcriptional activator. Two models are presented that can account for both outputs of Hh signaling. Two separate steps of Hh regulation could be required for the generation of Ci[act], the first one being the sparing of Ci-155 from degradation to Ci-75, and the second one being an activation step for nonprocessed Ci to acquire activator activity. One component of the Hh signaling cascade that could be responsible for the Hh-dependent activating step of Ci is the Ser/Thr kinase Fused (Fu). In wing imaginal discs, however, Fu kinase function does not appear essential for many aspects of Hh signaling. Although anterior fu mutant cells fail to express late en, they still transcribe ptc (albeit at lower levels than wild-type cells) and exhibit nearly normal levels of dpp expression. Thus, Fu activity can not fully account for the potent activation of ptc and dpp expression by Ci in response to Hh. Moreover, in the absence of Su(fu), Fu kinase activity is dispensable. Because fu;Su(fu) double mutant animals develop virtually like wild-type animals, it can be inferred that the Ci activator function is regulated properly by Hh in the absence of Fu. Together, these observations suggest that while Fu activity may be involved in some second-order level of regulation permitting the induction of less sensitive Hh targets by Ci (such as late en), the primary mechanism by which Hh signaling regulates the formation of Ci[act] does not depend on the catalytic function of Fu (Methot, 1999 and references).

Another model to explain these experimental results is based on the premise that Hh signaling governs the fate of Ci primarily by controlling its cytoplasmic association with multiprotein complexes containing the kinesin-related protein Cos2. If complex formation is a prerequisite for targeting Ci to its site of proteolytic processing, the main function of Hh signaling might be to prevent this association and thereby indirectly also spare Ci-155 from being processed by the proteolytic cleavage machinery. A prediction of this model would be that a cleavage-resistant form of Ci would still be incorporated into the microtubule-associated complex and thus still be subjected to Hh control. CiU fulfills this criterion (Methot, 1999 and references).

The function of Hh in controlling growth and pattern of the wing primordium is mediated to a large extent by the local expression of Dpp, which is secreted from a subset of anterior cells in response to Hh signaling. Dpp acts directly, at long range, and in a concentration-dependent manner to convey positional information to wing cells along the anteroposterior axis. Thus, the precise domain in which dpp is expressed and the absolute levels of Dpp secreted are consequential for the morphogenesis of the wing. It may not be coincidence, therefore, that it is precisely the dpp gene that is subject to both modes of Ci control. In the simultaneous absence of ci and en function, dpp is expressed at a constitutive basal level in all wing cells. From this is surmised the existence of a ubiquitous enhancer (B, for basal) that stimulates dpp transcription by default. The results presented in this paper indicate further that both regulatory inputs, Ci[act] and Ci[rep], act on dpp, and it is proposed that their superimposition serves to 'sharpen' the Dpp morphogen source. Two consequences can be invoked from the combination of the two regulatory mechanisms: (1) a narrowing of the dpp stripe, and (2) an increase in dpp expression levels. Finally, it is noted that the dual control of dpp expression by Ci necessitates a mechanism to prevent dpp transcription in P cells that contain neither form of Ci and would thus express dpp by default. This complication appears to be solved by subjecting dpp expression to repression by En. The result of all these regulatory measures is an exquisitely controlled system in which Dpp is secreted at high levels by a narrow strip of cells located along the A/P compartment boundary in the center of the wing primordium (Methot, 1999).

The cis-regulatory logic of Hedgehog gradient responses: key roles for gli binding affinity, competition, and cooperativity

Gradients of diffusible signaling proteins control precise spatial patterns of gene expression in the developing embryo. This study used quantitative expression measurements and thermodynamic modeling to uncover the cis-regulatory logic underlying spatially restricted gene expression in a Hedgehog (Hh) gradient in Drosophila. When Hh signaling is low, the Hh effector Gli, known as Cubitus interruptus (Ci) in Drosophila, acts as a transcriptional repressor; when Hh signaling is high, Gli acts as a transcriptional activator. Counterintuitively and in contrast to previous models of Gli-regulated gene expression, this study found that low-affinity binding sites for Ci were required for proper spatial expression of the Hh target gene decapentaplegic (dpp) in regions of low Hh signal. Three low-affinity Ci sites enabled expression of dpp in response to low signal; increasing the affinity of these sites restricted dpp expression to regions of maximal signaling. A model incorporating cooperative repression by Ci correctly predicted the in vivo expression of a reporter gene controlled by a single Ci site. This work clarifies how transcriptional activators and repressors, competing for common binding sites, can transmit positional information to the genome. It also provides an explanation for the widespread presence of conserved, nonconsensus Gli binding sites in Hh target genes (Parker, 2011).

The enhancers of dpp and ptc exhibit a regulatory logic opposite that predicted by the activator threshold model. ptc is regulated by Ci sites that match the optimal binding sequence (GACCACCCA), whereas dpp is regulated by nonconsensus sites of low predicted affinity. Competitive electrophoretic mobility shift assays (EMSAs) were used to measure the relative in vitro affinities of Ci sites in the ptc and dpp enhancers, and it was found that Ciptc sites in the ptc enhancer have considerably higher affinity than Cidpp sites. The predicted superior affinity of Ciptc sites, relative to Cidpp sites, is conserved across 12 Drosophila species. Thus, the regulation of dpp and ptc in the wing is opposite to that predicted by a simple activator threshold model. ptc, which is restricted to the region of highest Hh signal, is regulated by high-affinity sites. In contrast, dpp, which responds more broadly in a zone of lower Hh signaling, is regulated by low-affinity sites (Parker, 2011).

To investigate the developmental role of the low-affinity sites in the dpp enhancer, all three sites were altered to match the high-affinity Ci binding sequence found in the ptc enhancer, a change of only seven nucleotides. Transgenic lines were created containing an extra Flp-inducible copy of dpp, driven by the dpp disc (dppD) enhancer containing either wild-type low-affinity (Ciwt) or altered high-affinity (Ciptc) sites. An extra copy of dpp driven by the low-affinity dppD-Ciwt enhancer had no effect on development or survival, whereas the high-affinity Ciptc enhancer caused lethal developmental defects that resemble the effects of dpp misexpression in imaginal discs, including severe head and limb deformities and pupal lethality resulting from overgrowth fusion, and patterning defects in antenna and leg discs. These results indicate that the conserved low affinity of the dppD enhancer for Ci is functionally relevant (Parker, 2011).

A quantitative reporter gene assay was developed to further explore the role of low-affinity Ci binding sites in the dpp wing disc enhancer. Transgenic fly lines were constructed carrying two reporter genes: dppD-Ciptc-RFP, consisting of the high-affinity version of the dpp enhancer driving expression of a red fluorescent protein (RFP), and one of several dpp enhancers driving green fluorescent protein (GFP). By measuring GFP fluorescence across a transect of the wing pouch and normalizing to peak RFP expression in each disc, a quantitative readout was obtained of both the position and the intensity of GFP reporter activity (Parker, 2011).

Using this assay, the activity was compared of different versions of the dppD enhancer driving GFP expression, containing either three low-affinity sites (dppD-Ciwt) or three high-affinity sites (dppD-Ciptc). Ci-independent, 'basal' expression was also measured from a construct (dppD-CiKO) in which all three Ci sites were mutated to abolish Ci binding. This basal expression captures the effects of all factors other than Ci on dpp, including Engrailed, which directly represses dpp near the anterior/posterior boundary. This basal construct enabled direct measurement of both activation and repression by Ci, by comparing the activity of the low- or high-affinity enhancers against that of dppD-CiKO. The results show that the response of dpp to Hh cannot be explained by an activator threshold model. High-affinity Ciptc sites caused a posterior shift in stripe position toward the region of strongest Hh signal, whereas low-affinity Ciwt sites produced stronger activation in regions of moderate Hh signal. When the basal dppD-CiKO-GFP expression was subtracted from that of dppD-Ciptc-GFP and dppD-Ciwt-GFP, it was observed that within the zone of moderate Hh signal, low-affinity sites produced activation, whereas high-affinity sites conferred repression. This observation shows that CiREP plays a substantial role in the response to moderate Hh signal, a finding that directly contradicts the assumptions of the activator threshold model. Thus, an alternate biophysical model is required to explain the regulatory logic of the dpp response to Hh (Parker, 2011).

It is concluded that spatial information in the wing disc Hh gradient is interpreted by a cis-regulatory logic that relies on activator-repressor competition, which is modulated by binding site affinity and cooperative repression. In previous studies of Hh target genes, the role of the affinity of the Gli or Ci binding site has been neglected or has been assumed to play a role opposite to what the current data show. Moreover, the currently accepted activator threshold model of the transcriptional response to Hh assumes that the role of Gli and Ci repressors is limited to regions of little or no Hh signal. No previously described model of Hh response, including the activator threshold model, can account for the observations described in this study. The data show that substantial repression can occur even at moderate Hh signal and suggest that the transcriptional response in much of the Hh gradient depends on the outcome of a competition between CiACT and CiREP for enhancer binding. This new model of the cis-regulatory logic underlying Hh response integrates the effects of both CiACT and CiREP along the entire Hh gradient and explains the importance of low-affinity Gli binding sites in the positioning of gene expression (Parker, 2011).

The results suggest that the low affinity of the dpp enhancer for Ci can be explained by the need to mitigate the effects of cooperative repression in a region of the gradient where substantial amounts of CiREP are present, while still allowing activation by CiACT. Within the context of this model, repressor cooperativity is defined as any interaction that makes the binding of additional CiREP more favorable when one CiREP is already bound. Cooperativity could arise from direct interactions between CiREP or from interactions of CiREP with other transcription factors, cofactors, or histones. In principle, CiREP cooperativity at the enhancer could be attenuated by various cis-regulatory strategies besides lowering binding affinity, such as reducing the number of Ci sites or increasing their spacing. However, these alternative strategies may not be equally able to maintain activation by CiACT in regions of low Hh signaling (Parker, 2011).

The posterior-to-anterior gradient of Hh in the wing disc establishes opposing gradients of CiACT and CiREP. High amounts of CiACT are present at the anterior/posterior boundary, whereas more anterior regions feature high amounts of CiREP. Within the intermediate zone of the gradient, mixed amounts of CiACT and CiREP compete for enhancer binding, with activation and repression determined by the ratio of bound CiACT to bound CiREP. In the repressor cooperativity model, repressors outcompete activators for binding at high-affinity enhancers, but not at low-affinity enhancers, in this region of the gradient. Several morphogen signaling pathways have the potential to produce reciprocal gradients of repressors and activators competing for common binding sites. This study has presented the first detailed mechanistic model that explains how reciprocal gradients of Gli activators and repressors are transcriptionally interpreted. A similar regulatory logic may inform responses to other morphogens that control transcriptional switches, particularly those whose target genes are regulated by low-affinity sites (Parker, 2011).

The model provides an explanation for the widespread presence of evolutionarily conserved, nonconsensus Gli or Ci binding sites in the enhancers of Hh target genes. With the exception of ptc, all known direct targets of Hh in Drosophila are regulated by nonconsensus Ci sites with predicted low affinity The results indicate that low-affinity sites are necessary to position a stripe of expression in the middle of a Hh gradient, because high-affinity sites induce repression outside the zone of strongest signaling. Because mammalian Gli target genes are also regulated by nonconsensus sites, the conclusions may also apply to vertebrate Hh targets: Such targets may acquire weak-affinity Gli sites to minimize cooperative repression in a Gli cross-gradient (Parker, 2011).

In a few documented cases, low-affinity transcription factor binding sites have important spatiotemporal patterning functions. Two well-studied examples are the response to the morphogen Dorsal in Drosophila and the temporal control of developmental gene expression. In these cases, low-affinity sites set a high threshold for activator concentration, restricting activation to cells or times in which activator concentration is maximal. In the case described in this study, an opposite cis-regulatory logic applies: Low-affinity sites are specifically required for activation in cells receiving lower amounts of signal. This is a consequence of the fact that in this region of the gradient, activators and repressors compete for the same genomic binding sites (Parker, 2011).

The results suggest that most current biochemical and computational approaches to identifying Hh target genes, which typically focus on the highest-affinity Ci or Gli sites, may overlook a large proportion of important Hh target genes. More generally, transcriptional cooperativity may play an important cis-regulatory role in enhancers with conserved low-affinity binding sites (Parker, 2011).

The Hippo pathway coactivator Yorkie can reprogram cell fates and create compartment-boundary-like interactions at clone margins

During development, tissue-specific patterns of gene expression are established by transcription factors and then stably maintained via epigenetic mechanisms. Cancer cells often express genes that are inappropriate for that tissue or developmental stage. This study shows that high activity levels of Yki, the Hippo pathway coactivator that causes overgrowth in Drosophila imaginal discs, can also disrupt cell fates by altering expression of selector genes like engrailed (en) and Ultrabithorax (Ubx). Posterior clones expressing activated Yki can down-regulate en and express an anterior selector gene, cubitus interruptus (ci). The microRNA bantam and the chromatin regulator Taranis both function downstream of Yki in promoting ci expression. The boundary between Yki-expressing posterior clones and surrounding wild-type cells acquires properties reminiscent of the anteroposterior compartment boundary; Hedgehog signaling pathway activation results in production of Dpp. Thus, at least in principle, heterotypic interactions between Yki-expressing cells and their neighbors could activate boundary-specific signaling mechanisms (Bairzin, 2020).

Human cancers are characterized by multiple genetic lesions, a subset of which are driver mutations that are thought to be responsible for their tumorous characteristics. It is estimated that most cancers have two to eight driver mutations. This makes it difficult to evaluate the contribution of each mutation to any particular characteristic of the tumor. This study has taken advantage of the ability of single-gene manipulations to cause overgrowth in Drosophila imaginal discs to assess the ability of three different oncogenes to destabilize established patterns of selector gene expression; yki, the Drosophila ortholog of Yap and Taz, is especially potent in doing so. The patterns of expression of En, Ci, and Ubx are established relatively early in embryogenesis and maintained stably in imaginal discs during the larval stages of development. These patterns of expression can be disrupted in clones expressing an activated form of Yki. Expression of a wild-type form of Yki is capable of disrupting these expression patterns in combination with other genetic manipulations such as overexpression of ban or tara. This latter scenario is more likely to apply to human cancers; increased Yap or Taz activity has been described in multiple human cancers, which often also have other genetic lesions (Bairzin, 2020).

These studies show that sd, ban, and tara make important contributions to the pathway by which YkiCA destabilizes gene expression; reducing the expression of any of these in clones expressing YkiCA greatly reduces ectopic Ci expression, and increasing expression of both genes can cause ectopic Ci expression. It is likely that other mechanisms function in parallel to destabilize selector gene expression since combined overexpression of ban and tara increased ectopic Ci expression but did not reduce En expression (Bairzin, 2020).

Changing selector gene expression within an overgrowing clone can create interactions at the clone margin that are reminiscent of compartment boundaries and result in the production of morphogens. A recent study showed that forced expression of En in lgl clones can elicit similar phenomena in anterior clones. In addition, YkiCA clones are often extruded, consistent with previous observations that heterotypic interactions caused by overexpressing patterning genes also promotes extrusion. Previous work found that ci RNA levels were increased in wts mutant tissue, this study did not see ectopic Ci protein expression in wts mutant clones or wild-type Yki-overexpressing clones. This work shows therefore that sustained expression of very high Yki levels is necessary to destabilize expression of selector genes. However, even under these conditions, the effect on ectopic Ci expression is Sd dependent. Moreover, this study shows that even wild-type Yki can, in combination with increased expression of ban or tara, induce ectopic Ci expression. While these changes in gene expression are most obvious with above-physiological levels of Yki, they nevertheless reflect a previously unknown ability of this pathway to alter patterning gene expression and furthermore to change the growth characteristics of neighboring wild-type cells. Differences in selector gene expression between human cancers or precancerous lesions and their wild-type neighbors have received relatively little attention, and the current results call attention to tumor margins as sites where heterotypic interactions could create signaling centers that affect the behavior of tumor cells (Bairzin, 2020).

Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. This study shows that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, evidence is provided that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill (Roberto, 2022).

This study examined the roles of three potential negative regulators of Hh signal transduction, two of which are themselves encoded by Hh target genes. In each case interesting new aspects about the pathway's regulation. Anterior expression of en likely extends the range of the Hh gradient were discovered (Roberto, 2022).

Anterior expression of en in the wing imaginal disc was first observed 30 years ago and en is the Hh target gene requiring the highest level of Hh signaling. Its domain of expression exactly correlates with a region of lower full-length Ci protein levels. It had been proposed that the lower Ci protein levels are a consequence of Ci being particularly active and labile in this region. This study shows that the lower levels of Ci are not primarily due to it being particularly labile, but rather are a consequence of negative transcriptional regulation by En. The role of this negative feedback loop appears to be to modulate the Hh gradient by downregulating the expression of ptc in addition to its effects on dpp. This leads to Hh signaling extending further into the anterior compartment, with a corresponding anterior shift in the location of LV3 and the expression of dpp. A model is prefered in which the attenuation of ptc expression by anterior en is indirect via Ci, but in principle en could also directly negatively regulate ptc. This is thought less likely as, 'flip-out' clones expressing Ci activate high levels of ptc in the posterior compartment in the presence of en. The anterior expression of en occurs late in third instar larvae, which correlates with the downregulation of ci expression as visualized using the UAS-TT transcriptional timer and the refinement of wing vein specification (Roberto, 2022).

Ci function is modulated by two feedback loops acting at different levels. Anterior expression of the En protein attenuates Ci activity directly adjacent to the compartment boundary of the wing disc by downregulating the expression of the ci gene. Rdx and Su(fu) act at the protein level modulating the competition between the full-length (Ci FL) and repressor forms (Ci R) of Ci. Rdx specifically targets full-length Ci, whereas Su(fu) partially protects full-length Ci from Rdx-mediated degradation. Rdx degradation of full-length Ci appears to help downregulate Hh target genes in cells no longer receiving the Hh signal (Roberto, 2022).

Why did this mechanism evolve to modulate the Hh gradient? Morphogen gradients, by virtue of their central roles in the development of multiple tissues, must be robust and resistant to perturbation. Therefore, to specifically expand the range of the Hh gradient in the wing disc a new component was added, anterior expression of the ci repressor en (Roberto, 2022).

The lack of the C-terminal domain in the Ci repressor has multiple consequences. It loses the binding site for the co-activator CBP, and it loses C-terminal binding sites for Su(fu), Cos2 and Rdx. As a consequence, the Ci repressor is not sequestered in the cytoplasm by Cos2 in the absence of Hh signaling and enters the nucleus without Su(fu), whereas full-length Ci enters the nucleus only in the presence of Hh signaling and as a complex with Su(fu) (Roberto, 2022).

In order to better understand the roles of Su(fu) and Rdx, animals heterozygous for the ciCe2 mutation were examined. In this context, overexpression of rdx or loss of Su(fu) function leads to a complete fusion between LV3 and LV4. In addition, clones mutant for Su(fu) show dramatic reduction in the expression of the Hh target genes ptc and dpp. These results show that Su(fu) has a potential novel positive role in Hh signal transduction, improving the ability of full-length Ci to compete with the repressor form. A positive role for Su(fu) has also been found in mammals where Su(fu) appears to function as a chaperone for the full-length Gli proteins, but not the repressor forms, and is required for full activation of Gli target genes. The requirement for Drosophila Su(fu) is obviated in the absence of Rdx, suggesting that Rdx primarily targets full-length Ci and not Ci repressor, even though the repressor is not protected by Su(fu). These results are analogous to what is seen with the mammalian homologue of Rdx, SPOP, indicating that this mechanism has been conserved during evolution. SPOP is opposed by Su(fu) and degrades the full-length forms of the mammalian GLI2 and GLI3 but not the GLI3 repressor form. The competition between Rdx and Su(fu) appears to be rather finely balanced as either increasing the expression of rdx or reducing the expression of Su(fu) enhances the ability of CiCe2 to compete with full-length Ci. This function of protecting full-length Ci from Rdx presumably takes place in the nucleus, as this is where the Rdx protein primarily localizes (Roberto, 2022).

However, the functional relevance of rdx being an Hh target gene has been unclear. Zygotic loss of rdx in the embryo has no visible effect on segmental patterning of the cuticle and, unlike en, knockdown of rdx along the compartment boundary in the wing disc has little effect on wing patterning. Perhaps its role is to clear full-length Ci from cells that were once within the domain of Hh signaling and have moved outside the domain of Hh signaling. Perdurance of Rdx could target full-length Ci in the nucleus allowing the Ci repressor to shut off Hh target genes. This is the situation in the eye disc with the progression of the morphogenetic furrow. Cells that recently received high level Hh signaling and activated Ci must now downregulate Ci to allow proper differentiation of the ommatidia. Rdx appears to be important for this process, as loss of rdx leads to defects in the eye. A similar situation may exist in other tissues. Looking at the temporal regulation of ptc expression with UAS-TT, cells removed from the compartment boundary in the wing disc have lower levels of destabilized GFP relative to RFP and appear to be in the process of shutting off ptc. This distinction is lost following downregulation of rdx by RNAi (Roberto, 2022).

In the domain of modest level Hh signaling (in which dpp is expressed), both full-length Ci and Ci repressor must be present in some form of reciprocal gradients. In this domain, enhancers with perfect Ci consensus binding sites are silent due to binding of Ci repressor. The dpp enhancer with imperfect Ci binding sites is expressed, and for it to be completely active, full-length Ci must be bound. Why is full-length Ci able to better compete with Ci repressor for the imperfect binding sites? Full-length Ci and the Ci repressor share the same DNA binding domain, and it would be expected that the repressor would outcompete full-length Ci for binding to target sites because the repressor is primarily nuclear, whereas full-length Ci is primarily cytoplasmic, even in the presence of Hh signaling, due to a strong nuclear export signal (NES). I suggest that cooperativity between Ci repressor proteins at perfect Ci binding sites can account for this distinction. Another potential mechanism for preferentially recruiting full-length Ci to imperfect binding sites might be suggested by the different protein interactions observed with full-length Ci and CiCe2. Full-length Ci enters the nucleus with Su(fu) while the Ci repressor is not bound to Su(fu). In addition, the Ci repressor is missing the CBP binding site. As a consequence, full-length Ci could engage in protein-protein interactions with other transcription factors that are not available to the Ci repressor. This added affinity to other proteins within the enhanceosome could allow the preferential recruitment of full-length Ci to enhancers with imperfect Ci binding sites. Differential protein-protein interactions may also explain why full-length Ci is still able to activate ptc-lacZ expression along the compartment boundary in ciCe2/+ heterozygotes (Fig. S4) but not the artificial enhancer 4bs-lacZ. The ptc-lacZ enhancer is a bona fide Drosophila enhancer and is likely to recruit a constellation of proteins that could interact with full-length Ci, whereas protein-protein interactions are likely to be much less robust at 4bs (Roberto, 2022).

In conclusion, these results highlight the complexity of Hh signal transduction and its modulation. Expressing en in the anterior compartment of the wing pouch modulates the Hh gradient, whereas Su(fu) has a surprising positive role in the pathway, acting to partially protect full-length Ci from the E-3 ligase Rdx that Ci activates (Roberto, 2022).


GENE STRUCTURE

cDNA clone length - 4852

Bases in 5' UTR - 665

Exons - four

Bases in 3' UTR - 339


PROTEIN STRUCTURE

Amino Acids - 1282

Structural Domains

The protein encoded by the CI transcript contains a domain of five tandem amino acid repeats that have sequence similarity to the zinc-finger repeats of the Xenopus transcription factor TFIIIA and share the highest degree of identity with the human zinc-finger protein GLI, which has been found to be amplified in several human glioblastomas (Orenic, 1990).

The zinc finger domain of CI is homologous to the nematode tra-1 gene (Slusarski, 1995).

The patched-hedgehog-ci pathway is conserved in mammals, including expression of protein kinase A and the vertebrate homolog of Cubitus interruptus (Goodrich, 1996).


cubitus interruptus : Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised:  25 July 2022

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