Gene name - cubitus interruptus
Synonyms - ci dominant
Cytological map position - 102A3
Function - putative transcription factor
Symbol - ci
Genetic map position - 4-0.0
Classification - zinc finger
Cellular location - cytoplasmic
|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
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
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
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
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.
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).
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).
Bases in 5' UTR - 665
Exons - four
Bases in 3' UTR - 339
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).
date revised: 12 December 96
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