capicua: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - capicua

Synonyms -

Cytological map position - 92D1-2

Function - transcription factor

Keywords - terminal gene in the Torso pathway, dorsal pathway

Symbol - cic

FlyBase ID: FBgn0262582

Genetic map position -

Classification - HMG box protein

Cellular location - nuclear

NCBI link: Entrez Gene
cic orthologs: Biolitmine
Recent literature
Futran, A. S., Kyin, S., Shvartsman, S. Y. and Link, A. J. (2015). Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking. Proc Natl Acad Sci U S A 112: 8590-8595. PubMed ID: 26124095
Extracellular signal-regulated kinase (ERK; Rolled in Drosophila) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. This study used a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. The ERK-binding region of human Cic was also identified, and it was shown to bind to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, the amino acids involved in human Cic binding to ERK were mapped using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.

Fores, M., Simon-Carrasco, L., Ajuria, L., Samper, N., Gonzalez-Crespo, S., Drosten, M., Barbacid, M. and Jimenez, G. (2017). A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer. PLoS Genet 13(3): e1006622. PubMed ID: 28278156
HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This study reports that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. Contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. This binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors.
Goyal, Y., Levario, T. J., Mattingly, H. H., Holmes, S., Shvartsman, S. Y. and Lu, H. (2017). Parallel imaging of Drosophila embryos for quantitative analysis of genetic perturbations of the Ras pathway. Dis Model Mech [Epub ahead of print]. PubMed ID: 28495673
The Ras pathway patterns the poles of the Drosophila embryo by downregulating the levels and activity of a DNA-binding transcriptional repressor Capicua (Cic). This study demonstrates that the spatiotemporal pattern of Cic during this signaling event can be harnessed for functional studies of the Ras-pathway mutations from human diseases. The approach relies on a new microfluidic device that enables parallel imaging of Cic dynamics in dozens of live embryos. Although the pattern of Cic in early embryos is complex, it can be accurately approximated by a product of one spatial profile and one time-dependent amplitude. Analysis of these functions of space and time alone reveals the differential effects of mutations within the Ras pathway. Given the highly-conserved nature of Ras-dependent control of Cic, this approach opens a new way for functional analysis of multiple sequence variants from developmental abnormalities and cancers.
Papagianni, A., Fores, M., Shao, W., He, S., Koenecke, N., Andreu, M. J., Samper, N., Paroush, Z., Gonzalez-Crespo, S., Zeitlinger, J. and Jimenez, G. (2018). Capicua controls Toll/IL-1 signaling targets independently of RTK regulation. Proc Natl Acad Sci U S A 115(8): 1807-1812. PubMed ID: 29432195
The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. This study characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-kappaB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, these results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.
Weissmann, S., Cloos, P. A., Sidoli, S., Jensen, O. N., Pollard, S. and Helin, K. (2018). The tumor suppressor CIC directly regulates MAPK pathway genes via histone deacetylation. Cancer Res. Pubmed ID: 29844126
Oligodendrogliomas (ODG) are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with ODG; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the Drosophila melanogaster orthologue of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. This study addresses the mechanism by which CIC represses transcription and assesses the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on the results, it is suggested that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in ODG tumors prevented CIC from binding its target genes. Taken together, the current results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth.
Keenan, S. E., Blythe, S. A., Marmion, R. A., Djabrayan, N. J., Wieschaus, E. F. and Shvartsman, S. Y. (2020). Rapid dynamics of signal-dependent transcriptional repression by Capicua. Dev Cell. PubMed ID: 32142631
Optogenetic perturbations, live imaging, and time-resolved ChIP-seq assays in Drosophila embryos were used to dissect the ERK-dependent control of the HMG-box repressor Capicua (Cic), which plays critical roles in development and is deregulated in human spinocerebellar ataxia and cancers. It was established that Cic target genes are activated before significant downregulation of nuclear localization of Cic, and their activation was demonstrated to be preceded by fast dissociation of Cic from the regulatory DNA. Both Cic-DNA binding and repression are rapidly reinstated in the absence of ERK activation, revealing that inductive signaling must be sufficiently sustained to ensure robust transcriptional response. This work provides a quantitative framework for the mechanistic analysis of dynamics and control of transcriptional repression in development.
Patel, A. L., Zhang, L., Keenan, S. E., Rushlow, C. A., Fradin, C. and Shvartsman, S. Y. (2021). Capicua is a fast-acting transcriptional brake. Curr Biol. PubMed ID: 34166605
Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. This study addresses the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases. The speed limit for Cic-dependent gene repression is reported based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered. Measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic.

capicua (cic), meaning head-and-tail in Catalan, acts as a repressor of tailless (tll) and huckebein (hkb) in both the anterior and posterior domains of the early Drosophila embryo. Torso signaling acts to relieve repression of the tll and hkb Capicua-directed repression. In addition, cic mediates ventral repression of the dorsally expressed gene zerknullt (zen), a process that also requires Groucho and the transcripton factor Dorsal, and which is also inhibited by Torso signaling at the embryonic termini. cic encodes a putative transcription factor with a DNA-binding domain of the HMG box class. The Cic protein interacts with Gro in vitro, suggesting that both factors function in the same protein complex. Thus Gro and Cic form part of a repressor complex specifically inactivated by Tor signaling at the embryo poles (Jimenez, 2000).

Tor activation occurs exclusively at the embryonic poles via a ligand produced locally through the action of torso-like, fs(1)polehole (also known as fs(1)M3), fs(1)Nasrat and trunk genes. Trunk is likely to be the ligand for Tor, Torso-like is an accessory protein permitting Trunk to function, and both fs(1)Nasrat and fs(1)polehole have yet to be cloned. Tor signaling proceeds via the Ras/Raf/MAPK pathway to regulate expression of the zygotic genes tll and hkb, which are specifically expressed at each pole of the embryo. These genes encode transcription factors that initiate the developmental programs leading to differentiation of head and tail structures. Tor signaling does not activate terminal gene expression directly; rather, it functions by antagonizing at the poles a uniformly distributed repressor activity, allowing other maternal factors to activate transcription locally. Evidence for this view comes from the identification of regulatory elements in the tll promoter (called tor response elements, tor-REs) that confer terminal-specific expression and that, when mutated, cause severe derepression of tll transcription (Jimenez, 2000 and references therein).

Additional evidence for the regulation of tll and hkb by relief of repression derives from the role of the Groucho (Gro) corepressor in this process. Gro is a nuclear WD-repeat protein that does not bind DNA but interacts with a variety of DNA-bound transcriptional repressors. These associations recruit Gro to target promoters, bringing about transcriptional repression. Gro has been shown to participate in terminal development by restricting the expression of tll and hkb to the embryonic termini: embryos deprived of maternal Gro function show derepression of tll and hkb toward the middle of the embryo (Jimenez, 2000 and references therein).

What is the actual target of Tor signal inactivation at the embryonic poles? The Drosophila Yan Ets-like repressor factor is known to be degraded in response to RTK signaling during eye development. Thus, it is possible that the target of Tor signaling is similarly inactivated at the embryonic poles. The Gro protein is uniformly distributed in the blastoderm embryo and does not show down-regulation at the termini. Also, Gro corepressor activity during sex determination is not inhibited by Tor signaling, arguing that Gro is not the target of the Tor signal. To monitor the pattern of Cic distribution in embryos, a polyclonal antibody was raised against an HMG box-containing fragment of the protein. This antibody reveals a distinctive nuclear signal in wild-type but not cic1 blastoderm embryos, confirming both that Cic is a nuclear protein and that the cic1 allele is a strong loss-of-function mutation. Remarkably, Cic is distributed asymmetrically in blastoderm embryos, being present in nuclei from the presumptive trunk but absent at each embryonic pole. Because CIC mRNA is uniformly distributed in the embryo, the exclusion of the protein from the poles argues that Cic is under negative post-transcriptional regulation by the Tor signal transduction pathway (Jimenez, 2000).

To test this idea, the distribution of Cic was examined in tor mutant embryos. In such embryos, the Cic protein is detected not only in medial regions of the embryo but also at the termini, implying that Tor signaling inhibits accumulation of Cic protein at the embryo poles. These results suggest that Cic is the target inactivated by the Tor signal, possibly via MAPK phosphorylation and subsequent degradation of the protein, as in the case of Yan. Consistent with this idea, the Cic protein sequence includes 14 consensus MAPK phosphorylation sites. Future studies should define the mechanism by which Tor signaling regulates Cic accumulation and the functional domains of the protein involved in this control (Jimenez, 2000).

How does Cic mediate repression of terminal and dorsoventral genes? Because reporter constructs carrying the tor-RE of tll and zen ventral response element (VRE) are derepressed in cic mutant embryos, the simplest model suggests that Cic binds to these regulatory elements and recruits Gro for repression of these genes. Consistent with this idea, there are striking similarities between the consensus DNA-binding site for HMG box proteins and sequence elements within the tor-RE and VRE known to mediate transcriptional repression. However, although these elements are bound by control HMG box proteins, specific binding of Cic to them has not been detected. Perhaps Cic has a very low affinity for DNA and/or requires the presence of accessory factors for efficient DNA binding. Several HMG box proteins rely on interactions with partner proteins to increase their affinity for DNA. Clearly, identification of the precise molecular mechanism of Cic function will require further analyses of its ability to interact with target sequences in terminal and dorsal-specific genes (Jimenez, 2000).

That Cic functions in association with other factors is consistent with studies that have implicated several proteins in Gro-dependent repression of terminal and dorsal-specific genes. For example, it has been shown that Dri and Cut are two of the cofactors required for repression by Dorsal through the zen VRE (Valentine, 1998), and the current results indicate that Cic also contributes to switching Dorsal from an activator to a repressor of transcription. Similarly, the dramatic effects of Cic on terminal patterning indicate that both Cic and Gro are essential components in the repression of terminal genes. It is still not understood how the activity of all these factors is coordinated in vivo. Nevertheless, these results showing that Cic is under negative post-transcriptional control by the Tor RTK pathway, suggest that it functions as the regulatory element that links Tor signaling to the mechanism of repression (Jimenez, 2000).

Localized activation of the Ras/Raf pathway by epidermal growth factor receptor (Egfr) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the Egfr signal regulates transcriptional responses during the vein/intervein cell fate decision. Evidence is provided that Egfr signaling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of Egfr targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, Egfr signaling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some Egfr targets such as rhomboid appears not to be under direct control of Cic, suggesting that Egfr signaling branches out in the nucleus and controls different targets via distinct mediator factors. These results support the idea that localized inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway (Roch, 2002).

The expression pattern of cic in wild-type wing discs was examined. In situ hybridization of third instar discs shows uniform distribution of cic transcripts. By contrast, staining of similar discs with a specific Cic antibody reveals a complex pattern of protein accumulation: Cic accumulates at high levels in the wing pouch and in the primordial hinge region, but not in the notum region. At this stage, Cic levels begin to drop in the presumptive third longitudinal vein and in two rows of cells running along the D/V boundary that correspond to the future wing margin. Moreover, the remaining Cic protein in those cells is cytoplasmic, whereas in other regions of the wing pouch (and in the adjacent peripodial cells), Cic is clearly nuclear. During pupariation [from 6 to 34 hours after puparium formation (APF)], Cic levels also decline in all presumptive longitudinal wing veins and crossveins. This specific accumulation of Cic in intervein sectors is consistent with its role as a negative regulator of vein differentiation (Roch, 2002).

There are two key aspects of Cic function as a developmental regulator: its ability to repress specific target genes in defined territories, and its inhibition by the Ras/Raf pathway to allow expression of those targets in complementary positions. In the blastoderm embryo, Cic is required for development of trunk body regions and represses genes mediating differentiation of terminal structures. Torso RTK activation at each pole of the embryo alleviates Cic-dependent repression and initiates the terminal gene expression program. A similar model is proposed for cic function during specification of vein versus intervein fate in the wing. Loss of cic function in the wing causes formation of ectopic vein tissue, implying that Cic mediates intervein specification by restricting vein formation to appropriate regions. In intervein territories, Cic behaves as a repressor of vein-specific genes such as argos and vvl but does not seem to affect directly the expression of blistered, which is required for the specification of intervein fates. Finally, Egfr signaling leads to downregulation of Cic protein levels in vein nuclei, thus relieving Cic-mediated repression and promoting vein development (Roch, 2002).

Nevertheless, several data suggest a more complex regulation of vein specification compared to terminal patterning: (1) it has been shown that expression of rho, a positive target of Egfr signaling in the wing and other tissues, is not affected by cic during third larval instar and early pupariation. This suggests that Egfr signaling can mediate activation of some targets in the wing disc by mechanisms other than Cic inhibition. (2) Similarly, the Egfr pathway has been shown to repress bistered expression in presumptive vein cells, a process that is independent of Cic. These results imply that different transcription factors act downstream of the Egfr cascade to direct changes in gene expression during patterning of wing veins. Indeed, recent results indicate that Egfr signaling activates certain target genes via direct phosphorylation of Fos protein (Roch, 2002).

Moreover, vein differentiation is not a mere result of Egfr activation but depends on other signals such as Dpp and Notch, and on the distribution of additional transcription factors that contribute to wing patterning. For example, the Collier/Knot nuclear factor has been shown to induce high levels of Bs expression between veins L3 and L4, promoting intervein development in this region. All these inputs are linked in a complex circuit of intercellular signaling and gene regulation that progressively refines vein determination during late larval and pupal development. This signaling network could provide an explanation for the observed non-autonomy of cic phenotypes during vein specification. Thus, although cic represses aos expression in a cell-autonomous manner, this and other cic targets are likely to participate in signaling mechanisms that affect adjacent cells. Consistent with this idea, it has been found that cic mutant cells express ectopic Dpp product, a diffusible factor that promotes vein differentiation (Roch, 2002).

In cic mutant wings, many cells differentiate, acquiring morphological features that are intermediate between those observed in either vein and intervein cells. In these wings, most cells co-express Bs and Vvl proteins, which are normally restricted to vein and intervein cells, respectively, suggesting that vein/intervein fate specification may result from a balance of these factors rather than on a simple binary switch. In this context, the concerted activities of signaling cascades such as Dpp, Notch and Ras/Raf pathways may regulate cell differentiation by modulating the balance of nuclear factors that act in a dose-dependent way. This hypothesis provides a mechanism that could explain the enormous variability observed in the cell morphologies of different insect wings (Roch, 2002).

Cic acts in wing development in a way similar to that previously described in the early embryo. Moreover, the fact that mutant clones for the Groucho repressor display extraveins, similarly to cic clones, indicates that these two proteins could interact as partners during wing development, as is the case during embryonic development. Indeed, weak genetic interactions have been observed between different cic and gro alleles during wing development. Thus, Cic and Gro could be part of a conserved repressor complex downregulated by the Ras/Raf molecular cassette in different cellular contexts. In this regard, the phenotype of bullwinkle mutations (bullwinkle is allelic to cic) suggests that Cic may also function as a target of other RTK signals during patterning of the eggshell in the ovary. However, it should be noted that cic does not seem to act in all developmental processes mediated by Ras/Raf signaling in Drosophila. For example, the eyes of cic mutant flies appear normal, even though the Ras/Raf pathway controls several aspects of cell fate specification and patterning in this tissue. These observations support the idea that the Ras/Raf pathway can regulate cell specification in a cic-independent way depending on the cell context (Roch, 2002).

Previous work has shown that during patterning of ovary follicle cells, the expression of rho is controlled by the Ras/Raf pathway via another transcriptional repressor, the CF2 protein. CF2 is tagged for cytoplasmic retention and degradation after direct phosphorylation by MAPK. The Cic protein also has consensus sites for phosphorylation by MAPK, suggesting that Cic levels could be regulated post-transcriptionally in a way similar to CF2. This indicates that localized downregulation of specific repressors is a common mechanism for the activation of target genes by the Ras/Raf pathway. The identification of highly conserved cic homologs in mice and humans suggests that regulation of gene expression by RTK signaling in vertebrates may also involve relief of Cic-dependent repression (Roch, 2002).

Mutual repression by bantam miRNA and Capicua links the EGFR/MAPK and Hippo pathways in growth control

The epidermal growth factor receptor (EGFR) and Hippo signaling pathways control cell proliferation and apoptosis to promote tissue growth during development. Misregulation of these pathways is implicated in cancer. Understanding of the mechanisms that integrate the activity of these pathways remains fragmentary. This study identifies bantam microRNA as a common target of these pathways and suggests a mechanistic link between them. The EGFR pathway acts through bantam to control tissue growth. bantam expression is regulated by the EGFR pathway, acting via repression of the transcriptional repressor Capicua. Thus EGFR signaling induces bantam expression by alleviating the effects of a repressor. bantam in turn acts in a negative feedback loop to limit Capicua expression. bantam appears to be a transcriptional target of both the EGFR and Hippo growth control pathways. Feedback regulation by bantam on Capicua provides a means to link signal propagation by the EGFR pathway to activity of the Hippo pathway and may play an important role in integration of these two pathways in growth control (Herranz, 2012).

The ability of the EGFR pathway to drive tissue growth resides in its ability to coordinately stimulate cell proliferation and suppress apoptosis. Understanding how coordinated control is achieved depends on identification of the effector mechanisms that mediate these outputs along with the connections to other growth regulatory pathways. The results show that the bantam miRNA is a critical target of the EGFR pathway. Further, a mechanism is outlined by which bantam serves as a link between the EGFR and Hippo pathways (Herranz, 2012).

In Drosophila, EGFR pathway effectors include the transcription factors Pointed, Tramtrack, and Yan, and the HMG-box repressor Capicua. Capicua has an important role in early embryonic patterning and as a negative growth regulator. Although several Capicua targets involved in embryonic patterning have been identified, how Capicua regulates tissue growth was unknown. These results identify bantam as an important target of Capicua required to mediate EGFR-dependent tissue growth (Herranz, 2012).

A key finding of this study is the regulatory feedback relationship between bantam and Capicua. Each represses the activity of the other. Viewed from the perspective of the EGFR/MAPK pathway alone, the outcome of this relationship would be signal amplification, with downregulation of Capicua levels by bantam reinforcing direct MAPK-induced turnover of Capicua protein. This adds a new mechanism to the repertoire of positive and negative feedback loops affecting EGFR pathway activity. These feedback mechanisms are thought to be important in disease, and their regulation is complex. Relatively little is known about Capicua in cancer, although one recent study reports mutants in the human Cic protein in oligodendroglioma (Bettegowda, 2012; Herranz, 2012 and references therein).

An alternative logic for the relationship between bantam and Capicua may be seen in the fact that it links the output of the EGFR pathway to the output of the Hippo pathway, mediated through transcriptional regulation of bantam by Yorkie. EGFR signaling via MAPK and bantam cooperate to downregulate Capicua protein levels. Thus the transcriptional output of the Hippo pathway via Yorkie can be seen as potentiating EGFR signaling by 'lowering' the effective threshold of MAPK activity needed to reduce Capicua to a given level. Alternatively, the lack of sufficient Yorkie activity would lower bantam activity and thereby raise the threshold of EGFR activity required to reach an effective level of Capicua downregulation. This provides a mechanism to ensure coordination of the growth regulatory pathways. Signaling via the Hippo pathway has also been shown to induce the EGFR ligand amphiregulin to promote tissue growth in a nonautonomous manner. Thus, there appear to be multiple levels of crosstalk between these pathways (Herranz, 2012).

Considerable evidence is emerging linking miRNAs to robustness of regulatory feedback networks. It is intriguing that miRNAs are now implicated in regulation of all three of the known transcriptional effectors of EGFR signaling. miR-7 acts in two feed-forward loops downstream of EGFR to control photoreceptor specification and differentiation in the Drosophila eye. EGFR acts via the transcription factors Yan and Pointed. Yan is a direct target of miR-7. Yan also represses miR-7 transcription directly as well as indirectly. In the same cells, the ETS-1 factor Pointed-P1 activates miR-7 to repress Yan as well as acting directly to repress Yan. Use of interlinked motifs is thought to provide stability to the cell differentiation program controlled by EGFR. The current findings link bantam to regulation of a third EGFR transcriptional effector, Capicua, in addition to its regulation by the Hippo pathway. Coordination of diverse growth control inputs by miRNAs might contribute to robustness (Herranz, 2012).

EGFR/Ras signaling controls Drosophila intestinal stem cell proliferation via Capicua-regulated genes

Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. This study demonstrates that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs (Jin 2015).

It is well established that EGFR signaling is essential to drive ISC growth and division in the fly midgut. However, the precise mechanism via which this signal transduction pathway activates ISCs has remained a matter of inference from experiments with other cell types. Moreover, despite a vast literature on the pathway and ubiquitous coverage in molecular biology textbooks, the mechanisms of action of the pathway downstream of the MAPK are not well understood for any cell type. From this study, a model is proposed (see Model for Cic control of Drosophila ISC proliferation). Multiple EGFR ligands and Rhomboid proteases are induced in the midgut upon epithelial damage, which results in the activation of the EGFR, RAS, RAF, MEK, and MAPK in ISCs. MAPK phosphorylates Cic in the nucleus, which causes it to dissociate from regulatory sites on its target genes and also translocate to the cytoplasm. This allows the de-repression of target genes, which may then be activated for transcription by factors already present in the ISCs. The critical Cic target genes identified in this study include the cell cycle regulators stg (Cdc25) and Cyclin E, which in combination are sufficient to drive dormant ISCs through S and M phases, and pnt and Ets21C, ETS-type transcriptional activators that are required and sufficient for ISC activation (Jin 2015).

Upon damage, activated EGFR signaling mediates activation of ERK, which phosphorylates Cic, and relocates it to the cytoplasm. As a result, stg, CycE, Ets21C and pnt transcription are relieved from Cic repression, and induce ISC proliferation (Jin 2015).

Although this study found more than 2000 Cic binding sites in the ISC genome, not all of the genes associated with these sites were significantly upregulated, as assayed by RNA-Seq, upon Cic depletion. One possible explanation for this is that Cic binding sites from DamID-Seq were also associated with other types of transcription units (miRNAs, snRNAs, tRNAs, rRNAs, lncRNAs) that were not scored for activation by the RNA-Seq analysis. Indeed a survey of the Cic binding site distributions suggests this. This might classify some binding sites as non-mRNA-associated. However, it is also possible that many Cic target genes may require activating transcription factors that are not expressed in ISCs. Such genes might not be strongly de-repressed in the gut upon Cic depletion (Jin 2015).

In other Drosophila cells MAPK phosphorylation is thought to directly inactivate the ETS domain repressor Yan, and to directly activate the ETS domain transcriptional activator Pointed P2 (PNTP2). In fact Pnt and Yan have been shown to compete for common DNA binding sites on their target genes. Thus, previous studies proposed a model of transcriptional control by MAPK based solely on post-translational control of the activity of these ETS factors. However, this study found that Pnt and Ets21C were transcriptionally induced by MAPK signaling, and could activate ISCs if overexpressed, and that depleting yan or pntP2 had no detectable proliferation phenotype. In addition, overexpression of PNTP2 was sufficient to trigger ISC proliferation, suggesting either that basal MAPK activity is sufficient for its post-translational activation, or that PNTP2 phosphorylation is not obligatory for activity. Moreover, pntP2 loss of function mutant ISC clones had no deficiency in growth even after inducing proliferation by P.e. infection, which increases MAPK signaling. These observations indicate that the direct MAPKā†’PNTP2 phospho-activation pathway is not uniquely or specifically required for ISC proliferation. These results suggest instead that transcriptional activation of pnt and Ets21c via MAPK-dependent loss of Cic-mediated repression is the predominant mode of downstream regulation by MAPK in midgut ISCs (Jin 2015).

In addition to activating ISCs for division, EGFR signaling activates them for growth. Previous studies showed loss of EGFR signaling prevented ISC growth and division, and that ectopic RasV12 expression could accelerate the growth not only of ISCs but also post-mitotic enteroblasts. Similarly, this study shows that loss of cic caused ISC clones to grow faster than controls, by increasing cell number as well as cell size. For instance, increased size of GFP+ ISCs and EBs was observed when cic-RNAi was induced by the esgts or esgtsF/O systems. Therefore, in a search for Cic target genes probable growth regulatory genes such as Myc, Cyclin D, the Insulin/TOR components InR, PI3K, S6K and Rheb, Hpo pathway components, and the loci encoding rRNA, tRNAs and snRNAs were specifically checked. It was found that Cic bound to the InR, Akt1, Rheb, Src42A and Yki loci. However, of these only InR mRNA was significantly upregulated in Cic-depleted progenitor cells. In surveying the non-protein coding genome, it was found that Cic had binding sites in many loci encoding tRNA, snRNA, snoRNA and other non-coding RNAs, though not in the 28S rRNA or 5S rRNA genes. Due to the method used for RNA-Seq library production, RNA expression profiling experiments could not detect expression of these loci, and so it remains to be tested whether Cic may regulate some of those non-coding RNA's transcription to control cell growth. It is also possible that Cic controls cell growth regulatory target genes indirectly, for instance via its targets Ets21C and Pnt, which are also strong growth promoters in the midgut. But given that no conclusive model can be drawn from the data regarding how Cic restrains growth, one must consider the possibility that ERK signaling stimulates cell growth via non-transcriptional mechanisms, as proposed by several studies (Jin 2015).

The critical role of Cic as a negative regulator of cell proliferation in the fly midgut is consistent with its tumor suppressor function in mammalian cancer development. Also consistent with the current findings are the observations that the ETS transcription factors ETV1 and ETV5 are upregulated in sarcomas that express CIC-DUX, an oncogenic fusion protein that functions as a transcriptional activator, and that knockdown of CIC induces the transcription of ETV1, ETV4 and ETV5 in melanoma cells. Moreover the transcriptional regulation by ETS transcription factors is important in human cancer development. Their expression is induced in many tumors and cancer cell lines. For example, ERG, ETV1, and ETV4 can be upregulated in prostrate cancers, and ETV1 is upregulated in post gastrointestinal stromal tumors and in more than 40% of melanomas. In addition, the mRNA expression of these ETS genes was correlated with ERK activity in melanoma and colon cancer cell lines with activating mutations in BRAF (V600E), such that their expression decreased upon MEK inhibitor treatment. Furthermore, overexpression of the oncogenic ETS proteins ERG or ETV1 in normal prostate cells can activate a Ras/MAPK-dependent gene expression program in the absence of ERK activation. These cancer studies imply that there is an unknown factor that links Ras/Mapk activity to the expression of ETS factors, and that some of the human ETS factors might act without MAPK phosphorylation, as does Drosophila PntP1. Combining the knowledge of Cic with what was previously known about CIC in tumor development, it is proposed that CIC is the unknown factor that regulates ETS transcription factors in Ras/MAKP-activated human tumors (Jin 2015).

In summary, this study has elucidated a mechanism wherein Cic controls the expression of the cell cycle regulators stg (Cdc25) and Cyclin E, along with the Ets transcription factor Pnt, and perhaps also Ets21C, by directly binding to regulatory sites in their promoters and introns. Using genetic tests it was shown that these interactions are meaningful for regulating stem cell proliferation. Therefore, it is suggested that human CIC may also lead to the transcriptional induction of cell cycle genes and ETS transcription factors in RAS/MAPK activated- or loss-of-function-CIC tumors such as brain or colorectal cancers (Jin 2015).


The cic gene has been identified by positional cloning. cic is mapped to chromosomal position 92D1-2 using standard recombination and deficiency tests. DNA polymorphisms in this region specific to the cic1 chromosome were sought and the cic1 allele was found to be associated with the insertion of a 1.5-kb hobo transposon. This transposon maps ~300 bp away from a previously described P element, P(PZ) bwk8482, which causes the female sterile mutation bullwinkle (bwk; Rittenhouse, 1995). P(PZ) bwk8482 produces a phenotype different from cic1 and complements cic1, indicating that the two mutations affect different genetic functions. The cic1-specific hobo element is inserted in the 5' untranslated region of a novel gene, which corresponds to EST clone LD05430 from the Berkeley Drosophila Genome Project. Several lines of evidence confirm that this gene is cic. (1) Molecular analyses show that the hobo insertion disrupts the cic transcript. (2) The cic1 mutation is not complemented by small deficiencies (<500 bp) that span the hobo insertion site. (3) The cic phenotype is rescued by P-element transformation with a genomic fragment containing the cic gene. This fragment does not rescue the bwk phenotype, again showing that bwk and cic represent separate gene functions. Elucidation of the relationship between cic and bwk at the molecular level will require the cloning of bwk (Jimenez, 2000).

cDNA clone length - 5175

Bases in 5' UTR - 183

Bases in 3' UTR - 780


Amino Acids - 1393

Structural Domains

Sequencing of cic cDNA clones shows that they encode a putative transcription factor with a DNA-binding domain of the HMG box class. HMG box proteins are thought to function as architectural factors that induce bending of the target DNA and facilitate the assembly of multiprotein regulatory complexes at promoters. The Cic HMG box domain contains several amino acid residues shared by a group of HMG box factors that bind sequence specifically to DNA, such as the LEF-1/TCF-1 factors, SRY, and the SRY-related Sox proteins. Similarity searches against protein databases reveal that Cic defines a new subfamily within this group, which includes two related HMG box proteins from humans and Caenorhabditis elegans. The similarity between Cic and these proteins is particularly strong in the HMG box domain (75% and 67% identity with the human and C. elegans sequences, respectively), but also extends to other regions of the proteins, suggesting that they represent true orthologs (Jimenez, 2000).


In children, the majority of brain tumors arise in the cerebellum. Medulloblastomas, the most common of these, are believed to originate from the granule cell lineage. A mammalian gene, capicua (Cic), the ortholog of a Drosophila gene implicated in c-erbB (Egfr) signaling, is predominantly expressed during mouse granule cell development. Its expression in medulloblastoma is therefore of particular interest. This study anayzes the expression of human CIC in medulloblastoma. In silico SAGE analysis demonstrated that medulloblastomas exhibits the highest level of CIC expression and expression is most common in tumors of the CNS in general. The expression of CIC does not correlate with other markers, such as neurofilament, GFAP and Mib-1. In postnatally developing cerebellum a strong correlation was found between Cic expression and the maturation profile of cerebellar granule cell precursors. Expression of CIC is therefore a feature shared between immature granule cells and the tumors derived from them. Cic has been implicated as a mediator of ErbB signaling and this pathway has been associated with a poor prognosis for medulloblastomas. Therefore, further analysis of the role of Cic is likely to provide valuable insight into the biology of these tumors. Additionally, study of genes such as CIC should provide criteria by which to categorize these tumors into subgroups that might allow better allocation into specific treatment regimes (Lee, 2005).

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. Soluble protein complexes were examined from mouse cerebellum and it was found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions (Lam, 2006).

To establish whether CIC also interacts with mutant forms of ATXN1, immunoprecipitation was performed on lysates from HeLa cells transiently transfected with variants of FLAG-tagged ATXN1 containing 2Q, 82Q, or 82Q with a S776A mutation (which abolishes the interaction of ATXN1 with 14-3-3) or 2Q with a deletion of the AXH domain. Both endogenous CIC isoforms coimmunoprecipitated with each of these ATXN1 variants, except the one lacking the AXH domain. It is concluded that the ATXN1-CIC interaction requires the AXH domain and is independent of 14-3-3 binding (Lam, 2006).

To identify the domains responsible for interaction between the two proteins, yeast two-hybrid interaction assays were performed using a series of ATXN1 and CIC deletion constructs. Two N-terminal fragments of CIC-S (amino acids 1-300 and 1-205) interact with the C-terminal half of ATXN1, and the AXH domain of ATXN1 is sufficient for its interaction with CIC. To further map the ATXN1 binding domain of CIC, serial deletions were generated at the N terminus of mouse CIC-S for pull-down assays with GST-tagged full-length wild-type ATXN1. The interaction region was narrowed to 31 amino acids (amino acids 16-46) of CIC-S. Comparison of this 31-amino acid sequence across species revealed a conserved stretch of eight amino acids present in both CIC-S and CIC-L isoforms with the consensus sequence WXX(L/I)(V/L)PX(L/M). It was then confirmed in vitro that human ATXN1 binds to Drosophila Cic through the consensus eight amino acids. ATXN1 and CIC thus appear to be in vivo binding partners that interact directly through evolutionarily conserved domains (Lam, 2006).

CIC contains a Sox-like high mobility group (HMG) box and likely acts as a transcriptional repressor. Given that ATXN1 lacks sequence-specific DNA binding activity, CIC could be involved in directing ATXN1 to gene targets for repression. These studies clearly show that ATXN1 has a synergistic effect on the transcriptional repressor activity of CIC that is partially compromised by the polyglutamine expansion. One possible explanation is that polyglutamine expansion alters the conformational state of the ATXN1 protein, which in turn alters the conformational or functional state of the ATXN1-CIC complex. Identifying transcriptional targets of the ATXN1-CIC complexes, particularly in the selectively vulnerable Purkinje cells, will likely reveal specific pathways that are critical for disease pathogenesis (Lam, 2006).

Ewing's family tumors (EFTs) are highly malignant tumors arising from bone and soft tissues that exhibit EWS-FLI1 or variant EWS-ETS gene fusions in more than 85% of the cases. CIC, a human homolog of Drosophila capicua which encodes a high mobility group box transcription factor, is fused to a double homeodomain gene DUX4 as a result of a recurrent chromosomal translocation t(4;19)(q35;q13). This translocation was seen in two cases of soft tissue sarcoma diagnosed as Ewing-like sarcoma. CIC-DUX4 exhibits a transforming potential for NIH 3T3 fibroblasts, and as a consequence of fusion with a C-terminal fragment of DUX4, CIC acquires an enhanced transcriptional activity, suggesting that expression of its downstream targets might be deregulated. Gene expression analysis identified the ETS family genes, ERM/ETV5 and ETV1, as potential targets for the gene product of CIC-DUX4. Indeed, CIC-DUX4 directly binds the ERM promoter by recognizing a novel target sequence and significantly up-regulates its expression. This study clarifies the function of CIC and its role in tumorigenesis, as well as the importance of the PEA3 subclass of ETS family proteins in the development of EFTs arising through mechanisms different from those involving EWS-ETS chimeras. Moreover, the study identifies the role of DUX4 that is closely linked to facioscapulohumeral muscular dystrophy in transcriptional regulation (Kawamura-Saito, 2006).

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine tract in ataxin-1 (ATXN1). SCA1 pathogenesis studies support a model in which the expanded glutamine tract causes toxicity by modulating the normal activities of ATXN1. To explore native interactions that modify the toxicity of ATXN1, a targeted duplication of the mouse ataxin-1-like (Atxn1l, also known as Boat) locus, a highly conserved paralog of SCA1, was generated, and the role of this protein in SCA1 pathology was tested. Using a knock-in mouse model of SCA1 that recapitulates the selective neurodegeneration seen in affected individuals, it was found that elevated Atxn1l levels suppress neuropathology by displacing mutant Atxn1 from its native complex with Capicua (CIC). These results provide genetic evidence that the selective neuropathology of SCA1 arises from modulation of a core functional activity of ATXN1, and they underscore the importance of studying the paralogs of genes mutated in neurodegenerative diseases to gain insight into mechanisms of pathogenesis (Bowman, 2007).

Loss of Capicua alters early T cell development and predisposes mice to T cell lymphoblastic leukemia/lymphoma

Capicua (CIC) regulates a transcriptional network downstream of the RAS/MAPK signaling cascade. In Drosophila, CIC is important for many developmental processes, including embryonic patterning and specification of wing veins. In humans, CIC has been implicated in neurological diseases, including spinocerebellar ataxia type 1 (SCA1) and a neurodevelopmental syndrome. Additionally, mutations in CIC have been reported in several cancers. However, whether CIC is a tumor suppressor remains to be formally tested. This study found that deletion of Cic in adult mice causes T cell acute lymphoblastic leukemia/lymphoma (T-ALL). Using hematopoietic-specific deletion and bone marrow transplantation studies, it was shown that loss of Cic from hematopoietic cells is sufficient to drive T-ALL. Cic-null tumors show up-regulation of the KRAS pathway as well as activation of the NOTCH1 and MYC transcriptional programs. In sum, this study demonstrates that loss of CIC causes T-ALL, establishing it as a tumor suppressor for lymphoid malignancies. Moreover, mouse models lacking CIC in the hematopoietic system were shown to be robust models for studying the role of RAS signaling as well as NOTCH1 and MYC transcriptional programs in T-ALL (Tan, 2018).

capicua: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 7 October 2021

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