Suppressor of fused


Drosophila Suppressor of fused [Su(fu)] encodes a novel 468-amino-acid cytoplasmic protein that, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. This study describes the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) [hSu(fu)]. Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52 amino-acid extension at the carboxy terminus. Both isoforms are expressed in multiple embryonic and adult tissues, and exhibit a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus maps to chromosome 10q24-q25, a region that is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) represses activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and physically interacts with Gli, Gli2 and Gli3 as well as with Supernumerary limbs (Slimb), an F-box containing protein that, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homolog. Coexpression of Slimb with Su(fu) potentiates the Su(fu)-mediated repression of Gli. Taken together, these data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein that links Gli to the Slimb-dependent proteasomal degradation pathway (Stone, 1999).

The human Suppressor-of-Fused (SUFUH) complementary DNA has been identified and the gene product has been shown to interact physically with the transcriptional effector GLI-1. SUFUH can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signaling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a GLI-1 nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signaling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses (Kogerman, 1999).

To test whether vertebrate Sufu is expressed in a pattern consistent with a potential role in mediating Shh signaling during embryogenesis, whole-mount in situ hybridization was used to analyse Sufu expression in mouse embryos at days 8.5 to 15.5 of development. Throughout the entire period signals were observed in the neural tube and, at the later stages, in the neural tube derivatives -- the brain and spinal cord. The somites express Sufu at all stages; the vibrissae field stain positively for Sufu from day 12.5 and onwards, with the vibrissae themselves being spared. The Sufu expression pattern during limb-bud development appears to be separated into two distinct phases, with strong homogeneous staining all over the limb buds being observed from their emergence at 9.5 days, whereas at 12.5 days only the interdigital mesenchyme of the limbs stain positively. This expression pattern partially overlaps with the expression of Ptch and the Ci homologs Gli 1-3, and is compatible with a conserved role for Sufu in Shh signaling (Kogerman, 1999).

To substantiate this observation in more detail and in the human system, the expression of SUFUH and PTCH1 was analyzed in the developing limb of a 12-week-old human embryo by radioactive in situ hybridization. The results show marked SUFUH expression in the osteoblasts of the perichondrium, where PTCH1 is also highly expressed. These findings are consistent with earlier observations in the avian and murine systems, in which Ptch1 and Gli1 are highly expressed in the same type of cells in response to Ihh secretion by prehypertrophic chondrocytes. Taken together, these results show that SUFUH is preferentially expressed in cells that receive a Hedgehog signal, and indicate that, during embryogenesis, SUFUH may be co-regulated with PTCH1 and GLI1 (Kogerman, 1999).

The retention of GLI-1 in the cytoplasm by SUFUH when nuclear export is compromised, and the similar SUFUH-mediated retention in the cytoplasm of an otherwise constitutively nuclear GLI-1 variant (truncated so that it lacks the NES) indicates that SUFUH could block nuclear entry of GLI-1, possibly by masking a nuclear-localization signal, and thereby inhibit transcriptional activation of target genes. Consistent with this idea, a truncated SUFUH variant unable to repress GLI1-induced transcriptional activation is also unable to modify the subcellular localization of GLI-1. What remains an interesting question for future studies is whether or not binding of SUFUH to GLI-1 on DNA, or elsewhere in the nuclear compartment, actually acts to repress or block activation of transcription, alone or in combination with cytoplasmic retention of GLI-1. The expression of Sufu in cells next to Shh- or Ihh-producing cells during mouse and human embryogenesis, coupled with the ability of Sufu to inhibit Gli-mediated transcriptional activation, indicates that an important function of Sufu may be to act in an intracellular negative feedback mechanism and to impose thresholds on the responsiveness of cells to Shh and Ihh. A similar role for D-Axin has been proposed as regards Wingless signaling in Drosophila (Kogerman, 1999).

The Suppressor of fused [Su(fu)] gene of Drosophila encodes a protein containing a PEST sequence [a sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] that acts as an antagonist to the serine-threonine kinase Fused in Hedgehog (Hh) signal transduction during embryogenesis. The Su(fu) gene isolated from a distantly related Drosophila species, D. virilis, shows significantly high homology throughout its protein sequence with its D. melanogaster counterpart. These two Drosophila homologs of Su(fu) are functionally interchangeable in enhancing the fused phenotype. Mammalian homologs of Su(fu) have been isolated. The absence of the PEST sequence in the mammalian Su(fu) protein suggests a different regulation for this product between fly and vertebrates. Using the yeast two-hybrid method, the murine Su(fu) protein has been shown to interact directly with the Fused and Cubitus interruptus proteins, known partners of Su(fu) in Drosophila. Su(fu) could be regulated posttranslationally in the fly and at another level in vertebrates. A similar divergence is observed for the regulation of the ci gene and its homologs, the Gli genes: in Drosophila, there is only one ci gene whose product is regulated posttranslationally; in vertebrates, there are three ci-related genes (Gli, Gli2 and Gli3) that are regulated at a transcriptional level (Delattre, 1999).

The Hedgehog (Hh) signaling pathway has critical functions during embryogenesis of both invertebrate and vertebrate species; defects in this pathway in humans can cause developmental disorders as well as neoplasia. Although the Gli1, Gli2, and Gli3 zinc finger proteins are known to be effectors of Hh signaling in vertebrates, the mechanisms regulating activity of these transcription factors remain poorly understood. In Drosophila, activity of the Gli homolog Cubitus interruptus (Ci) is likely to be modulated by its interaction with a cytoplasmic complex containing several other proteins, including Costal2, Fused (Fu), and Suppressor of fused [Su(fu)], the last of which has been shown to interact directly with Ci. Mouse Suppressor of fused [mSu(fu)] has been cloned, and its 4.5 kb transcript has been detected throughout embryogenesis and in several adult tissues. In cultured cells, mSu(fu) overexpression inhibits transcriptional activation mediated by Sonic hedgehog (Shh), Gli1 and Gli2. Co-immunoprecipitation of epitope-tagged proteins indicates that mSu(fu) interacts with Gli1, Gli2, and Gli3, and that the inhibitory effects of mSu(fu) on Gli1's transcriptional activity are mediated through interactions with both amino- and carboxy-terminal regions of Gli1. Gli1 is localized primarily to the nucleus of both HeLa cells and the Shh-responsive cell line MNS-70; co-expression with mSu(fu) results in a striking increase in cytoplasmic Gli1 immunostaining. These findings indicate that mSu(fu) can function as a negative regulator of Shh signaling and suggest that this effect is mediated by interaction with Gli transcription factors (Ding, 1999).

Suppressor of fused (Sufu) is a negative regulator of the Hedgehog pathway both in Drosophila and vertebrates. The genomic organization of the mouse Sufu gene (mSufu) comprises 11 exons spanning more than 30 kb, encoding a protein with a putative PEST sequence. DNA-consensus sequences recognized by basic helix-loop-helix (bHLH) proteins, referred to as E-box motifs, are found in the 5' flanking region. Analyses employing single-strand conformation polymorphism and radiation hybrids have positioned the Sufu locus to the distal end of mouse Chr 19 between D19Mit102 and D19Mit9, near the Fgf8 and dactylin genes. Mouse Sufu is expressed in various tissues, particularly in the nervous system, ectoderm, and limbs, throughout the developing embryo. Sufu binds with all three Gli proteins, with different affinities. This report, in conjunction with recent studies, points out the importance of Sufu in mouse embryonic development (Simon-Chazottes, 2000).

Hedgehog (Hh) proteins are secreted factors that control cell proliferation and cell-fate specification. Hh signaling is mediated in vertebrates by the Gli zinc-finger transcription factors (Gli1, Gli2 and Gli3) and in Drosophila by the Gli homolog Cubitus interruptus (Ci). However, the mechanisms that regulate Gli/Ci activity are not fully understood. Genetic studies in Drosophila have identified a putative serine-threonine kinase, Fused (Fu), and a new protein, Suppressor of Fused [Su(fu)], as modulators of Ci activity. A human homolog of Drosophila Fu, hFu, regulates the activity of Gli1 and Gli2 on several levels. hFu converts Gli2 from a weak to a strong transcriptional activator; antagonizes the repressive effect of the human Su(fu) homolog [hSu(fu)] on Gli1 and Gli2, and promotes nuclear localization of Gli1 and Gli2 (Murone, 2000).

To identify possible regulators of Gli proteins, complementary DNAs were isolated encoding hFu, which shares a significant level of homology with Drosophila Fu in the kinase domain (55%), but only a limited amount of homology over the remaining 1,052 amino acids. The gene encoding hFu was mapped to chromosome 2q35, close to the PAX3 gene, which is implicated in the Klein-Waardenburg syndrome. PAX3 is a target of Sonic hedgehog (Shh) and it has been suggested that additional loci in the 2q35 region may regulate the PAX3 locus, thereby influencing the Klein-Waardenburg phenotype. Northern-blot analysis has shown that a single 5-kb hFu transcript is expressed at low levels in most fetal tissues and adult ovaries, and at high levels in adult testes, where it is localized in germ cells with other components of the Hh pathway. Examination by in situ hybridization of a mouse embryo at day 13.5 of development shows that mouse Fu (mFu) mRNA is widely distributed in Shh-responsive tissues, including the forebrain, midbrain, hindbrain, spinal cord, somites, developing limb buds and skin (Murone, 2000).

To determine whether hFu can regulate Gli activity, hFu was cotransfected with a Gli-binding-site (Gli-BS) luciferase reporter in the Hh-responsive cell line C3H10T1/2. hFu alone is capable of weakly inducing transcription of the Gli-BS reporter, indicating that it may be a positive regulator of the Hh pathway. Although hFu contains a putative kinase domain, no substantial kinase activity for hFu was detected; a similar lack of kinase activity has been reported for Drosophila Fused (Murone, 2000).

To determine the function of the kinase domain of hFu, a putative catalytically dead version of hFu [hFu(K33R)] was constructed by mutating a conserved lysine residue in the ATP-binding site at position 33. This residue is crucial to the catalytic activity of all kinases, and the corresponding mutation in Drosophila leads to a fu phenotype. hFu(K33R) is able to activate the Gli-BS reporter as efficiently as wild-type hFu, indicating that the putative kinase activity of hFu may not contribute significantly to Gli activation under these conditions. A similar result has been obtained for a hFu construct [hFu(270-1,315)] lacking the entire kinase domain (amino acids 1-269). The activity of hFu was tested in combination with various Gli-family members. Whereas human Gli1 alone strongly induces the luciferase reporter, mouse Gli2 exhibits only weak activity and human Gli3 shows no activity at all. hFu does not affect the activity of Gli1 and Gli3, but strongly synergizes with Gli2. Moreover, activation of Gli2 by hFu is antagonized by hSu(fu). In contrast, Gli1 is constitutively active and its ability to activate the Gli-BS reporter is inhibited by hSu(fu) and restored in the presence of hFu (Murone, 2000).

To investigate further the mechanisms by which hFu regulates Gli activity, it had to be determined whether hFu forms a physical complex with hSu(fu) or the various Gli proteins. Cultured cells were cotransfected with epitope-tagged versions of hFu, hSu(fu), Gli1, Gli2 and Gli3 and the resulting interactions were observed. hFu co-immunoprecipitates with hSu(fu) and with Gli1, Gli2 and Gli3. In vertebrates, Su(fu) represses Gli1 function in part by tethering it in the cytoplasm. In contrast, hFu and hFu(K 33R) promote nuclear localization of Gli1. An assessment was made of whether hFu could influence the subcellular localization of Gli1 when co-expressed with hSu(fu). In the presence of hSu(fu), roughly 3% of cells exhibit nuclear staining of Gli1. In contrast, when both hSu(fu) and hFu are present, 20% of cells possess nuclear Gli1. Identical results are obtained for Gli2. Overall, these results indicate that hFu controls the activity of Gli1 and Gli2 by opposing the effect of hSu(fu). Whereas hSu(fu) constrains Gli1 and Gli2 in the cytoplasm, hFu promotes their nuclear localization. Gli2 also requires an additional function of hFu to become transcriptionally active, since Gli2 transfected in the absence of hSu(fu) is unable to activate transcription unless hFu is present, despite the fact that it enters the nucleus. The mechanisms by which hFu activates Gli2 remain to be elucidated but may include a hFu-mediated modification of Gli2 to mask the inhibitory Gli2 amino-terminal domain (Murone, 2000).

The activity of hFu described here does not seem to require a functional kinase domain, since overexpression of kinase-mutant forms of Fu are as active as wild-type forms. Catalytically dead versions of other serine-threonine kinases, such as the RIPs8 and IRAKs14, show comparable activity to their wild-type counterparts in (respectively) inducing apoptosis or activating NFkappaB. Although some Drosophila kinase-domain fu mutants suffer a complete lack of induction of Hh target genes in the embryo, they show only a partial fu phenotype in the wing discs, indicating that there may be different requirements for the kinase activity of Fu in different cellular contexts (Murone, 2000).

The Suppressor of Fused [Su(fu)] protein plays a conserved role in the regulation of Gli transcription factors of the hedgehog (Hh) signaling pathway that controls cell fate and tissue patterning during development. In both Drosophila and mammals, Su(fu) represses Gli-mediated transcription, but the mode of its action is not completely understood. Recent evidence suggests that Su(fu) physically interacts with the Gli proteins and, when overexpressed, sequesters Gli in the cytoplasm. However, Su(fu) also traverses into the nucleus under the influence of a serine-threonine kinase, Fused (Fu), and has the ability to form a DNA-binding complex with Gli, suggesting that it has a nuclear function. This study reports that the mouse homolog of Su(fu) [mSu(fu)] specifically interacts with SAP18 (see Drosophila Sap18), a component of the mSin3 and histone deacetylase complex. In addition, mSu(fu) functionally cooperates with SAP18 to repress transcription by recruiting the SAP18-mSin3 complex to promoters containing the Gli-binding element. These results provide biochemical evidence that Su(fu) directly participates in modulating the transcriptional activity of Gli (Cheng, 2002).

Sonic hedgehog signaling plays a critical role during development and carcinogenesis. While Gli family members govern the transcriptional output of Shh signaling, little is known how Gli-mediated transcriptional activity is regulated. The actin-binding protein Missing in Metastasis (MIM) has been identified as a new Shh-responsive gene. MIM is a member of the Wiskott-Aldrich Syndrome family of proteins and contains a conserved coiled-coil protein interaction domain and a C-terminal WH2 domain. Previous independent studies have shown that MIM binds monomeric actin through its WH2 domain and bundles F-actin using its N-terminal coiled-coil domain. Together, Gli1 and MIM recapitulate Shh-mediated epidermal proliferation and invasion in regenerated human skin. MIM is part of a Gli/Suppressor of Fused complex and potentiates Gli-dependent transcription using domains distinct from those used for monomeric actin binding. These data define MIM as both a Shh-responsive gene and a new member of the pathway that modulates Gli responses during growth and tumorigenesis (Callahan, 2004).

Suppressor of fused is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein was determined. Several mSufu variants were identified, some of which were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed the identification of several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. The mSufu-SAP18 and mSufu-Galectin3 interactions were confirmed by independent biochemical assays. Using a cell transfection assay, it was also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. These findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus (Paces-Fessy, 2004).

Suppressor of fused is a negative regulator of Hh signaling. Targeted disruption of the murine suppressor of fused gene (Sufu) leads to a phenotype that includes neural tube defects and lethality at mid-gestation (9.0-10.5 dpc). This phenotype resembles that caused by loss of patched (Ptch1), another negative regulator of the Hh pathway. Consistent with this finding, Ptch1 and Sufu mutants display excess Hh signaling and resultant altered dorsoventral patterning of the neural tube. Sufu mutants also had abnormal cardiac looping, indicating a defect in the determination of left-right asymmetry. Marked expansion of nodal expression in 7.5 dpc embryos and variable degrees of node dysmorphology in 7.75 dpc embryos suggest that the pathogenesis of the cardiac developmental abnormalities is related to node development. Other mutants of the Hh pathway, such as Shh, Smo and Shh/Ihh compound mutants, also have laterality defects. In contrast to Ptch1 heterozygous mice, Sufu heterozygotes have no developmental defects and no apparent tumor predisposition. The resemblance of Sufu homozygotes to Ptch1 homozygotes is consistent with mouse Sufu being a conserved negative modulator of Hh signaling (Cooper, 2005).

Genetic elimination of Suppressor of Fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. This study shows that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at ~E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu−/− embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu−/− fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu+/− mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. These data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway (Svärd, 2006).

Sufu knockouts are embryonic lethal and show strong similarities with Ptch1 knockouts, and Hh signaling is strongly activated in a ligand-independent manner in Sufu−/− cells. Moreover, Sufu+/− mice develop a skin phenotype with many features found in Gorlin syndrome. Furthermore, no support for a direct role of Sufu in the Wnt pathway was found (Svärd, 2006).

There is a remarkable agreement between the Sufu−/− and Ptch1−/− embryos in terms of the spatiotemporal expression pattern of all the markers examined both in the whole-mount embryo and neural tube analysis. High-level expression of the Ptch1 and Gli1 target genes in the Sufu−/− embryos suggests that Sufu is endowed with a strong repressor activity, removal of which causes ectopic activation of the Hh pathway. It should be emphasized that in the ventralized spinal cord from the Sufu−/− embryos, prominent ectopic expression of target genes such as FoxA2 and Nkx2.2, which require the highest levels of Hh signaling, is evident. Evidently, this high Hh activity is dominant in patterning the neural tube over the dorsalizing signals mediated by the Bmp proteins, which normally act in an antagonizing manner to Shh in the neural tube. Taken together, this indicates that mammalian Sufu, in striking contrast to the situation in Drosophila, is an equally strong repressor of the Hh pathway, as Ptch1 and removal of either one is sufficient to induce a similar high level of cell autonomous Hh signaling (Svärd, 2006).

Based on studies of the effects of activators and inhibitors (SAG and cyclopamine, respectively) of Smo activity in the Sufu−/− MEFs, it appears that these cells have uncoupled the upstream ligand-dependent activation of Smo from the downstream Gli activity. This observation further suggests that constitutive activation of Hh signaling in cells lacking Sufu is determined by their intrinsic competence for Hh response and not actual exposure to Hh ligands. This independence of Hh ligand is further demonstrated in mouse Sufu−/−; Shh−/− double mutant embryos, which appear morphologically similar to the Sufu−/− mutants. It has similarly been shown that downregulation of Sufu levels by RNAi in NIH-3T3 or Smo−/− cells results in activation of Hh signaling (Svärd, 2006).

The severe functional consequences and the high level of Hh signaling caused by deleting the Sufu gene in the mouse was surprising given the overall conservation of the Hh pathway during evolution and the lack of an altered phenotype in the corresponding Drosophila mutants. Recently, another unanticipated result was revealed when a possible mouse ortholog of Drosophila Fu was targeted and no apparent Hh-dependent phenotypes during embryonic development were seen. In contrast, Drosophila fu mutants are embryonic lethal, and, in zebrafish, MO knockdown of Fu abrogates Hh-dependent specification of myotome cell types. Moreover, genetic inactivation or MO knockdown of Sufu in zebrafish results in a detectable Hh-related phenotype, but the effect is much weaker than that caused by elimination of Ptc. In contrast, the data presented here indicate that, in the mouse, null mutations of Sufu and Ptch1 produce equally strong perturbations of the Hh pathway. This suggests that not only has the Hh pathway evolved differently in vertebrates compared to invertebrates, but also within vertebrates, since mammals and teleosts show divergence in the pathway. Another such example of differences in the Hh pathway is illustrated by the recent finding that intraflagellar transport (IFT) proteins are required for transduction of the Hh signal in the mouse, whereas analysis of IFT mutants in zebrafish and Drosophila did not reveal any Hh-dependent phenotypes. This fundamental divergence in regulatory mechanisms has several important implications. In the fly, it is presently believed that Su(fu) together with the atypical kinesin Cos2 represent intracellular negative regulators of Hh signaling and that Cos2 plays a major role by tethering the transcriptional effector Ci to microtubules and, in the absence of ligand, promotes processing of Ci to its truncated repressor form. Upon ligand stimulation, a direct interaction between the C-terminal tail of Smo and Cos2 is enhanced, leading to inhibition of Ci processing and nuclear availability of activated full-length Ci. Su(fu), on the other hand, resides mostly in a separate intracellular complex also containing Ci and functions both to tether Ci in the cytoplasm and to inhibit activated full-length Ci. However, even in the absence of Su(fu), Cos2 is able to sequester Ci in the cytoplasm and maintain normal regulation of Hh signaling, whereas, in the absence of Cos2, constitutive activation of Ci ensues. In the mouse, Sufu loss alone leads to complete activation of the Hh signaling pathway, and mice therefore may not utilize a Cos2-like activity that in Drosophila is sufficient to maintain Hh pathway regulation in the absence of Sufu. Thus, when Sufu is removed, unrestrained Gli-mediated transcriptional activation is allowed by eliminating repression mechanisms normally inhibiting Gli activity. In mammalian cells, Gli1 and Gli2 serve mainly as positive transcriptional regulators, whereas Gli3 is processed and, for the most part, acts as a repressor. Since Sufu can interact with the N-terminal part of Gli proteins and the Sufu binding motif is retained in the Gli3 repressor form, it appeared likely that Sufu also exerts a negative influence on the repressor activity of Gli3. Since a strong activation of Hh signaling was found in cells lacking Sufu in vivo and in vitro, it is proposed that Gli-mediated transcriptional activation is dominant over Gli-mediated repression. Alternatively, this result may be due to the fact that Sufu, in analogy to the situation with Gli1, must interact with two different domains on Gli3, one of which is missing in the Gli3 repressor form, to achieve effective repression. This result may also be due to differential expression domains for the activating and repressive Gli isoforms (Svärd, 2006).

The results presented in this study highlight an important evolutionary divergence in the basic regulatory circuits controlling Hh signaling, a pathway having a key role in development and human disease, including cancer, thereby illustrating the need to understand in detail the function of the pathway in mammalian systems. The unanticipated role of Sufu as a specific and potent repressor of Hh signaling also opens new therapeutic avenues to control deregulated Hh pathway activation, for example, by development of Sufu mimetics (Svärd, 2006).

Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved

A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. This study focussed on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveals that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. It is proposed that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop (speckle-type POZ protein), a conserved Gli-degrading factor [a homolog of the Drosophila MATH and BTB domain-containing protein Hib (Roadkill or Rdx). Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. A novel positive role of Sufu in regulating Hh signaling was uncovered, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species (Chen, 2009).

Studies on Sufu provide important mechanistic insight into how Sufu regulates Hh signaling. Largely based on physical interactions between Sufu and Gli proteins, the traditional model proposed that Sufu tethers Gli protein in the cytoplasm, preventing nuclear translocation and subsequent activation of target genes. This study showed that Sufu antagonizes Spop, preventing degradation of full-length Gli2 and Gli3. The process of Sufu-Spop antagonism is evolutionarily conserved since Drosophila Sufu protects Ci from Hib-mediated degradation through competitive binding to Ci (Zhang, 2006). As a result, loss of Sufu affects production of Gli2/Gli3 activator and repressor forms, which are both derived from full-length proteins. This is achieved by Hib/Spop forming a complex with Ci/Gli2/Gli3 and Cul3, thus promoting Ci/Gli ubiquitination through the Cul3-based E3 ubiquitin ligase and resulting in complete degradation by the 26S proteasome (Chen, 2009).

Drosophila Sufu is able to partially restore the defects in Gli2/Gli3 protein levels, ciliary localization, and Hh pathway activation in Sufu-/- MEFs, supporting conservation of this process. Interestingly, overexpression of Drosophila Sufu in imaginal discs inhibits Hh target gene expression in anterior cells that receive the Hh signal, but activates Hh target gene expression in the most anterior region that does not receive the Hh signal. This is consistent with a dual role of fly Sufu and whether a conserved mechanism underlies these effects needs to be further investigated (Chen, 2009).

Nevertheless, important differences in the Sufu-Spop-Gli circuit exist between flies and mammals. Gli1, unlike Gli2 and Gli3, does not appear to be subject to Spop regulation. Furthermore, while sufu mutant flies are viable, Sufu-/- mice die during early embryogenesis. Therefore, the gain-of-function phenotype in Sufu-null mice may result from increased levels of Gli1, triggered by Spop-mediated degradation of full-length Gli2/Gli3. Gli1 may have lost a requisite Spop-interacting domain, allowing it to escape regulation by Spop. Identification of domains in Gli2 and Gli3 that interact with Spop will further clarify this issue. Notably, full-length Ci and Ci repressor levels appear to be proportionately reduced in sufu mutant flies, implying that sufu affects Ci protein stability. Duplication of the ancestral Ci/Gli gene, coupled with subfunctionalization (including the distribution of activator and repressor function) and evolution of negative and positive transcriptional regulatory loops, may account for the vastly different effects of loss of Sufu in insects and vertebrates (Chen, 2009).

Regulation of Gli protein stability is a key step in controlling Hh pathway activity, and multiple, distinct degradation signals have been identified in the three Gli proteins. For instance, two degradation signals are present in Gli1, one of which contains recognition sequences for the β-TrCP adapter protein, and two β-TrCP-binding motifs also exist in Gli2. This allows utilization of the β-TrCP adapter protein for Gli1/2 proteolysis via the Cul1-based E3 ligase, distinct from Spop-mediated Gli2/3 degradation through the Cul3-based E3 ligase.β-TrCP is also required for limited proteolysis of Gli3 into a truncated repressor form. A critical unresolved issue is to understand how multiple degradation signals in Gli proteins are used to regulate full-length protein stability as well as generation of repressor forms. Further investigation is required to determine if the role of Sufu is specific in antagonizing Spop-mediated degradation, or if it is capable of opposing additional degradative pathways. It is also formally possible that Sufu has a direct effect on Gli repressor stability (Chen, 2009).

Sufu was postulated to function in both the nucleus and the cytoplasm, as overexpressed Sufu protein in cultured cells could be detected in both compartments. Furthermore, Sufu can be coimmunoprecipitated with all three Gli proteins and was shown to cooperate with SAP18-Sin3 corepressor complex in repressing transcription from a multimerized Gli-binding site luciferase reporter. Thus, it was proposed that Sufu may have a direct role in repressing Gli-mediated transcription in the nucleus in addition to sequestering Gli proteins in the cytoplasm. Recent work has challenged Sufu's cytoplasmic function by demonstrating that an overexpressed Gli1-eGFP fusion protein has a similar cytoplasmic distribution in wild-type or Sufu-deficient MEFs; in both cell types, Gli1 is largely cytoplasmic and becomes predominantly nuclear when nuclear export is blocked. However, the distributions of overexpressed Gli proteins may fail to reflect those of endogenous Gli proteins. Importantly, conclusions based on Gli1 studies may not be applicable to Gli2 and Gli3 given their distinct properties. This study observed that knockdown of Spop in Sufu-/- MEFs partially restored levels of cytoplasmic Gli2 and Gli3, resembling the wild-type nuclear-cytoplasmic distribution. While the data suggest that Sufu may have minimal effect on shuttling Gli1, Gli2, and Gli3, potential alternations in kinetics of Gli trafficking or possible post-transcription degradation events cannot be ruled out at this time. Contrary to previous reports, this stidu failed to observe any discernable effects of SAP18 either singly or in conjunction with other Hh pathway components on Hh pathway activity in MEFs. Nevertheless, although these studies highlight a major function of Sufu in regulating cytoplasmic Gli protein levels, potential minor roles in the nucleus cannot be conclusively excluded (Chen, 2009).

The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins

The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. The interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. It is proposed that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer (Humke, 2010).

Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors

Gli2 and Gli3 are primary transcriptional regulators that mediate hedgehog (Hh) signaling. Mechanisms that stabilize and destabilize Gli2 and Gli3 are essential for the proteins to promptly respond to Hh signaling or to be inactivated following the activation. This study show that loss of suppressor of fused (Sufu; an inhibitory effector for Gli proteins) results in destabilization of Gli2 and Gli3 full-length activators but not of their C-terminally processed repressors, whereas overexpression of Sufu stabilizes them. By contrast, RNAi knockdown of Spop (a substrate-binding adaptor for the cullin3-based ubiquitin E3 ligase) in Sufu mutant mouse embryonic fibroblasts (MEFs) can restore the levels of Gli2 and Gli3 full-length proteins, but not those of their repressors, whereas introducing Sufu into the MEFs stabilizes Gli2 and Gli3 full-length proteins and rescues Gli3 processing. Consistent with these findings, forced Spop expression promotes Gli2 and Gli3 degradation and Gli3 processing. The functions of Sufu and Spop oppose each other through their competitive binding to the N- and C-terminal regions of Gli3 or the C-terminal region of Gli2. More importantly, the Gli3 repressor expressed by a Gli3 mutant allele (Gli3Delta699) can mostly rescue the ventralized neural tube phenotypes of Sufu mutant embryos, indicating that the Gli3 repressor can function independently of Sufu. This study provides a new insight into the regulation of Gli2 and Gli3 stability and processing by Sufu and Spop, and reveals the unexpected Sufu-independent Gli3 repressor function (Wang, 2010).

moothened transduces Hedgehog signal by forming a complex with Evc/Evc2

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis in species ranging from Drosophila to mammals. The Hh signal is transduced by Smoothened (Smo), a seven-transmembrane protein related to G protein coupled receptors. Despite a conserved mechanism by which Hh activates Smo in Drosophila and mammals, how mammalian Hh signal is transduced from Smo to the Gli transcription factors is poorly understood. This study provides evidence that two ciliary proteins, Evc and Evc2, the products of human disease genes responsible for the Ellis-van Creveld syndrome, act downstream of Smo to transduce the Hh signal. Loss of ciliary protein complex Evc/Evc2 does not affect Sonic Hedgehog-induced Smo phosphorylation and ciliary localization but impedes Hh pathway activation mediated by constitutively active forms of Smo. Evc/Evc2 are dispensable for the constitutive Gli activity in Sufu(-/-) cells, suggesting that Evc/Evc2 act upstream of Sufu to promote Gli activation. Furthermore, it was demonstrated that Hh stimulates binding of Evc/Evc2 to Smo depending on phosphorylation of the Smo C-terminal intracellular tail and that the binding is abolished in Kif3a(-/-) cilium-deficient cells. It is propose that Hh activates Smo by inducing its phosphorylation, which recruits Evc/Evc2 to activate Gli proteins by antagonizing Sufu in the primary cilia (Yang, 2012).

This study has obtained evidence that Hh stimulates the binding of Kif7 to Smo, which is facilitated by Evc/Evc2. Increased binding of Cos2 to activated Smo has also been observed in Drosophila. It has been shown that binding of Cos2 to activated Smo promotes Fu dimerization, phosphorylation and activation, and activated Fu then regulates Ci by inhibiting its repressor form and stimulating its activator form. The mammalian Fu homolog is dispensable for Hh signaling; however, it is possible that another kinase(s) may substitute for the Fu function in the mammalian Hh pathway. It has been shown that Hh stimulates Gli3 phosphorylation, which correlates with formation of Gli3. Therefore, it would be interesting to determine whether Hh-induced Gli3 phosphorylation is affected by loss of Evc/Evc2. It is tempting to speculate that the activated Smo/Evc/Evc2 complex may recruit one or more kinases to phosphorylate Gli proteins and promote their activation (Yang, 2012).

Neuropilins are positive regulators of Hedgehog signal transduction

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. This study shows that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, it was shown that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish; morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system (Hillman, 2011).

This study investigated the mechanism of Nrp action in the Hh pathway to the extent allowed by contemporary understanding of pathway biology. The molecular mechanisms of well-studied pathway components such as Smo and SuFu are only beginning to be understood, making it likely that a more detailed understanding of Nrp function will emerge concomitantly with increases in understanding of Hh pathway biology. Nrps could affect Hh signal transduction in one or more of several distinct ways: action as a coreceptor for ligand or as a downstream transducer and integrator of external stimuli, or by affecting a basic cell property such as cilium formation, adhesion, or intracellular trafficking. The experiments narrow the possibilities. First, it is thought unlikely that Nrps control Hh reception by acting in a coreceptor capacity for Hh ligands, since activation of Hh signal transduction by ligand-independent methods (Ptc1 mutation, SAG stimulation) is sensitive to Nrp loss of function. Similarly, the inhibition of Hh signaling caused by Nrp RNAi is not simply due to generalized cellular derangement in fibroblasts, since canonical Wnt signaling is intact in the absence of Nrp function (Hillman, 2011).

No evidence was found that Nrps act as signal integrators, because there appears to be no convergence between VEGF or Sema signals and the Hh pathway. In the cultured fibroblasts where Hh signal transduction was observed to require Nrp function, the Semas that interact with Nrp had no effect on Hh transduction and the VEGF receptor is not expressed. Possible interactions between Hh and these other pathways remain an open question for other cell types, but they do not explain the requirement for Nrps in cultured fibroblasts (Hillman, 2011).

Primary cilia are required for Hh transduction, based on two combined lines of evidence. Mutations in components of cilia interfere with Hh transduction, and several pathway components are found located in cilia, some of them dynamically in response to ligand or to drugs that affect Hh transduction. Mutations that alter cilia lead to altered Hh transduction, so Nrp inhibition could affect cilia and, thus, Hh signals. Therefore cilia structure were monitored after Nrp inhibition and no change was seen in frequency or size of cilia. As a more precise measure of cilia function, the trafficking of Smo and Gli2, two proteins whose concentration in cilia is a reflection of Hh ligand received, were examined. Both were unchanged following Nrp1+2 RNAi. Therefore, gross changes in cilia function or Hh pathway component localization are not responsible for the connection of Nrps to Hh transduction. As more is learned about how cilia process and transmit Hh transduction steps, additional tests of Nrp effects will be important. It is always possible that Nrps affect a subtle post-translational modification of a Hh pathway component that awaits elucidation (Hillman, 2011).

Last, the mechanism of Nrp action could be to influence the Hh pathway by altering general cell properties. Overexpression of Nrp1 in fibroblasts can result in increased cell-cell adhesion, likely involving an interaction between Nrp1 and a second, unknown cell surface protein. Subsequent work clarified the region of Nrp1 that mediates this adhesion but did not identify the putative interacting partner. This study found that Nrp1 protein in NIH3T3 fibroblasts is present on the cell surface and exists predominantly outside the primary cilium. Nrp-mediated cell-cell adhesion could contribute to a cytoskeletal scaffold important for an as-yet-uncharacterized step in Hh signal transduction (Hillman, 2011).

Kif7 promotes hedgehog signaling in growth plate chondrocytes by restricting the inhibitory function of Sufu

Proper regulation of Indian hedgehog (Ihh) signaling is vital for chondrocyte proliferation and differentiation in the growth plate. Its dysregulation causes skeletal dysplasia, osteoarthritis or cartilaginous neoplasia. This study shows that Suppressor of fused (Sufu) and Kif7 are essential regulators of Ihh signaling. While Sufu acts as a negative regulator of Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in the turnover of Sufu and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dose of Sufu restores normal hedgehog pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory activity of Sufu. Furthermore, Kif7 also inhibits Gli transcriptional activity in the chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and -independent mechanisms (Hsu, 2011).

Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. A systems-level genetic dissection of adiposity regulation was performed using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo; hedgehog signaling was the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, fat-specific hedgehog-activation mutant mice were generated. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals (Pospisilik, 2010).

To assess the in vivo relevance of hedgehog signaling in mammalian adipogenesis, fat-specific Sufu knockout animals (aP2-SufuKO) were generated. Sufu is a potent endogenous inhibitor of hedgehog signaling in mammals. Sufuflox/flox mice were crossed to the adipose tissue deleting aP2-Cre transgenic line, and the resulting aP2- SufuKO animals were born healthy and at Mendelian ratios. PCR amplification revealed target deletion in both white adipose tissue (WAT) and brown adipose tissue (BAT). aP2-SufuKO mice displayed an immediate and obvious lean phenotype. MRI analysis revealed a significant and global reduction in white adipose tissue mass, including subcutaneous, perigonadal, and mesenteric depots. Intriguingly, though, in contrast to the gross loss of WAT, cross-sectional examination of the interscapular region revealed fully developed BAT depots of both normal size and lipid content. Direct measurement of WAT and BAT depot weights corroborated the divergent WAT/BAT phenotype, with an ~85% reduction in perigonadal fat pad mass in aP2-SufuKO mice concomitant with unaltered BAT mass. Tissue weight and histological analyses confirmed lack of any remarkable phenotype in multiple other tissues including pancreas and liver (no indication of steatosis), and muscle mass was unaffected. Cutaneous adipose was also markedly diminished. Whereas the morphology of Sufu-deficient BAT depots was largely indistinguishable from that of control animals, examination of multiple WAT pads revealed marked and significant reductions in both adipocyte size and total numbers in mutant animals. Of note, qPCR showed elevated Gli1, Gli2, and Ptch2 expression in both WAT and BAT verifying the intended pathway activation in both tissues. Thus, deletion of Sufu in fat tissue results in a markedly decreased white fat cell number and, remarkably, in normal brown adipose tissue (Pospisilik, 2010).

When the literature was cross-referenced focusing on adipogenesis, an impressive 18 of 65 key regulators of adipogenesis were found to be described as Gli targets in other systems. Intriguingly, when examined in 3T3-L1 preadipocytes, hedgehog activation induced a coordinated downregulation of the proadipogenic targets Bmp2, Bmp4, Egr2/Krox20, Sfrp1, and Sfrp2 by an average of ~50% after only 24 hr. In contrast, quantification of the antiadipogenic target set showed upregulation of the multiple critical repressors (Nr2f2, Gilz, Hes1, and Ncor2); the negative regulators Jag1 and Pref1 remained unchanged at this time point. Analysis of the master regulatory machinery downstream of these effectors revealed critical reductions in Pparg, Cebpb, and Cebpd and increases in the antiadipogenic factors Cebpg and Ddit3. Outside of this dramatic antiadipogenic profile, elevated levels of Cebpa were observed. Importantly, a similar coordinate downregulation of Pparg, Cebpb, Cebpd, as well as Cebpa was observed in WAT-derived primary murine adipocyte progenitors (stromal vascular cell, SVC, preparations) following genetic activation of hedgehog signaling (Pospisilik, 2010).

To establish a direct link between hedgehog activation and adipogenic block in white adipose, in silico predictions were used to identify clusters of probable Gli-binding sites in the highly SAG-responsive genes Ncor2, Nr2f2, Sfrp2, and Hes1. To assess the functionality of these putative binding sites, the relevant promoter fragments were cloned and luciferase reporter assays were performed. Gli1 and Gli2 induced activation of all Ncor2 and Nr2f2 reporter constructs, with the binding site clusters Ncor2_B, Nr2f2_A, and Nr2f2_B showing responses comparable to the hallmark target Ptch. Further, chromatin immunoprecipitations on 3T3-L1 preadipocytes using Gli2- and Gli3-specifc antibodies revealed increases in Gli2 and Gli3 binding within the endogenous Ncor2, Hes1, Nr2f2, and Sfrp2 regulatory regions following SAG treatment. Together, these findings demonstrate endogenous Gli2/Gli3 binding to multiple adipogenic loci and implicate direct modulation of Ncor2 and Nr2f2 in the dysregulation of adipogenesis (Pospisilik, 2010).

The power of D. melanogaster RNAi transgenics to probe gene function on a genome-wide scale has allowed screening of ~78% coverage of the Drosophila genome. One significant advantage of this inducible approach is the ability to interrogate the fat regulatory potential of the ~30% of the Drosophila genome that is developmentally lethal under classic mutation conditions. Indeed, the result that cell differentiation genes scored as the most enriched ontology subcategory substantiates the inducible strategy employed and identifies a large number of developmentally lethal genes with strong lipid storage regulatory potential. Consistent with a previous feeding-induced RNAi C. elegans screen, the fraction of candidate genes resulting in decreased fat content upon knockdown (360 of 516; 70%) exceeded that of obesity-causing candidates (216 of 516; 30%), which is consistent with the hypothesis that the major evolutionary pressures for animals have been to favor nutrient storage. The screen identified a large number of genes already known to play a key role in mammalian fat or lipid metabolism, including enzymes of membrane lipid biosynthesis, fatty acid and glucose metabolism, and sterol metabolism. Further, the whole-genome screen has uncovered a plethora of additional candidate genes of adiposity regulation, a large proportion of which had no previous annotated biological function. Moreover, multiple genes were identified that either positively or negatively regulate whole fly triglyceride levels when targeted specifically in neurons, the fly liver (oenocyte), the fat body, or muscle cells. Analyses of the hits allowed definition of either gene sets that function globally in all these tissues or others that display coordinate regulation of adiposity when targeted in metabolically linked organs such as the fat and the liver. Since >60% of the candidate genes are conserved across phyla to humans, this data set is a unique starting point for the elucidation of novel regulatory modalities in mammals (Pospisilik, 2010).

The top-scoring signal transduction pathway in the GO-based enrichment analysis was the hedgehog pathway. Tissue-specificity assessment revealed further that this enrichment was primarily derived from a pronounced fat-body restriction in function. Hedgehog signaling has been previously implicated in adipose tissue biology. In Drosophila larvae, hedgehog activation reduces lipid content consistent with what was found in adult flies and the fat-specific fly knockdown lines (Suh, 2006). Similarly, knockdown of the C. elegans equivalent of the inhibitory hedgehog receptor Ptch results in a prominent adiposity reducing phenotype in a feeding-based RNAi screen. Therefore this study homed into the hedgehog pathway to provide proof of principle for the fly screen and to translate Drosophila results directly into the mammalian context (Pospisilik, 2010).

Several reports exist describing systemic manipulation of hedgehog signaling, either by injection of ligand-depleting antibody or through examination of a systemic hypomorphic mutant, the Ptchmes/mes mouse. Indeed Ptchmes/mes mice display largely normal white adipose tissue depots albeit reduced in size (Li, 2008). Hedgehog signaling plays a crucial role in multiple organs systems including at least one intimately involved in nutrient storage and the etiologies of obesity and insulin resistance, namely, the pancreatic islet. In vitro and in vivo data using the adipose-specific Sufu mutant mice clearly show that hedgehog activation results in a complete and cell-autonomous inhibition of white adipocyte differentiation. The residual white adipose tissue observed in aP2-SufuKO mice is most likely due to late inefficient deletion and/or is due to developmental timing effects. Indeed, aP2 (and thus aP2-Cre) are expressed relatively late during adipocyte differentiation. The remarkable finding was that genetic activation of hedgehog signaling in vivo and in vitro blocks only white but not brown adipocyte differentiation (Pospisilik, 2010).

Fat is mainly stored in two cell types: WAT, which is the major storage site for triglycerides, and BAT, which, through the burning of lipids to heat (through uncoupling of mitochondrial oxidative phosphorylation), serves to regulate body temperature. Recent PET-CT data have revealed that adult humans contain functional BAT and that the amount of BAT is inversely correlated with body mass index. These new data in humans rekindle the notion that a functional BAT depot in humans could represent a potent therapeutic target in the context of obesity control. Lineage tracking and genetic studies have shown that WAT and interscapular BAT cells derive from two different but related progenitor pools. The current genetic data now demonstrate both in vitro and in vivo that hedgehog activation results in a virtually complete block of WAT development but leaves the differentiation process of brown adipocytes wholly intact. These data further support the concept that white and brown adipocytes are derived from distinct precursor cells (Pospisilik, 2010).

aP2-SufuKO mice are the first white adipose-specific lipoatrophic mice with a fully functional BAT depot over the long-term and normal glucose tolerance and insulin sensitivity. The capacity of an intact BAT depot to burn energy in aP2-SufuKO mice likely underlies, at least in part, their lack of ectopic lipid accumulation and insulin resistance. This largely normal metabolic picture highlights the potent regulatory capacity of brown adipose tissue and should prove invaluable in understanding the distinct roles of brown and white adipose tissues (Pospisilik, 2010).

Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci

Hedgehog (Hh) signalling regulates embryonic development and adult tissue homoeostasis. Mutations of its pathway components including Suppressor of Fused (Sufu) and Gli/Ci predispose to cancers and congenital anomalies. The Sufu-Gli protein complex occupies a central position in the vertebrate Hh signalling pathway, especially in mammals. Structures of full-length human and Drosophila Sufu, the human Sufu-Gli complex, along with normal mode analysis and FRET measurement results, reveal that Sufu alternates between 'open' and 'closed' conformations. The 'closed' form of Sufu is stabilized by Gli binding and inhibited by Hh treatment, whereas the 'open' state of Sufu is promoted by Gli-dissociation and Hh signalling. Mutations of critical interface residues disrupt the Sufu-Gli complex and prevent Sufu from repressing Gli-mediated transcription, tethering Gli in the cytoplasm and protecting Gli from the 26S proteasome-mediated degradation. This study thus provides mechanistic insight into the mutual recognition and regulation between Sufu and Gli/Ci (Zhang, 2013).

Sufu is a crucial regulator of the Gli/Ci family of transcription factors in both vertebrates and invertebrates, and the Sufu-Gli protein complex forms the core of the vertebrate Hh signalling pathway downstream of Smoothened. Crystal structures of FL hSufu, FL dSufu and the hSufuΔ60-hGli1 (112-128) complex, as well as the normal mode analysis, reveal that the Sufu protein possesses an intrinsic conformational flexibility, with the arrangement of its NTD and CTD domains alternating between 'open' and 'closed' states. Binding to Gli stabilizes Sufu in the 'closed' state, with the β-strand of Gli acting as a 'glue' to bring strands β5 of Sufu-NTD and β9 of Sufu-CTD together. In response to Hh, the conformation of Sufu relaxes to the 'open' state and it is dissociated from Gli. Therefore, the 'closed' state of Sufu is favored when it is associated with Gli and when the Hh signal is absent, and the 'open' conformation of Sufu prevails when it dissociates from Gli and is promoted by Hh signalling (Zhang, 2013).

The Sufu-binding fragment of Gli/Ci, the 'SYGHLS' motif, is completely surrounded by conserved residues from Sufu. It was reported that two of the major HIB-binding sites on Ci are at residues 216-227 and 368-376, which are nearby its 'SYGHIS' motif, residues 255-260. In the case of Gli3, it is only known that its N-terminal region (residues 242-477 of mouse Gli3) contains a Spop-binding site (Spop/HIB is a negative regulator of the HH pathway), but its exact location has not been pinpointed. It is likely that the complex formation between Sufu and Gli/Ci creates a steric hindrance for Spop/HIB, thus prohibiting the Spop/HIB-mediated ubiquitination and degradation of Gli/Ci. Moreover, Spop/HIB has been reported to form a dimer and make multivalent interactions with Gli/Ci, therefore the complex formation between Sufu and Gli/Ci would indeed leave barely enough space for a Spop/HIB dimer to occupy two adjacent binding sites on Gli/Ci at the same time. It would be worthwhile to investigate further the underlying mechanism(s) of how Sufu inhibits Gli/Ci degradation, which could be Spop/HIB-dependent or -independent (Zhang, 2013).

Previous investigations on the molecular mechanism of interaction between Sufu and Gli/Ci have obtained somewhat contradictory results. Part of the reason might be that Sufu uses both its NTD and CTD to clamp Gli/Ci in the middle, hence any attempt using the deletion mapping approach might be hampered by the pitfall of damaging the structural integrity of FL Sufu and thus yield misleading results. Studies conducted by mutation-based approaches also need to be carefully evaluated so as not to be over-interpreted. For example, the highly conserved 'HGRHFTYK' motif (residues 391-398 in hSufu) is reported to be required for stable interaction with Gli/Ci, and mutation of this motif disrupted the binding and inhibition of Gli by Sufu. However, structure of the hSufu-hGli1 complex shows that these residues are not in direct contact with Gli. This fragment forms strand β13 of hSufu-CTD and juxtaposes strand β9, which engages hGli1 by β-sheet interaction. Mutation of the 'HGRHFTYK' motif to alanines would potentially affect the structural integrity of β13, which would in turn obstruct the formation of β9 and even the entire Sufu-CTD. Without the participation of Sufu-CTD, especially that of β9, a stable complex between Sufu and the 'SYGHLS' motif of Gli would not be achieved (Zhang, 2013).

ITC and Ni2+ column pull-down results suggested that the NTD or CTD of Sufu alone was not sufficient for stable complex formation with the 'SYGHLS' motif of Gli/Ci). However, it was also reported that Sufu-NTD and -CTD had reduced but detectable interactions with FL Gli1 and Sufu-NTD was sufficient for the inhibition of Gli1- and Gli2-dependent transcriptional activation. Gli/Ci proteins contain a second Sufu-binding site in their C-terminal halves, but its precise location has not been pinpointed. Determination of the exact position of this C-terminal Sufu-binding site on Gli/Ci and elucidation of its role in the regulation of Gli/Ci by Sufu deserve further attention (Zhang, 2013).

In vertebrates, especially in mammals, primary cilium has an important role in Hh signalling. Many key Hh signalling components such as Patched and Smoothened are localized to primary cilia and their ciliary localizations are regulated by Hh. Sufu translocates to cilia coordinately with Gli and its ciliary localization depends on Gli. The regulation of Gli by Sufu is independent of cilia, but the Hh/Smo-induced release of Sufu from Gli requires cilia. It is conceivable that in response to Hh, some kind of cilia-specific post-translational modification event such as phosphorylation happens on Gli or Sufu, resulting in the dissociation of Gli from Sufu in cilia and the subsequent translocation of Gli to the nucleus. Intriguingly, there are many potential phosphorylation sites within and surrounding the Sufu-binding 'SYGHLS' motif in Gli/Ci. It might be warranted to investigate whether any Hh-induced and cilia-dependent phosphorylation occurs on Gli or Sufu and whether there exists any cilia-localized kinase specifically mediating these phosphorylation events (Zhang, 2013).

Mutations of the human Sufu gene predispose individuals to cancers. The tumour suppressor function of Sufu is considered mainly as working through restraining the activities of the Gli transcription factors, whose mutations are also correlated with tumours and congenital malformations. Understanding the complex interplay between Sufu and Gli at the structural level would deepen understanding of the molecular mechanism of how pathogenic mutations of Sufu and Gli work. For example, a frameshift mutation IVS8+1G->A was found in both medulloblastoma and Gorlin syndrome patients. This mutation resulted in a truncated Sufu-Δex8 protein (residues 1-322, instead of WT hSufu residues 1-484), in which the majority of the CTD domain of hSufu was removed. This result is fully consistent with the finding that Sufu-CTD is indispensible in the recognition and regulation of Gli proteins. The Gli transcription factor represents a potential therapeutic target, and understanding of the Sufu-Gli protein complex might also inspire the development of pharmaceutical approaches to rein in aberrant activities of Gli in cancers (Zhang, 2013).

Suppressor of fused: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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