CA2252965A1 - Novel suppressor of fused (sufu) gene - Google Patents

Abstract

A novel mammalian polynucleotide and the suppressor of fused (Sufu) protein it encodes are described. The polynucleotide is useful for the screening and isolation of other mammalian Sufu and Sufu-related polynucleotides, while the Sufu protein itself is useful in the study of many physiological conditions including tumorigenesis.

Description

NOVEL SUPPRESSOR OF FUSED (Sufis) GENE
Field of the Invention The present invention relates to a novel mammalian suppressor of fused (Sufis) gene and the regulatory protein it encodes.
Background of the Invention Hedgehog (Hh) signaling is involved in many developmental processes and disease conditions, including cancer. Current knowledge of Hh signaling has been derived from genetic studies in Drosophila (Ingham, EMBO J (1998)17:3505-3511; Ming et al., Molecular Medicine Today (1998) 4:343-349) in which two transmembrane proteins, Patched (Ptc) and Smoothened (Smo), have been identified as opposing partners in the Hh receptor (Chen &
Struhl, Cell (1996) 87:553-563). The current model suggests that Ptc functions as a negative regulator of Hh signaling by inhibiting the activity of Smo. When Hh binds to Ptc, repression of Smo is released.
Derepressed Smo then signals through a Gli zinc forger protein, Cubitus interruptus (Ci), to activate the transcription of target genes. Ci is believed to form a mufti-protein complex including a serine-threonine protein kinase, Fused (Fu) (Therond et al., Genetics (1996) 142:
1181-1198); a kinesin-like molecule, Costal 2 (Cos2)(Sisson et al., Cell (1997) 90:235-245); and a novel PEST-domain containing protein, Suppressor of fused (Su(fu))(Pham et al., Genetics (1995) 140:587-598; Monnier et al., Current Biology (1998) 8:583-586). While the role of Su(fu) is still unclear, Fu and Cos2 function, respectively, as positive and negative regulators of Hh signaling.
In Drosophila, Su(fu) was identified using a genetic screen for suppressors of fu phenotypes (Preat, Genetics (1992) 132:725-736). It encodes a novel PEST-domain containing protein and is expressed both maternally and zygotically (Pham et a1.,1995).
Although loss-of function Su(fu) mutations are viable and display no visible phenotype, they can fully suppress all the known phenotypes associated with loss of Fu function, including the segmentation defects, wing defects, ovarian tumors and pupal lethality (Preat, 1992). Su(fu) antagonizes Fu in a dose-dependent manner (Preat, 1992). Loss of one dose of Su(fu) can partially suppress the Fu phenotype and removal of both copies of Su(fu) fully rescues the Fu phenotype.
Conversely, the Fu phenotype is enhanced by a gene duplication of Su(fu). While Su(fu) mutations can suppress the phenotype of null alleles (Fu0) and Fu[I] mutants (mutations in the kinase domain), double mutants of Su(fu) and Fu[II] (mutations in the regulatory domain) display a novel phenotype which is similar to Cos2 mutants (Preat et al., Genetics (1993) 135:1047-1062;
Therond et al., 1996). It has also been shown that both Fu[II] and Su(fu) mutations can enhance the phenotype of Cos2 mutants (Preat et al., 1993). Taken together, these observations suggest that there is a tight relationship between Su(fu), Fu and Cos2 throughout Drosophila development and in Hh signaling.
It has recently been demonstrated that Su(fu) can bind directly to the regulatory domain of Fu and the N-terminal region of Ci (Monnier et al., 1998). In yeasts, Fu cannot bind Ci but Su(fu) can link Fu to Ci. Immunoprecipitation experiments further show that Su(fu) and Ci interact in Drosophila embryos. These biochemical data indicate that Su(fu) is part of the multi-protein complex involved in the transduction of Hh signal and can serve as a linker protein bringing Fu and Ci together. It has been suggested that Fu activation triggers the degradation of Su(fu) through the phosphorylation of its PEST sequence (Monnier et al., 1998). Therefore, the level of Su(fu) might affect the phosphorylation of Ci by Fu. The proteolysis of Ci is known to be inhibited in Cos2-deficient Drosophila embryos (Sisson et al., 1997). Since Su(fu) behaves like Cos2 in the absence of Fu (Preat et al., 1993), it may also play an indirect role in the control of Ci proteolysis. These observations suggest that homologs of Su(fu) as well as Fu and Cos2 are potentially important regulatory components in mammalian Hh signaling.
Mammalian Hh signaling is more complex and involves at least two Ptc genes (Motoyama et al., Nature Genetics (1998) 18:104-106) and three Gli genes (Hui et al., Developmental Biology (1994) 162:402-413). Through mutational analysis in mice, the role of mammalian Gli proteins has been investigated. These analyses indicated that Gli2 functions as a major mediator of Shh (Sonic hedgehog) signaling while Gli3 appears to function as a repressor of Shh signaling (Hui & Joyner, Nature Genetics (1993) 3:241-246; Ding et al., Development (1998) 125:2533-2543). Furthermore, double mutant analysis demonstrates that Gli2 and Gli3 possess overlapping developmental functions (Mo et al., Development (1997) 124:113-123;
Hardcastle et al., 1998; Motoyama et al., Nature Genetics (1998) 20:54-57). In contrast, Glil has been shown to be dispensable with regard to developmental functions (unpublished observations). A neuroepithelial cell line MNS-70 (Nakagawa et al., Development (1996) 122:2449-2464) has also been used to investigate the roles of the three Gli transcription factors.
Using this cell line, both Gli 1 and Gli2 activate Gli binding site-dependent transcription, whereas Gli3 acts as a transcriptional repressor (Sasaki et al., 1997). Consistent with genetic analysis, Gli2 is seen to be primarily responsible for the transduction of Shh signal in this cell line.
The human Gli 1 gene was initially identified as an amplified oncogene in glioblastoma and several mesenchymal tumors (Kinzler et al., Nature (1988) 332:371-374;
Ruppert et al., Mol.
Cell Biol (1991) 11:1724-1728). Recently, it was also shown to be overexpressed in basal cell carcinomas (BCCs) and medulloblastomas (MBs) (Dahmane et al., Nature (1997) 389:876-881;
Reifenberger et al., Cancer Research (1998) 58:1798-1803). Interestingly, although forced Glil expression in early frog embryos lead to skin tumors (Dahmane et al., 1997), BCCs could not be induced in transgenic mice with Gli 1 overexpression in the skin (Andrzej Dlugosz, personal communication). In transgenic mice, Glil overexpression itself is apparently not sufficient for BCC formation. While the roles of Gli2 and Gli3 in tumorigenesis remain unclear, it has recently been observed that Gli2+~- and Gli2+/-/Gli3+~- mutants exhibit a higher incidence of spontaneous tumors, including BCCs.
Through genetic manipulation, forced Shh signaling has been shown to lead to BCC
formation. Furthermore, a putative activating mutation in Shh has also been identified in one BCC, one MB and one breast carcinoma although its role in tumorigenesis remains unclear (Oro et al., Science (1997) 276;817-821). It is worth noting that BCCs represent the most common cancer in humans with more than 750,000 new cases diagnosed annually and MBs (malignant primitive neuroectodermal tumors of the cerebellum) occur predominantly in childhood with an incidence of about five per million children. About one-third of sporadic BCCs and 10-15% of sporadic MBs are now estimated to have mutations in Ptc (Hahn et al., 1996;
Johnson et al., Science (1996) 272:1668-1671; Reifenberger et al., 1998). Recently, several activating Smo mutations have also been found in both BCCs and MBs (Reifenberger et al., 1998; Xie et al.
Nature (1998) 391:90-92). Together, these observations indicate that Shh signaling is an oncogenic pathway in the developing skin and cerebellum, and that Ptc and Smo are important targets for genetic alterations in BCCs and MBs.
Aberrant Hh signaling is likely involved in other tumors as Ptc mutations have been found in a variety of tumors (Ming et al., 1988). In addition to MBs, Ptc+~-mutant mice also exhibit a high incidence of spontaneous tumors such as rhabdomyosarcomas (Goodrich et al., 1997; Hahn et al., 1998). In Drosophila, the diffusion of Hh signal is controlled by Tout-velu (Bellaiche et al., Nature (1998) 394:85-88). Tout-velu was recently found to be a homolog of the multiple exotoses syndrome gene, EXT 1. This latter observation further indicates that aberrant Hh signaling is involved in other types of tumors and suggests that mutations in other components of Hh signaling, such as Fu, Cos2 and Su(fu) homologs, might cause BCCs, MBs as well as other tumors.
It is clear that Fu, Cos2 and Su(fu) are significant factors in the regulation of Hh signaling and that Hh signaling is involved in many developmental processes and disease conditions, particularly cancer. It would be desirable, thus, to isolate mammalian homologs of these factors in order to better understand their influence in mammalian systems, and to provide tools useful for screening compounds having potential as therapeutic agents.
Summary of the Invention Accordingly, in one aspect of the present invention, an isolated polynucleotide, consisting of either DNA or RNA, that encodes a mammalian suppressor of fused protein is provided, as well as the novel suppressor of fused protein itself.
In other aspects of the present invention, there are provided cells that have been genetically engineered to produce mammalian suppressor of fused protein and methods for producing suppressor of fused protein from such cells. In related aspects of the present invention, recombinant DNA constructs are provided as well as antibodies to suppressor of fused.
Other aspects of the present invention will become apparent from the following detailed description, and from the accompanying drawings in which:
Brief Reference to the Drawings Figure 1 is the nucleotide sequence of a Sufu-encoding polynucleotide in accordance with the present invention;
Figure 2 is the amino acid sequence of a Sufu protein encoded by the polynucleotide of Fig. l;
Figure 3A is a Northern blot analysis of mouse embryonic and adult RNA
indicating localization of Sufu;

Figure 3B illustrates the results of RNA in situ hybridization to determine Sufu localization;
Figure 4 illustrates the results of a co-immunoprecipitation assay to determine the interaction between Sufu and Gli proteins;
Figure 5 is a bar graph indicating the effect of Sufu overexpression on Gli-dependent transactivation;
Figure 6A identifies amino- and carboxyl-terminal Gli2 mutants and the results of a co-immunoprecipitation assay to determine Sufu/Gli2 mutant binding;
Figure 6B depicts a yeast hybrid assay scheme to confirm the Sufu/Gli2 mutant binding set out in Fig. 6A;
Figure 7 is a Northern blot analysis of Hh signaling components in C3H10T1/2 cells;
Figure 8 is a bar graph illustrating the results of Gli-dependent transactivation of BGlixB-BS luciferase transcription in C3H10T1/2 cells;
Figure 9 is a bar graph illustrating the results of Shh-dependent transactivation of BGlix8-BS luciferase transcription in C3H10T1/2 cells; and Figure 10 illustrates the subcellular localization of Sufu, Glil and Gli2 in COS cells.
Detailed Description of the Invention A polynucleotide encoding mammalian suppressor of fused (Sufu) has been isolated.
Such a polynucleotide is useful for the screening and isolation of other Sufu and Sufu-related mammalian genes, including human Sufu genes, the proteins encoded by which are implicated in the suppression of tumor growth. Sufu is a regulatory protein, in particular, a negative regulator of hedgehog (Hh) signalling and is characterized by its ability to bind Gli transcription factors as determined in assays of conventional design, such as the assays herein described.
As used herein, the term "Sufu" is meant to refer to the protein herein referred to as "suppressor of fused", and specifically to mammalian suppressor of fused.
"Mammalian" as it is used with respect to Sufu is meant to encompass Sufu of any mammal, including human.
The sequence of a particular isolated mammalian Sufu polynucleotide is set out in SEQ
ID NO: 1 (Fig.l). The polynucleotide encodes mammalian Sufu consisting of 483 amino acid residues in its mature form, as identified by three-letter code in SEQ ID NO:
2, and single-letter code in Fig.2. The protein contains several PEST regions, regions which are rich in proline, glutamine, serine and threonine residues.
In order to make mammalian Sufu, techniques of genetic engineering may be applied to prepare a mammalian cell line that produces mammalian Sufu in functional form as a heterologous product. The construction of such cell lines is achieved by introducing into a selected host cell a recombinant DNA construct in which DNA coding for mammalian Sufu is associated with expression controlling elements that are functional in the selected host to drive expression of Sufu-encoding DNA, thus elaborating the desired Sufu protein.
The particular cell type selected to serve as host for production of Sufu can be any of several cell types currently available in the art, including both prokaryotic and eukaryotic cell types.
Chinese hamster ovary (CHO) cells for example of K1 lineage (ATCC CCL 61) including the Pros variant (ATCC CRL
1281); the fibroblast-like cells derived from SV40-transformed African Green monkey kidney of the CV-1 lineage (ATCC CCL 70), of the COS-1 lineage (ATCC CRL 1650) and of the COS-7 lineage (ATCC CRL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658), murine C127 cells, human embryonic kidney cells of the 293 lineage (ATCC CRL 1573), human carcinoma cells including those of the HeLa lineage (ATCC CCL 2), and neuroblastoma cells of the lines IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11 ) all represent examples of suitable cell types for the production of mammalian Sufu.
A variety of gene expression systems have been adapted for use with these hosts and are now commercially available. Any one of these systems can be selected to drive expression of the Sufu-encoding DNA. These systems, available typically in the form of plasmidic vectors, incorporate expression cassettes the functional components of which include DNA constituting expression controlling sequences, which are host-recognized and enable expression of Sufu-encoding DNA when linked 5' thereof. Such Sufu-encoding DNA is referred to herein as being incorporated "expressibly" into the system, and incorporated "expressibly" in a cell once successful expression from a cell is achieved. These systems further incorporate DNA sequences which terminate expression when linked 3' of the receptor-encoding region.
Thus, for expression in the selected mammalian cell host, there is generated a recombinant DNA
expression construct in which the Sufu-encoding DNA is linked with expression controlling DNA
sequences recognized by the host, and which include a region 5' of the Sufu-encoding DNA
to drive expression, and a 3' region to terminate expression.
Included among the various recombinant DNA expression systems that can be used to achieve mammalian cell expression of the Sufu-encoding DNA are those that exploit promoters of viruses that infect mammalian cells, such as the promoter from cytomegalovirus (CMV), the Rous sarcoma virus (RSV), simian virus (SV40), murine mammary tumor virus (MMTV) and others. Also useful to drive expression are promoters such as the LTR of retroviruses, insect cell promoters, including those isolated from Drosophila which are regulated by temperature, as well as mammalian gene promoters such as those regulated by heavy metals, i.e. the metallothionein gene promoter, and other steroid-inducible promoters.
The plasmidic vector harbouring the expression construct typically incorporates a marker to enable selection of stably transformed recombinant cells. The marker generally comprises a gene conferring some survival advantage on the transformants allowing for the selective growth of successful transformants in a chosen medium. For example, common gene markers include genes which code for resistance to specific drugs, such as tetracycline, ampicillin and neomycin.
Thus, transformants which have successfully taken up the plasmid DNA will incorporate both the gene of interest, i.e. the Sufu gene and the marker gene, e.g. gene for drug resistance such as neomycin, and will survive culturing in media containing the drug which they could otherwise not tolerate.
For incorporation into the recombinant DNA expression vector, DNA coding for Sufu can be obtained by applying selected techniques of gene isolation or gene synthesis. As described in more detail in the examples herein, Sufu can be obtained by careful application of conventional gene isolation and cloning techniques. This typically will entail extraction of total messenger RNA from a fresh source of mammalian tissue, followed by conversion of message to cDNA and formation of a cDNA library e.g. a bacteriophage cDNA library. Such bacteriophage harboured fragments of the human DNA are grown by plating on a lawn of susceptible E. coli bacteria such that individual phage plaques or colonies can be isolated. The DNA carried by the phage colony is then immobilized on a nitrocellulose or nylon-based hybridization membrane, and then hybridized, under carefully controlled conditions, to a labelled, e.g. radioactively or otherwise labelled, probe sequence to identify the particular phage colony carrying the DNA

fragment of particular interest, in this case a mammalian Sufu gene. The phage carrying the gene of interest is then isolated from contaminating phages in order that the gene may be more easily characterized. For convenience, the gene or a portion thereof is generally subcloned into a plasmidic vector at this stage.
Having herein provided the nucleotide sequence of a mammalian Sufu protein and a Sufu-encoding gene, it will be appreciated that automated techniques of gene synthesis and/or amplification can be performed to generate Sufu-encoding DNA. In this case, because of the length of the Sufu-encoding DNA, application of automated synthesis may require staged gene construction in which regions of the gene up to about 300 nucleotides in length are synthesized individually and then ligated in correct succession via designed overlaps.
Individually synthesized gene regions can then be amplified by PCR.
With appropriate template DNA in hand, the technique of PCR amplification may be used to directly generate all or part of the final gene. In this case, primers are synthesized which will prime the PCR amplification of the final product, either in one piece, or in several pieces that may subsequently be ligated together via step-wise ligation of blunt ended, amplified DNA
fragments, or preferentially via step-wise ligation of fragments containing naturally occurnng restriction endonuclease sites. Both cDNA or genomic DNA are suitable as templates for PCR
amplification. The former may be obtained from a number of sources including commercially available cDNA libraries, single- or double-stranded cDNA, or cDNA constructed from isolated messenger RNA from a suitable tissue sample. Human genomic DNA may also be used as a template for the PCR-based amplification of the gene; however, the gene sequence of such genomic DNA may contain unwanted intervening sequences.
Once obtained, the Sufu-encoding DNA is incorporated for expression into any suitable expression vector, and host cells are transfected therewith using conventional procedures, such as DNA-mediated transformation including calcium phosphate precipitation, protoplast fusion, microinjection, lipofection and electroporation. Expression vectors may be selected to provide transformed cell lines that express the Sufu-encoding DNA in a stable manner.
Suitable expression vectors will typically harbour a gene coding for a product that confers on the transformants a survival advantage to enable their subsequent selection. Genes coding for such selectable markers include the _E. coli gpt gene which confers resistance to mycophenolic acid, the neon gene from transposon Tn5 which confers resistance to neomycin and to the neomycin analog 6418, the dhfr sequence from murine cells or E. coli which changes the phenotype of DHFR- cells into DHFR+ cells, and the tk gene of herpes simplex virus, which makes TK- cells phenotypically TK+ cells. Other methods of selecting for transformants may of course be used, if desired, including selection by morphological parameters, or detection of surface antigen or receptor expression. The latter can be monitored using specifically labelled antibodies and a cell-sorter, e.g. fluorescent activated.
As one of skill in the art will appreciate, Sufu-encoding DNA may be modified prior to its incorporation into an expression vector to enhance protein expression.
Specifically, modifications may be made to the 5' and 3' non-coding regions of Sufu-encoding DNA in order to increase the level of protein expression. For example, the 5' non-coding end of Sufu-encoding DNA may be modified to provide a 5' "translation-enhancing sequence" (TES).
Such modifications include truncating the 5' end of the Sufu-encoding DNA preceding the native translation-enhancing sequence. Alternatively, the DNA is truncated and the native translation-1 S enhancing sequence is replaced with a heterologous translation-enhancing sequence using conventional methods of restriction enzyme digestion followed by ligation techniques. By "heterologous" is meant a sequence that is not native to Sufu-encoding DNA.
Further, by "translation-enhancing sequence" is meant the 5' sequence which is required for translation to occur, and includes the translation initiation codon, i.e. ATG.
The present invention also provides, in another of its aspects, antibody to mammalian Sufu. To raise such antibodies, there may be used as immunogen either full-length Sufu or an immunogenic fragment thereof, produced in a microbial or mammalian cell host as described above or by standard peptide synthesis techniques. Regions of Sufu particularly suitable for use as immunogenic fragments include regions which are determined to have a high degree of antigenicity based on a number of factors, as would be appreciated by those of skill in the art, including for example, amino acid residue content, hydrophobicity/hydrophilicity and secondary structure. Specific examples of immunogenic fragments of Sufu suitable for generating antibodies include, but are not limited to, the region spanning residues 305 to residues 325, the region spanning residues 452 to residues 468.

The raising of antibodies to mammalian Sufu or to desired immunogenic fragments can be achieved, for polyclonal antibody production, using immunization protocols of conventional design, and any of a variety of mammalian hosts, such as sheep, goats and rabbits. Alternatively, for monoclonal antibody production, immunocytes such as splenocytes can be recovered from the immunized animal and fused, using hybridoma technology, to myeloma cells.
The fusion cell products, i.e. hybridoma cells, are then screened by culturing in a selection medium, and cells producing the desired antibody are recovered for continuous growth, and antibody recovery.
Recovered antibody can then be coupled covalently to a reporter molecule, i.e.
a detectable label, such as a radiolabel, enzyme label, luminescent label or the like, using linker technology established for this purpose, to form a specific probe for Sufu.
According to a further aspect of the present invention, DNA or RNA encoding mammalian Sufu, and selected regions thereof, may also be used in detestably labeled form, e.g.
radiolabeled form, as hybridization probes to identify sequence-related genes existing in the human or other mammalian genomes (or cDNA libraries) or to locate Sufu-encoding DNA in any other specimen. This can be done using the intact coding region, due to a high level of conservation expected between related genes, or by using a highly conserved fragment thereof, having radiolabeled nucleotides, for example, 32P nucleotides, incorporated therein.
Embodiments and aspects of the present invention will now be described by reference to the following specific examples which are not to be construed as limiting.
Example 1-Cloning of mouse Su~pressor of fused (Msuful eene In order to isolate a full length clone of the mouse homolog of suppressor of fused (Msufu), a partial cDNA clone (EST clone number 513730 containing sequences homologous to Drosophila suppressor of fused gene; Research Genetics) was radio-labeled and used as a probe to screen a lambda phage cDNA library constructed from E11.5 mouse embryo mRNA
using procedures well-known in the art such as those set out in Current Protocols in Molecular Biology (Whey).
A cDNA insert encoding mouse Sufu (Msufu) was isolated from the cDNA library and cloned into the plasmid vector, pBluescriptII (Stratagene), and the nucleotide sequence, as set out in SEQ ID NO: 1, was determined by DNA sequencing.

Example 2 - Expression of the Msufu eene In order to express the isolated Msufu gene, an expression vector was prepared as follows. A SaII restriction enzyme site was generated at the N-terminal end of the Sufu coding region of the isolated gene by PCR using procedures as set out in Current Protocols in Molecular Biology. The gene was then subcloned into the eukaryotic expression vector, pCMVS(3 (gift from Dr. Jeff Wrana of the Hospital for Sick Children, Toronto, Ontario; see P.A. Hoodless et al., Cell (1996) 85:489-500), downstream of a Myc-tag driven by the cytomegalovirus promoter.
The predicted open reading frame of Msufu encodes 483 amino acid residues. At the amino acid level, the overall sequence identity between the mouse and Drosophila proteins is 36%. Northern blot analysis of embryonic and adult mouse RNA revealed a 4.5 kb Msufu transcript (Fig. 3A). During mouse embryogenesis, Msufu is expressed from E8.5 to term.
Msufu transcripts are found in many adult tissues, including heart, brain and liver. RNA in situ hybridization revealed that Msufu transcripts are ubiquitous from E8.5 to E10.5 (Fig. 3B) and its expression becomes more restricted during organogenesis (data not shown).
Example 3 - Interactions between Msufu and Gli proteins To study potential interactions between Msufu and Gli proteins, eukaryotic expression vectors containing FLAG-, Myc- and HA-tagged versions of Msufu were generated by PCR, using the procedure described above in Example 2, for use in co-immunoprecipitation experiments. FLAG-tagged Gli 1 and GIi2 were generated by subcloning the protein coding region of Gli 1 and Gli2 downstream of a FLAG-tag in the eukaryotic expression vector, pCMVS(3 (gift of Hiroshi Sasaki, Osaka University, Japan; unpublished).
The co-immunoprecipation experiment generally used is as described in Current Protocols in Molecular Biology. FLAG-tagged GIi2 and Myc-tagged Sufu were co-transfected into HeLa and COS cells by lipofectamine treatment. Forty eight hours post-transfection, lysate was prepared from the cells. GIi2 or Sufu was precipitated from the cell lysate using FLAG- or Myc-antibodies (Sigma) respectively, and the immunoprecipitates were subjected to SDS-PAGE
analysis. The interactions between Gli2 and Sufu were then determined by Western blot analysis of FLAG-immunoprecipitate using Myc-antibodies and Myc-immunoprecipitate using FLAG-antibodies.
Similar results were obtained in both cells. As shown in Fig.4, the results of these experiments revealed that Msufu forms a protein complex with Gli2 upon transfection into COS
cells.
Similar co-immunoprecipitation experiments were conducted to determine if Msufu also interacted with Gli 1 and Gli3. The results of these experiments confirmed the formation of Msufu-Gli 1 and Msufu-Gli3 protein complexes.
Example 4 - Modulatorv Effect of Msufu on transcriptional activities of Gli proteins To determine the functional significance of the Msufu-Gli interactions, the transcriptional activities of the Gli proteins were assayed with and without Msufu.
Specifically, Glil and Gli2 are known to activate transcription in HeLa cells from a luciferase reporter gene (Gli-luc) via the Gli binding sites while Gli3 represses this transcription as previously described (Sasaki et al., 1997, Development 124, 1313-1322). Using this assay, the effect of Msufu overexpression on Gli-dependent transactivation was determined.
The assay was conducted as described (Sasaki, 1997). As shown in Fig. 5, although Msufu overexpression alone has no effect on the transcription of the luciferase reporter gene, it does affect Gli-dependent transactivation. In particular, Msufu overexpression dramatically decreases the transactivation by Gli2. In contrast, Msufu has very little or no effect on the transactivation by Glil . Since Gli2 is the primary target of Shh signaling, the ability of Msufu to differentially regulate the transcriptional activities of Glil and Gli2 suggests the regulatory role that Msufu plays with respect to Shh signaling. Moreover, Msufu overexpression converts the activity of Gli3 from repressor to activator. Although the physiological significance of this latter observation is unclear, it is important to note that Gli3 acts as a repressor of Shh signaling. In additional control experiments, Msufu overexpression was found to have an insignificant effect on the transactivation of a GAL4 reporter by a GAL4-VP 16 activator (data not shown) indicating the specificity of Msufu overexpression on Gli-dependent transactivation.
Example 5 - Interaction of Msufu with amino- and carboxyl-termini of Gli2 To determine the regions of Gli 2 involved in Msufu-Gli2 interactions, a series of amino-terminal and carboxyl-terminal deletion mutants of Gli2 (identified in Fig.
6A) were prepared and co-transfected with Msufu into COS cells. Msufu-Gli2 interaction was then determined by co-immunoprecipitation as described above in detail in Example 3. As set out in Fig. 6A, the results of the assay reveal that the N-terminal region (between amino acid residues 1 and 389) of Gli2 can form a stable complex with Msufu. Interestingly, Msufu can also interact with a carboxyl-terminal region (between residues 1184 and 1544) of Gli2.
To further examine these interactions, these truncated amino- and carboxyl-terminal regions of Gli2 were cloned in frame with the GAL4 activation domain in pGAD424 (Clontech) to generate, respectively, plasmids Gli2N-TA and Gli2C-TA. These were each assayed for protein-protein interactions in yeast with an Msufu-GAL4 DNA-binding domain fusion protein cloned in pAS2-1 (Clontech) identified as Msufu-DB. As shown in Fig. 6B, both regions can interact with Msufu in yeasts, indicating that Msufu forms a complex with Gli2 through two distinct regions.
Example 6 -Inhibition of Shh signaling in C3H10T1/2 cells by Msufu To examine the role of Msufu in Shh signaling, transcriptional assays in the pluripotent mouse mesenchymal cell lines, C3H10T1/2 (ATCC - CCL-226), were developed as previously described in Example 4. Since C3H10T1/2 cells are known to respond to Shh (Nakamura et al., 1997, Biochemical and Biophysical Research Communications 237, 465-469) and express high levels of Gli2 and Msufu as well as Ptch and Smo transcripts (see Fig.7), they can serve as a useful in vitro system for studying the molecular mechanism of Shh signaling, and, in particular, the interactions between Msufu and Gli2.
As shown in Fig.8, the activity of Gli-luc in C3H10T1/2 cells was found to be 3- to 4-fold more active than a control luciferase reporter lacking the Gli binding site suggesting that Gli2 can act as an activator of the Gli-luc reporter in the absence of exogenous Shh signal. In this cell line, Gli2 activity seems to be rate-limiting because Gli2 overexpression can further augment the activity of Gli-luc. Similar to the results observed in HeLa cells, the activity of Gli-luc, but not the control luciferase reporter, was activated by Glil overexpression and repressed by Gli3 overexpression. When C3H10T1/2 cells were treated with Shh, the transcription of Gli-luc, but not the control reporter, is enhanced (Fig.9). Furthermore, preliminary experiments demonstrate that this Shh-dependent enhancement is inhibited by cotransfection with Msufu (not shown). Together, these results indicate that Msufu overexpression can inhibit Shh signaling in C3H10T1/2 cells.
Example 7 - Subcellular localization of Msufu. Glil, Gli2 and Gli3 To study the subcellular distribution of Msufu and the three Gli proteins, FLAG-tagged cDNA expression vectors were introduced into COS and HeLa cells by lipofection and analyzed by immunofluorescence using a monoclonal anti-FLAG antibody.
Similar staining results were observed in both cell lines. The results obtained in COS
cells are illustrated in Fig.lO. Msufu was found to be uniformly distributed in the cytoplasm of transfected cells. Both Gli2 and Gli3 (data not shown) are distributed primarily in the cytoplasm of transfected cells although 10-20% of the transfected cells also show nuclear staining. In contrast, Gli 1 was found to be nuclear in the transfected cells. Preliminary experiments indicate a similar subcellular distribution of these proteins in C3H10T1/2 cells.
Example 8 - Generation of Sufu-specific Antibodies Full length Sufu as well as various subfragments of the N- and C- terminal regions of mouse Sufu were subcloned into a His-tagged bacterial expression vector (such as pQE30, pQE31 and pQE32 available from Qiagen). Purified His-tagged Sufu was injected into rabbits to raise polyclonal antibodies by using standard procedures such as those described in Harlow &
Lane (Antibodies: A laboratory manual. Cold Spring Harbor Laboratory, 1988).
The following Sufu-specific peptides were synthesized:
1) the region spanning residues 305 to residues 325; and 2) the region spanning residues 452 to residues 468 and used for raising antibodies. The specificity of the antibodies was determined using Western blot analysis of C3H10T1/2 cell extracts and mouse embryo extracts. The size of epitope-tagged Sufu has been determined to be about 53 kDa by Western blot analysis (see Fig.4). Specificity was also confirmed by the ability of the antibodies to immunoprecipitate Sufu as set out in Example 3. The immunoprecipitate was analyzed by Western blot analysis using a pan-Gli antibody (gift from Dr. David Markowitz, University of Michigan) which recognizes all three mouse Gli proteins. Interaction of endogenous Sufu and Gli2 proteins in C3H10T1/2 cells is determined by a 200 kDa band on the Western blot, while interactions between Sufu and Gli 1 in mouse embryos is determined by a 140 kDa band, Gli2 interaction is determined by a 200 kDa band and Gli3 interaction is determined by a 210-220 kDa band.
Example 9 - Mutagenesis of Sufu Mutagenesis of Sufu is conducted to determine the domains required for binding and repressing Gli2. Sequence analysis of Drosophila and mouse Sufu proteins have revealed three highly conserved regions. Within these regions, there are eight serine/threonine residues that can serve as potential phosphorylation sites. Based on this information, a series of amino-terminal, carboxyl-terminal and internal deletion mutants of Sufu were generated by PCR
as follows:
Mutant Gli2 Interaction Residues 1-105 -Residues 1-211 ++
Residues 1-308 +++
Residues 1-408 +
Residues 99-211 -Residues 99-308 +++
Residues 99-408 -Residues 99-482 +
Residues 244-482 ++
The mutants were cloned into an epitope-tagged expression vector, Myc-tagged mammalian expression vector, and the interactions of these mutant Sufu proteins with Gli2 were assayed. The interaction between each mutant and Gli2 is set out above.
Two different methods were used to determine the mutant Sufu/Gli2 interaction.
First, mutant Sufu was co-transfected with Gli2 into COS cells and their interactions were analyzed by co-immunoprecipitation assays as set out in Example 3. Second, radio-labeled mutant Sufu was generated by in vitro transcription and translation, and examined for the ability to bind GST

(glutathione-S-transferase) columns containing GST fusion proteins with either amino- or carboxyl-terminal regions of Gli2 in a GST-pull down assay.
It is possible for the results of these two assays to be different. For example, the GST-pull down assay could reveal a Gli2 binding region in Sufu that is missed in the co-immunoprecipitation assay because a mutant form of Sufu might form a complex with Gli2 through indirect interaction with a third protein in transfected cells.
Therefore, both regions of Sufu required so that (1) the formation of a stable complex Gli2 (co-immunoprecipitation assay) and (2) direct interactions with Gli2 (GST-pull down assay) can be determined.
To fine map the Gli2 binding region(s), additional mutants (internal deletions or smaller subfragments of Sufu) are constructed and examined as set out above.
Various mutants are also transfected into C3H10T1/2 cells and assayed for their abilities to repress Gli2 to determine whether stable complex formation and/or direct Gli2 binding are required for the repression of Gli2 transactivation.
The above-described assays can also be conducted to determine the effect of Sufu mutants in modulating Gli 1 and Gli3 transcriptional activities and to determine differential abilities in modulating Gli proteins.

SEQUENCE LISTING
(1) GENERAL
INFORMATION:

S

(i) APPLICANT:

(A) NAME: Chi-Chung Hui (B) STREET: 1023 St. Clarens Avenue (C) CITY: TORONTO

IO (D) STATE: ONTARIO

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(F) POSTAL CODE (ZIP): M6H 3X8 (A) NAME: Qi Ding IS (B) STREET: 1117 McIntyre Drive (C) CITY: Ann Arbor (D) STATE: Michigan (E) COUNTRY: USA

(F) POSTAL CODE (ZIP): 48105 (ii) TITLE OF INVENTION: Novel Mammalian Suppressor of Fused Gene (iii) NUMBER OF SEQUENCES: 2 ZS (iv) COMPUTER READABLE FORM:

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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION
FOR
SEQ
ID NO:
1:

(i) SEQUENCE CHARACTERISTICS:

3S (A) LENGTH: 1663 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear 4O (ii) MOLECULE TYPE: cDNA

4S(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 1:

SO

GTACTGGTTG

$

lO CCACCAACATGGCCAGCAGAGCTGATGCAGGGCCTAGCCCGATATGTCTTCCAGTCAGAG 600 1$

2$

3$

4$

(2) INFORMATION
FOR SEQ
ID NO:
2:

SO (i) SEQUENCE :
CHARACTERISTICS

(A) LENGTH:483 aminocids a (B) TYPE:
amino acid (C) STRANDEDNESS: e singl (D) TOPOLOGY:
linear $$

(ii) MOLECULE TYPE: protein (xi) SEQUENCE
DESCRIPTION:
SEQ
ID
NO:
2:

Met AlaGlu LeuArgPro SerValAla ProGlyPro AlaAlaPro Arg Ser GlyPro SerAlaPro ProAlaPhe AlaSerLeu PheProPro Gly Leu HisAla IleTyrGly GluCysArg ArgLeuTyr ProAspGln Pro 1$ 35 40 45 Asn ProLeu GlnValThr AlaIleVal LysTyrTrp LeuGlyGly Pro Asp ProLeu AspTyrVal SerMetTyr ArgAsnMet GlyCysPro Ser Ala AsnIle ProGluHis TrpHisTyr IleSerPhe GlyLeuSer Asp Leu TyrGly AspAsnArg ValHisGlu PheThrGly ThrAspGly Pro Ser GlyPhe GlyPheGlu LeuThrPhe ArgLeuLys ArgGluThr Gly Glu SerAla ProProThr TrpProAla GluLeuMet GlnGlyLeu Ala 3$ Arg TyrVal PheGlnSer GluAsnThr PheCysSer GlyAspHis Val Ser TrpHis SerProLeu AspAsnSer GluSerArg IleGlnHis Met Leu LeuThr GluAspPro GlnMetGln ProValArg ThrProPhe Gly Val ValThr PheLeuGln IleValGly ValCysThr GluGluLeu His Ser AlaGln GlnTrpAsn GlyGlnGly IleGlnGlu LeuLeuArg Thr $0 Val Pro Ile Ala Gly Gly Pro Trp Leu Ile Thr Asp Met Arg Arg Gly Glu Thr Ile Phe Glu Ile Asp Pro His Leu Gln Gln Glu Arg Val Asp Lys GlyIle GluThrAsp GlySerAsn LeuSerGly ValSerAla Lys Cys AlaTrp AspAspLeu SerArgLeu ArgArgMet LysArgIle Ala $ 275 280 285 Gly AlaSer AlaArgHis ThrProArg ArgLeuSer GlyLysAsp Thr Glu GlnIle ArgGluThr LeuArgArg GlyLeuGlu IleAsnSer Lys Pro ValLeu ProProIle AsnSerGln ArgGlnAsn GlyLeuThr His Asp ArgAla ProSerArg LysAspSer LeuGlySer AspSerSer Thr Ala IleIle ProHisGlu LeuIleArg ThrArgGln LeuGluSer Val His LeuLys PheAsnGln GluSerGly AlaLeuIle ProLeuCys Leu Arg GlyArg LeuLeuHis GlyArgHis PheThrTyr LysSerIle Thr Gly AspMet AlaIleThr PheValSer ThrGlyVal GluGlyAla Phe Ala ThrGlu GluHisPro TyrAlaAla HisGlyPro TrpLeuGln Ile Leu LeuThr GluGluPhe ValGluLys MetLeuGlu AspLeuGlu Asp Leu ThrSer ProGluGlu PheLysLeu ProLysGlu TyrSerTrp Pro Glu LysLys LeuLysVal SerIleLeu ProAspVal ValPheAsp Ser Pro Leu His SEQUENCE LISTING
(1) GENERAL INFORMATION:
S
(i) APPLICANT:
(A) NAME: Chi-Chung Hui (B) STREET: 1023 St. Clarens Avenue (C) CITY: TORONTO
IO (D) STATE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE (ZIP): M6H 3X8 (A) NAME: Qi Ding IS (B) STREET: 1117 McIntyre Drive (C) CITY: Ann Arbor (D) STATE: Michigan (E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 48105 (ii) TITLE OF INVENTION: Novel Mammalian Suppressor of Fused Gene (iii) NUMBER OF SEQUENCES: 2 2S (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
3S (A) LENGTH: 1663 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear 4O (ii) MOLECULE TYPE: cDNA
4S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CTATGTTAGC

CTACATCAGC

AACAGACGGA

GGAGTCTGCC

CCAGTCAGAG

CAGTGAGTCA

1$ GACACCCTTT

GGGGTAGTGA CTTTCCTCCA GATTGTTGGT GTCTGCACTG AGGAGTTACA 7g0 TTCAGCCCAA

TGGCGGTCCC

GCACCTGCAA

CGTCAGTGCC

GGAGACCCTG

TCAGCGACAG

CGACAGCTCC

GCATCTAAAA

GTCCACGGGA

CTGGTTACAA

TCTAACCTCT

(2) INFORMATION FOR SEQ ID N0: 2:
SO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
2:

Met Ala Glu Leu Arg Pro Ser Val Ala Pro Gly Pro Ala Ala Pro Arg Ser Gly Pro Ser Ala Pro Pro Ala Phe Ala Pro Pro Ser Leu Phe Gly Leu His Ala Ile Tyr Gly Glu Cys Arg Arg ProAsp Gln 1$ Leu Tyr Pro Asn Pro Leu Gln Val Thr Ala Ile Val Lys Trp LeuGly Gly Tyr Pro 20 Asp Pro Leu Asp Tyr Val Ser Met Tyr Arg Met GlyCys Pro Asn Ser Ala Asn Ile Pro Glu His Trp His Tyr Ile Phe GlyLeu Ser Ser Asp Leu Tyr Gly Asp Asn Arg Val His Glu Phe Gly ThrAsp Gly Thr Pro Ser Gly Phe Gly Phe Glu Leu Thr Phe Arg Lys ArgGlu Thr Leu Gly Glu Ser Ala Pro Pro Thr Trp Pro Ala Glu Met GlnGly Leu Leu Ala 3$ Arg Tyr Val Phe Gln Ser Glu Asn Thr Phe Ser Gly Cys Asp His Val Ser Trp His Ser Pro Leu Asp Asn Ser Glu Ser Arg Ile Gln His Met 40 16s 170 17s Leu Leu Thr Glu Asp Pro Gln Met Gln Pro Val Arg Thr Pro Phe Gly Val Val Thr Phe Leu Gln Ile Val Gly Val Cys Thr Glu Glu Leu His 4$ 195 200 205 Ser Ala Gln Gln Trp Asn Gly Gln Gly Ile Gln Glu Leu Leu Arg Thr $0 Val Pro Ile Ala Gly Gly Pro Trp Leu Ile Thr Asp Met Arg Arg Gly Glu Thr Ile Phe Glu Ile Asp Pro His Leu Gln Gln Glu Arg Val Asp $$ 245 250 255

Claims (12)

1. An isolated polynucleotide comprising a nucleotide sequence encoding mammalian suppressor of fused which exhibits binding to at least one Gli transcription factor.

2. A polynucleotide as defined in claim 1, wherein the nucleotide sequence encodes the amino acid sequence set out in SEQ ID NO: 2.

3. A polynucleotide as defined in claim 1, wherein the nucleotide sequence corresponds to that of SEQ ID NO: 1.

4. A polynucleotide as defined in claim 1, wherein the suppressor of fused exhibits binding to a Gli transcription factor selected from the group consisting of Gli1, Gli2 and Gli3.

5. A recombinant DNA construct having incorporated therein a polynucleotide as defined in claim 1.

6. A cell that has been engineered genetically to produce a mammalian suppressor of fused, said cell having incorporated expressibly therein a heterologous polynucleotide as defined in claim 1.

7. A cell as defined in claim 6, which is a mammalian cell.

8. A process for obtaining a substantially homogeneous source of a mammalian suppressor of fused, comprising the steps of culturing cells having incorporated expressibly therein a polynucleotide as defined in claim 1, and then recovering said cultured cells.

9. A mammalian suppressor of fused, in a form essentially free from other proteins of mammalian origin.

10. A mammalian suppressor of fused as defined in claim 9, encoded by a polynucleotide having the nucleotide sequence set out in SEQ ID NO: 1.

11. A mammalian suppressor of fused as defined in claim 8, having the amino acid sequence set out in SEQ ID NO: 2.

12. An antibody which binds a mammalian suppressor of fused.