Wnt genes of vertebrates and invertebrates encode a large family of secreted, cystein rich proteins that play key roles as intercellular signaling molecules in a wide variety of biological processes (for an extensive review see Wodarz and Nusse 1998). The first Wnt gene, mouse wnt-1, was discovered as a proto-oncogene activated by integration of mouse mammary tumor virus in mammary tumors (Nusse and Varmus 1982). Consequently, the involvement of the Wnt/Wg pathway in cancer has been an area of intensive study. With the identification of the Drosophila polarity gene wingless as a wnt-1 homologue (Cabrera, Alonso et al. 1987; Perrimon and Mahowald 1987; Rijsewijk, Schuermann et al. 1987), it became clear that Wnt genes are important developmental regulators. Thus, although at first glance dissimilar, biological processes, like embryogenesis and carcinogenesis, both rely on cell communication via identical signaling pathways.

In a current model of the pathway, the secreted Wnt protein binds to Frizzled (Fz) cell surface receptors and thereby activates the cytoplasmic protein Dishevelled (Dsh). Dsh then transmits the signal to a complex of several proteins, including the protein kinase GSK3-β/Shaggy (Sgg), the scaffold proteins Axin and Adenomatous Polyposis CoIi (APC), as well as β- catenin, the vertebrate homologue of Armadillo (Arm). In this complex, β-catenin is normally targeted for degradation after being phosphorylated by GSK3-β. In response to Wnt signaling and the resulting down-regulation of GSK3-β activity, β-catenin escapes from degradation and accumulates in the cytoplasm. Free cytoplasmic β-catenin can translocate to the nucleus, where it modulates gene transcription by binding the TCF/LEF family of transcription factors (Grosschedl 1999).

This setup, in which the key transducer is continuously held in check, is highly susceptible to mutations in its inhibitory components. The loss of any of the three elements of the β-catenin destruction complex, or mutations in β-catenin which render it insusceptible to degradation, lead to an increase in β-catenin levels and hence to the constitutive activation of the pathway. This can lead to cell fate changes, uncontrolled proliferation and tumorigenic behaviour, as occurring with loss of APC or Axin function (Barker 1999; Morin 1999; Potter 1999; Roose and Clevers 1999; Waltzer and Bienz 1999). There are several possibilities to counter the negative effects of uncontrolled Wnt signaling for therapeutic intervention, however, most potent would be interference with the transcriptional activator function of β-catenin.

- 3 -

Currently, there are no known therapeutic agents effectively inhibiting β-catenin transcriptional activation. Consequently, there is a dire need to improve our understanding of the Wnt pathway in order to better develop drugs against diseases where Wnt signaling is implicated.

In a study of Wnt/Wg signaling, the inventors identified the novel Drosophila gene hyrax (hyx) as an auxilliary nuclear Wg component. Overexpression and loss of function in vitro and in vivo influenced various Wnt/Wg signaling readouts downstream of the cytoplasmic β- catenin/Arm degradation complex, hyx encodes a highly conserved nuclear protein which is related to yeast Cdc73p, a factor of the Paf 1 complex that is involved in RNA Polymerase II (RNAPII)-associated histone modifications during transcription. Additionally, the product of the human tumor suppressor HRPT2, Parafibromin, is the direct functional ortholog of Drosophila Hyx. The N-terminus of Parafibromin/Hyx interacts directly with the C-terminal Armadillo (Arm) repeats and the proximal C-terminus of β-catenin/Arm in vitro and in vivo. Together, this data suggests the involvement of Parafibromin/Hyx in Wnt/Wg signaling via a nuclear interaction with β-catenin/Arm to transmit the Wnt/Wg signal.

- 4 -

SUMMARY OF THE INVENTION

The inventors have presented evidence that human Parafϊbromin and Drosophila Hyx are a novel nuclear element of the Wnt/Wg signaling cascade. In particular, they have demonstrated that Parafibromin is a novel β-catenin interacting protein, an interaction that is conserved between their respective orthologs Hyx and Arm in Drosophila. In addition, they have also identified minimal regions within β-catenin and Parafϊbromin that are required for their binding.

The invention relates to a method for screening for a substance that inhibits or enhances the binding between Parafibromin and β-catenin comprising the steps of:

a) bringing a candidate substance into contact with a (polypeptide with the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14 under conditions that permit binding of said substance to said (poly)peptide; or b) bringing a candidate substance into contact with a (polypeptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. No 12 extending from amino acid residue 637 to 781 or from 637 to 722, or with the homologue (polypeptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. No 14 under conditions that permit binding of said substance to said

(polypeptide fragment; or c) bringing a candidate substance into contact with a (polypeptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. No 7 extending from amino acid residue 1 to 343, from 200 to 343, from 200 to 263, from 200 to 250, from 218 to 343, from 218 to 263 or from 218 to 250, or with the homologue (poly)peptide fragment of the (polypeptide with the amino acid sequence of SEQ ID. Nos 8, 9, 10 or 11 under conditions that permit binding of said substance to said (polypeptide fragment; or d) bringing a candidate substance into contact with a derivate of the (polypeptide with the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or of the (polypeptide fragment according to b) or c) under conditions that permit binding of said substance to said derivate; or θ) bringing a candidate substance into contact with a (polypeptide which is at least 50 % homologous to the (polypeptide with the amino acid sequence of

- 5 -

SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or with a (ρoly)peptide fragment which is at least 50 % homologous to the (poly)peptide fragment as stated under b) or c) under conditions that permit binding of said substance to said (polypeptide or (poly)peptide fragment; and f) detecting if the candidate substance is having inhibitory activity or enhancing activity on the binding between Parafibromin and β-catenin or their respective orthologs.

This means that if the candidate substance binds to a (poly)peptide with the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or to a (polypeptide fragment as stated under b) or c), or to a derivate, (polypeptide or (poly)peptide fragment as stated under d) or e), respectively, it is detected if the candidate substance is having inhibitory activity or enhancing activity on the binding between Parafibromin and β-catenin or their respective orthologs.

A derivate as stated under d) is a (polypeptide with the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or a (poly)peptide fragment according to b) or c) with an arbitrary molecule attached to the N- or C-terminal part or to a side chain of an amino acid, and a candidate substance showing an affinity to said derivate which is at the most 50% increased or decreased compared to the affinity between the candidate substance and the corresponding unmodified (poly)peptide or (polypeptide fragment, respectively.

"Percent (%) homologous" with respect to the mentioned (polypeptide sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequences with SEQ ID. Nos 7, 8, 9, 10, 11, 12 und 14, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and not considering any conservative amino acid substitution as part of the sequence identity. The % identity values used herein can be generated by WU-BLAST-2, which was obtained from (Tatusova TA 1999). WU-BLAST-2 uses several search parameters, most of which are set to the default values.

In a similar manner, "percent (%) homologous" with respect to nucleic acid sequences with the SEQ ID. Nos 1, 2, 3, 4, 5, 6 and 13 is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the nucleic acid

- 6 -

sequences of Parafibromin, β-catenin or their respective orthologs. The identity values used herein can be generated using BLAST module of WU-BLAST-2 set to the default parameters.

In a preferred embodiment of the invention the (poly)peptide or the (polypeptide fragment stated under e) is at least 52 % homologous to the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or to the (poly)peptide fragment as stated under b) or c), preferably at least 55 % homologous, more preferably at least 60 % homologous, even more preferably at least 65 % homologous, yet even more preferably at least 70 % homologous.

In another preferred embodiment of the invention the (poly)peptide or the (poly)peptide fragment stated under e) is at least 75 % homologous to the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or to the (polypeptide fragment as stated under b) or c), preferably at least 80 % homologous, more preferably at least 85 % homologous, even more preferably at least 86 % homologous, yet even more preferably at least 87 % homologous.

In another preferred embodiment of the invention the (polypeptide or the (polypeptide fragment stated under e) is at least 88 % homologous to the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or to the (poly)peptide fragment as stated under b) or c), preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 % homologous.

In another preferred embodiment of the invention the (polypeptide or the (polypeptide fragment stated under e) is at least 93 % homologous to the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or to the (polypeptide fragment as stated under b) or c), preferably at least 94 % homologous, more preferably at least 95 % homologous, even more preferably at least 96 % homologous, yet even more preferably at least 97 % homologous.

In further preferred embodiment of the invention the (polypeptide or the (poly)peptide fragment stated under e) is at least 98 % homologous to the amino acid sequence of SEQ ID. Nos 7, 8, 9, 10, 11, 12 or 14, or to the (poly)peptide fragment as stated under b) or c), preferably at least 99 % homologous.

Further, the invention relates to an antibody which specifically binds, in part at least, to a (polypeptide domain of the (polypeptide with the amino acid sequence of SEQ ID. No 12

- 7 -

extending from amino acid residue 637 to 781 or from 637 to 722, or to the homologue (polypeptide domain of the (polypeptide with the amino acid sequence of SEQ ID. No 14.

Additionally, another aspect of the invention provides for an antibody which specifically binds, in part at least or fully, to a (poly)peptide domain of the (polypeptide with the amino acid sequence of SEQ ID. No 7 extending from amino acid residue 200 to 343, from 200 to 263, from 200 to 250, from 218 to 343, from 218 to 263 or from 218 to 250, or to the homologue (poly)peptide domain of the (polypeptide with the amino acid sequence of SEQ ID. Nos 8, 9, 10 or 11.

Further, the invention relates to a method for screening for a substance that inhibits or enhances the binding between Parafibromin and β-catenin comprising the steps of:

a) bringing a candidate substance into contact with a nucleic acid molecule with the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13 under conditions that permit binding of said substance to said nucleic acid molecule; or b) bringing a candidate substance into contact with a third molecule containing the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13 under conditions that permit binding of said substance to said nucleic acid molecule; or c) bringing a candidate substance into contact with a fourth molecule being a derivate of the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13 under conditions that permit binding of said substance to said nucleic acid molecule; or d) bringing a candidate substance into contact with a nucleic acid molecule which is at least 50 % homologue to the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13 under conditions that permit binding of said substance to said nucleic acid molecule; or e) bringing a candidate substance into contact with a nucleic acid molecule which contains a fragment of the nucleic acid sequence of SEQ ID. No 1, 2, 3, 4, 5, 6 or 13 under conditions that permit binding of said substance to said nucleic acid molecule, wherein the fragment comprises at least 240 nucleotides; and

- 8 -

f) detecting if the candidate substance is having inhibitory activity or enhancing activity on the binding between Parafibromin and β-catenin or their respective orthologs.

A derivate as stated under c) is a nucleic acid molecule with a nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13 with an arbitrary molecule attached to the said nucleic acid molecule, said derivate having the characteristic that a candidate substance showing an affinity to said derivate which is at the most 50% increased or decreased compared to the affinity between the candidate substance and the corresponding unmodified nucleic acid molecule.

In one embodiment of the invention, the nucleic acid molecule stated under d) is at least 52 % homologue to the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13, preferably at least 55 % homologous, more preferably at least 60 % homologous, even more preferably at least 65 % homologous, yet even more preferably at least 70 % homologous.

In another preferred embodiment, the nucleic acid molecule stated under d) is at least 75 % homologue to the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13, preferably at least 80 % homologous, more preferably at least 85 % homologous, even more preferably at least 86 % homologous, yet even more preferably at least 87 % homologous.

In a further preferred embodiment, the nucleic acid molecule stated under d) is at least 88 % homologue to the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5 6 or 13, preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 % homologous.

In a further preferred embodiment, the nucleic acid molecule stated under d) is at least 93 % homologue to the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13, preferably at least 94 % homologous, more preferably at least 95 % homologous, even more preferably at least 96 % homologous, yet even more preferably at least 97 % homologous.

In a further preferred embodiment, the nucleic acid molecule stated under d) is at least 98 % homologue to the nucleic acid sequence of SEQ ID. Nos 1, 2, 3, 4, 5, 6 or 13, preferably at least 99 % homologous.

- 9 -

In another aspect, the invention relates to a pharmaceutical composition for the treatment of cancer, bone or joint disorders or developmental disorders comprising a (poly)peptide fragment of the (polypeptide with the amino acid sequence of SEQ ID. No 12 extending from amino acid residue 637 to 781 or from 637 to 722 or the homologue (polypeptide fragment of the (polypeptide with the amino acid sequence of SEQ ID. No 14, or a functionally equivalent derivate thereof.

In a further aspect, the invention relates to a pharmaceutical composition for the treatment of cancer, bone or joint disorders or developmental disorders comprising a (poly)peptide fragment of the (polypeptide with the amino acid sequence of SEQ ID. No 7 extending from amino acid residue 1 to 343, from 200 to 343, from 200 to 263, from 200 to 250, from 218 to 343, from 218 to 263 or from 218 to 250, or the homologue (polypeptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. Nos 8, 9, 10 or 11, or a functionally equivalent derivate thereof.

A functionally equivalent derivate is a pharmaceutically acceptable salt of said (poly)peptide fragments or a said (poly)peptide fragment with an arbitrary molecule attached to the N- or C- terminal part or to a side chain of an amino acid, said derivate showing an affinity to Parafibromin or β-catenin, respectively, which is at the most 50% increased or decreased compared to the affinity between the unmodified (polypeptide fragment and Parafibromin or β-catenin, respectively.

Additionally, the invention relates to a pharmaceutical composition for the treatment of cancer, bone or joint disorders or developmental disorders comprising one of the mentioned antibodies.

Preferred cancer types are Wnt-dependent cancer types, preferably colorectal cancer, lung cancer, nasopharyngeal carcinoma, preferably Wnt-2 dependent nasopharyngeal carcinoma, small intestinal adenocarcinoma, fundic gland polyps (gastric), gastric carcinoma, gastric (intestinal-like), gastric adenoma (without associated adenocarcinoma), gastrointestinal carcinoid tumor, esophageal adenocarcinoma, juvenile nasopharyngeal angiofibromas, melanoma, pilamatricomas, lung adenocarcinomas, ovarian carcinoma, uterine cervix, uterine endometrial, breast fibromatoses, prostate, thyroid carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma associated with hepatitis C, medulloblastoma, desmoid

- 10 -

tumor, Wilm's tumor (kidney), pancreatic (non-ductal acinar cell carcinomas), pancreatoblastoma and synovial sarcoma.

Prefered types of bone or joint disorders are osteoarthritis or rheumatoid arthritis, respectively.

The invention relates further to an assay for studying diseases induced by a enhanced or reduced binding between Parafibromin and β-catenin or their orthologs or for drug screening comprising the use of a cell line or organism selected from the group comprising Drosophila, mice, rats, rabbits, chicken, frogs, pigs, fishes, worms or sheep, said organism showing enhanced or reduced binding between Parafibromin and β-catenin or their respective orthologs in at least one tissue or organ and said cell line showing enhanced or reduced binding between Parafibromin and β-catenin or their respective orthologs.

- 11 -

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 13 A: Hyx is an evolutionary conserved Cdc73 homology protein with homology and functional relationship to the human tumour suppressor protein Parafibromin. This figure depicts a dendrogram showing the evolutionary relationship of the

Parafibromin/Hyx homologs,

Fig. 13B: Hyx is an evolutionary conserved Cdc73 homology protein with homology and functional relationship to the human tumour suppressor protein Parafibromin. This figure depicts an alignment of Parafibromin, Hyx, and their homologues from D. rerio (NP_956642), C elegans (F35F11.1) and S. cerevisiae

(Cdc73p).

Fig. 14: hyx RNAi decreases Wg signaling downstream of axin/dAPC2 in S2 cells. This figure depicts the effects of RNAi against hyx on Wg reporter activation in Drosophila S2 cells.

Fig. 15: Somatic hyx loss of function clones show cell-autonomous decrease in Distal- less (DIl) expression and other developmental defects, but not general transcription impairment. This figure depicts the effect of somatic loss of function of hyx on the expression of Wg target genes and other unrelated genes.

Fig. 16: Identification Of hyx as a novel essential gene which by overexpression counter-acts the dominant-negative effect of overexpressed lgs ^7E . This figure depicts the reversion of the overexpression phenotype of a dominant negative inhibitor of Wg signaling, lgs ^17E , by co-overexpression of hyx. This overexpression has no general effect on transgene expression. Additionally, hypomorphic hyx loss of function rescue leads to leg development impairment.

Fig. 17: Parafibromin is a nuclear protein that can substitute Hyx in vivo and influences the TOP-Flash Wnt signaling reporter in 293T cells. This figure depicts experiments demonstrating that Hyx and Parafibromin can functionally replace each other in flies and in mammalian tissue culture, as well as the effect of

- 12 -

Parafibromin overexpression and reduction on Wnt signaling in mammalian cells.

Fig. 18: Parafibromin/Hyx directly interacts independently of its Cdc73 motif in the region between Arm repeat 12 and the proximal C-terminus of beta- catenin/Arm. This figure depicts the results of interaction studies demonstrating that Parafibromin and β-catenin can interact and the experiments beginning to delineate the minimal interaction domains.

Fig. 19: The Parafibromin N-terminus, and the β-catenin interaction domain (CID) region in particular, are sufficient for β-catenin binding. Numbers indicate amino acid residues of Parafibromin used to generate GST fusion proteins. GST alone, as well as the unrelated GST-GFP and GST-dTAB2 fusion proteins served as negative controls and GST-hTCF ^' as positive control.

- 13 -

DETAILED DESCRIPTION OF THE INVENTION

The Wnt signaling cascade is essential for the development of both vertebrates and invertebrates, and has been implicated in tumorigenesis. The Drosophila Wnt genes are one of the best characterized within the Wnt-protein family, which includes more than hundred genes across all species. In the Drosophila embryo, Wg is required for formation of parasegment boundaries and for maintenance of engrailed (en) expression in adjacent cells. The epidermis of embryos defective in Wg function shows only a rudimentary segmentation, which is reflected in an abnormal cuticle pattern. While the ventral cuticle of wild type larvae displays denticle belts alternating with naked regions, the cuticle of Wg mutant larvae is completely covered with denticles. During imaginal disc development, Wg controls dorso- ventral positional information. In the leg disc, Wg patterns the future leg by the induction of ventral fate (Struhl and Basler 1993). In animals with reduced Wg activity, the ventral half of the leg develops into a mirror image of the dorsal side (Baker 1988). Accordingly, reduced Wg activity leads to the transformation of wing to notal tissue, hence the name of the gene (Sharma and Chopra 1976). In the eye disc, Wg suppresses ommatidial differentiation in favour of head cuticle development, and is involved in establishing the dorso-ventral axis across the eye field (Heberlein, Borod et al. 1998).

Additional genes have been implicated in the secretion, reception or interpretation of the Wg signal. For instance, genetic studies in Drosophila revealed the involvement of frizzled (Dfz), dishevelled (dsh), shaggy/zeste-white-3 (sgg/zw-3), armadillo (arm), adenomatous polyposis coli (E-apc), axin, and pangolin (pan) in Wg signaling. The genetic order of these transducers has been established in which Wg acts through Dsh to inhibit Sgg, thus relieving the repression of Arm by Sgg, resulting in the cytoplasmic accumulation of Arm and its translocation to the nucleus. In the nucleus Arm interacts with Pan to activate transcription of target genes. Vertebrate homologues have been identified for all these components (for an updated review see Peifer and Polakis 2000), suggesting that novel identified members of the Drosophila signaling pathway most likely have vertebrate counterparts.

Mutations leading to nuclear accumulation of the mammalian homologue of Arm, β-catenin, and consequently to constitutive activation of the Wg/Wnt pathway have been observed in many types of cancer, including colon cancer, breast cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastomas, pilomatricomas, and

- 14 -

prostate cancer (Morin 1999; Polakis, Hart et al. 1999). It is now apparent that deregulation of β-catenin signaling is an important event in the genesis of these malignancies. However, there are still no known therapeutic agents effectively inhibiting β-catenin transcriptional activation. This is partly due to the fact that many of the essential components required for its full activation and nuclear translocation are still unknown.

With different tools the inventors showed that the Drosophila gene CGl 1990 (hyrax) and its human homolog HRPT2, encoding Parafibromin, are involved in Wg/Wnt signaling. More specifically, it was shown that Hyx and Parafibromin are novel components of the Wg/Wnt pathway and interact with a central Wg/Wnt pathway component, Armadillo/β-catenin. Further, minimal regions were identified required for this interaction, which provide the basis to screen for and develop novel therapeutics which modulate this interaction.

RNA interference (RNAi) in Drosophila Schneider 2 (S2) cells, combined with a Dual- Luciferase reporter readout, is an established system to analyse the impact of gene knockdown on signal transduction pathways (Boutros et al., 2004; Caplen et al., 2000; Clemens et al., 2000; Kiger et al., 2003; Lum et al., 2003). The inventors developed an S2-cell based, Wg-sensitive Luciferase reporter system based on the LEF-luc reporter system described previously (Schweizer and Varmus, 2003) to monitor Wg pathway activation. Targeting known positive Wg pathway components dsh, arm, lgs and pygo by RNAi strongly decreased the LEF-luc readout (Fig. 14 A and data not shown). Reciprocally, RNAi against the negative regulators daxin and APC2, but not the absent APC, increased the pathway readout, even without Wg-mediated induction (Fig. 14 A). RNAi against Hyx in this system, using dsRNA against different regions of the Hyx mRNA, severely decreased the LEF-luc readout, without significant impairment of the constitutive Renilla Luciferase levels (Fig. 14 A and data not shown). Moreover, independent Dual Luciferase reporter systems for heavy metal stress induction and brinker (brk) enhancer control (Mϋller et al., 2003) were not significantly affected by hyx RNAi (Fig. 14 B). Thus, the hyx RNAi results indicate that Wg reporter gene activity in S2 cells is crucially dependent on sufficient hyx transcript, providing evidence that hyx can be a positive component in the Wg/Wnt signaling cascade.

To epistatically map hyx in the Wg/Wnt pathway, combinatorial RNAi (Kuznicki et al., 2000; Lum et al., 2003; Schmid et al., 2002) was employed in the S2-cell reporter systems mentioned above, by targeting two genes simultaneously . When the positive downstream

- 1 -

component lgs was targeted together with AP C 2 or daxin in the S2-cell Wg reporter system, a reduction of the Luciferase readout levels was observed comparable to lgs RNAi alone, but not when using GFP dsRNA instead (Fig. 14 C). Co-administration of hyx RNAi decreased the elevated Luciferase levels caused by APC2 or daxin dsRNA as efficiently as lgs RNAi (Fig. 14 C). This suggests that hyx is, like lgs, epistatic to the cytoplasmic Arm degradation complex, indicating that Hyx acts as a positive, presumably nuclear Wg signaling component. The results of in vivo loss of function and overexpression experiments (presented below) in Drosophila are consistent with this model.

To gain more insight into hyx function, the inventors generated mutant alleles using a standard EMS mutagenesis screen approach, using reversion of the salE>lgs phenotype by EP -hyx (P 9) as the basis for an EMS mutagenesis screen (Fig. 16 A-F, see below). Two potential alleles were identified which upon sequencing of the hyx coding region revealed premature stop codons (R505X in hyx ¹ , and Q461X in hyx ² , Fig. 16 G). Subsequently the inventors identified the EP-insertion EY6898 (Bellen et al, 2004), which maps to the hyx 5' UTR as a third, recessive lethal allele which was designated hyx (Fig. 16 G).

One copy of act5c-Gal4 rescued the homozygous lethality of hyx ^EY to pharate adult stage with high efficiency (data not shown). However, these pharates are rarely eclosed from their pupal cases. Examination of fully eclosed animals and pharate pupae revealed severe leg defects, mostly in the second and third leg pairs. Although all segments were present, segments showed shortening, twists and potential polarization (Fig. 16 K and L). This phenotype is reminiscent of hypomorphic phenotypes of the established Wg components pan and pygo (Brunner et al., 1997; Parker et al., 2002; Thompson et al., 2002). This data suggests an important function for hyx during development.

To further analyze the effect of homozygous hyx loss of function on Wg signaling in vivo, the FRT/FLP system (Xu and Rubin, 1993) was used to generate somatic clones with the hyx alleles in Drosophila imaginal discs to monitor target gene expression by immunofluorescence. By monitoring clones mutant for hyx, it was found that the expression levels of the long-range Wg target Distal-less (DIl) (Neumann and Cohen, 1997; Zecca et al., 1996), were severely reduced or lost in a cell-autonomous fashion (Fig. 15 A-C). This downregulation of DIl was caused by an apparently direct effect on Wg signaling rather than impairing general transcription and viability, since expression of the Hedgehog (Hh) target

- 16 -

genes decapentaplegic (dpp) and patched (ptc), the Dpp target optomotor-blind (omb), and the selector gene engrailed (en) was not impaired in hyx mutant clones, as monitored by corresponding lacZ reporter expression (Fig. 15 D-I and data not shown). Taken together, these results show specific involvement of hyx in controlling the Wg target gene DU.

To further analyze hyx loss of function effects, the inventors wanted to monitor hyx mutant clones in adult structures. However, since such clones disappear from imaginal discs before reaching the adult stage, the inventors utilized the FRT/FLP system together with the Minute (Min) technique to generate mutant clones observable in adult structures. In this setup, adults were recovered showing a wide variety of phenotypes. Common abnormalities the inventors observed phenotypes associated with impaired Wg signaling: leg defects of the same type as found in the act5c-Gal4, hyx ^EY rescue, as well as notches around the wing blade margin (Fig. 15 J and K). Additionally, large hyx mutant clones in the notum region showed loss or severe impairment of thoracic dorsocentral macrochaetae with high penetrance (data not shown), a phenotype described for Wg signaling impairment (Gerlitz and Basler, 2002; Phillips and Whittle, 1993). However, presumably Wg-independent wing blade patterning defects in Hyx mutant clones were also noted, namely the formation of ectopic vein material (Fig. 15 J). Also noted were severe impairment of eye development and outgrowths when clones were induced selectively in the head region by eyeless (ey)-flp (data not shown), yet only minor defects in antenna development when compared to mutant clones of the Wg component pygo (data not shown). Thus, it was concluded from the in vivo loss of function analysis that a) hyx influences certain, but not all, Wg signaling readouts, and that b) its loss clearly also impacts other, Wg-independent developmental processes. Therefore, hyx does not fall into the category of core nuclear Wg signaling components such as arm, lgs or pygo, but rather represents an auxiliary, more pleiotropic component, similar to CBP/p300, BRG-1/brm, TIP48/49 or tsh (Barker et al, 2001; Bauer et al., 2000; Collins and Treisman, 2000; Gallet et al., 1999; Gallet et al., 1998; Hecht et al., 2000; Takemaru and Moon, 2000; Waltzer et al., 2001).

lgs ^17E is a mutant allele of the Wg component lgs which encodes a protein product with severely impaired Armadillo interaction capability, but normal binding potential to the nuclear factor Pygo (Kramps et al., 2002). Using the Gal4/UAS-system(Brand and Perrimon, 1993), the inventors overexpressed a UAS-lgs ^17E transgene using spalt major-enhancer GaH (salE-Gal4), a strong Gal4 driver derived from regulatory elements of salm (Barrio, 2004)

- 17 -

with activity in the area between the presumptive wing veins L2 and L5 during larval development (D. Nellen and K. B., data not shown). salE>lgs ^17E lead to margin notches in the adult wing, typical for late loss of Wg signal transduction (Fig. 16 A-F). This effect is probably caused by a dominant-negative action of lgs ^17E when overexpressed, presumably by competition with endogenous lgs and thus interfering with Armadillo-mediated target gene activation.

Afterwards, various double-headed EP-element insertions recovered in a screen for genes involved in wing patterning and growth (D. Nellen and K. B., data not shown) were tested to analyze their effect on the salE>lgs ^17E phenotype. EP -insertions (P9 and PlO) which map to the hyx locus as well as direct UAS constructs driving expression of hyx showed a complete phenotype reversion (Fig. 16 D and E). This reversion was specific, since overexpression of hyx did not lead to reversions of unrelated phenotypes in other pathway-specific screening systems and did also not impair general transgene expression (Fig. 16 H-J and data not shown).

In summary, the evidence suggests that hyx encodes a normally non-limiting product which is specifically able, when overexpressed, to overcome the dominant-negative effect of excess lgs ^17E on Wg signaling.

Analysis by motif and BLAST searches of the primary amino acid sequence of the hyx protein product revealed that it is part of a conserved group of proteins represented in every annotated metazoan and plant genome, as well as genomes of lower eukaryotes (Fig. 13A). Usually, only a single putative Hyx homolog is present in each species except in Drosophila, which has a second Cdc73 homology-encoding gene, CG6220 (Fig. 13A). It is unlikely that CG6220 interferes with redundancy as it did not have a detectable effect on the systems described below (data not shown). Moreover, its expression pattern is probably biased to the testis and thus unlikely to be of relevance in somatic tissue (Parisi et al., 2004).

With 59% protein sequence identity, the human Hyx homolog is Parafibromin, encoded is by the HRPT2 (clorf28) tumor suppressor gene involved in the Hyperparathyroidism- Jaw Tumor Syndrome (HPT-JT) (Carpten et al., 2002; Howell et al., 2003; Shattuck et al., 2003) (Fig. 13B). HPT-JT features predominantly parathyroid tumors, ossifying fibromas of the jaw, and

- 18 -

renal cysts, caused by loss of heterozygosity of HRPT2 in both familial and sporadic cases (Chen et al, 2003).

Although the exact molecular nature of Parafibromin function is unknown at present, a clue as to the potential function of Hyx/Parafibromin-related proteins was initially provided by the Saccharornyces cerevisiae Hyx homolog, Cdc73p (Fig. 13B) ₅ which is associated with the nuclear Polymerase-Associated Factor 1 (Pafl) complex (Chang et al., 1999; Krogan et al., 2003; Krogan et al., 2002; Mueller and Jaehning, 2002; Porter et al., 2002; Shi et al., 1997; Squazzo et al., 2002). The Pafl complex binds to initiating and elongating RNA polymerase II (RNAPII) directly via Cdc73p and coordinates histone modifications associated with transcriptional initiation and elongation (Krogan et al., 2003; Mueller et al., 2004; Rondon et al,, 2004; Wood et al., 2003). There are potential homologs for most Pafl complex components in all species, suggesting an evolutionary conservation of this complex. Recently, a mammalian Pafl complex was isolated and shown to contain Parafibromin, further suggesting that a role in transcriptional initiation and elongation is probably conserved (Rozenblatt-Rosen et al., 2005). Parafibromin and Hyx contain a metazoan-specific, long N- terminal region which is absent in Cdc73p. However, the C-terminal portion of all these proteins is highly homologous and presumably contains an evolutionarily conserved function.

Given the strong evidence implicating hyx as component of Wg signalling, the inventors examined if this role was evolutionarily conserved in mammals. More specifically, it was examined if the human homolog HRPT2, encoding Parafibromin, also positively influences Wnt signaling in human cells. Therefore, the inventors used the Wnt-responsive TOP -Flash Luciferase assay (van de Wetering et al., 1997) in HEK 293T cells and modified HRPT2 levels by overexpression and RNAi. In the TOP/FOP-Flash system , the TOP-Flash construct harbouring tandem TCF consensus binding sites in front of a Firefly Luciferase was transfected together with a constitutively transcribed Renilla Luciferase (CMV-RL) to monitor β-catenin-mediated Wnt pathway activity. To activate the Wnt pathway, the inventors treated the transfected cells 24h before Luciferase measurement with 25mM Lithium Chloride (LiCl), which potently inhibits GSK-3β and thus blocks β-catenin degradation, consequently inducing Wnt target genes (Jho et al., 2002).

When a CMV promoter-driven HA-HRPT2 or HA-hyx construct together with the TOP-Flash reporter was transfected, a two- to threefold increase in TOP-Flash activity was observed

- 19 -

when compared to transfection of an EGPF control after LiCl stimulation (Fig. 17 K). Parafϊbromin levels in this system were quickly saturated, since introducing half the amount of HA-HRPT2 construct resulted in a similar degree of activation, while four times less increased the induction by 50% (Fig. 17 K and data not shown). HA-hyx overexpression had a similar effect on the reporter readout (Fig. 17 K). This overexpression data suggests that Parafibromin positively synergizes with Wnt signaling components downstream of GSK-3β in activation of the TOP -Flash Luciferase.

To decrease HRPT2 levels, two independent small interfering RNAs (siRNAs) against HRPT2 were used and their RNAi effect after transfection on HRPT2 transcript levels in HEK

293T cells were monitored. Both siRNAs knocked the HRPT2 mRNA levels down to approximately 20% within 48h after transfection, as determined by RT-PCR, without any obvious deleterious effects on cell viability and control gene expression (P. Brugger, P.

Zipperlen, C. M. and K. B., data not shown). Next, the effect of siRNA-mediated HRPT2 knock-down on Wnt pathway activity was analysed. When monitored by the TOP -FLASH assay, the HRPT2 siRNAs significantly reduced the TOP -Flash readout 48h after transfection

(data not shown). The decrease was more pronounced when the cells were harvested 72h after transfection (Fig. 17 L). A similar decrease was observed when a siRNA against the known positive Wnt component hPYGO-2 was used, but not when using a GFP control siRNA (Fig. 17 L). This suggests that Parafibromin is a rather stable protein or present at high levels, and that its amount is crucial for Wnt signaling transmission in HEK 293T cells downstream of

GSK-3β.

It was reasoned that if the HRPT2 siRNA effect on the TOP -Flash readout was specific, it could be restored by supplying the Drosophila ortholog hyx into the system, analogous to the in vivo experiments (see below). The hyx mRNA sequence does not align to any of the siRNAs which were used to target HRPT2 and thus should not be targeted in these assays. When co-transfected with a HRPT2 siRNA, HA-hyx restored the relative Luciferase levels in the TOP -Flash assay to a level comparable to the expression HA-hyx alone (Fig. 17 L).

The converse question of whether HRPT2 could functionally replace hyx in Drosophila, and if yes, what is the functional sub-cellular localization of Parafibromin, was also investigated. In order to address these questions, the inventors assumed with respect to their previous findings that 1) overexpression of HRPT2 should reverse the dominant-negative effect of salE>lgs ^17E ,

- 20 -

analogous to hyx overexpression; 2) more directly, ubiquitously expressed HRPT2 should be able to rescue hyx loss of function; finally, 3) functional Parafibromin should localize to the nucleus, at least in Wg signal-receiving cells.

To address the first point, transgenic lines were generated carrying a UAS-HRPT2 construct containing the full Parafibromin-encoding open reading frame. Several independent insertion lines reversed the effects of sα/ϋ'-GαW-mediated overexpression of UAS-lgs ^17E (Fig 17 A-C and data not shown). Similar to overexpression of hyx, overexpression of HRPT2 with various Gal4 drivers did not generate any obvious phenotypes, besides a slightly distorted ommatidial pattern with GMR-GaU (data not shown). Therefore, these results fully recapitulated the hyx overexpression findings.

In order to address points 2 and 3, transgenic lines were established harbouring a construct encoding N-terminally HA-tagged Parafibromin controlled by the weak and ubiquitous tubulin-la promoter (tub Ia-HA-HRPTl). Using three different, independent transgenic insertions, the inventors were able to fully rescue all mutant hyx allele combinations as well as hyx ^EY homozygosity to adulthood without obvious phenotypic defects (Fig. 17 D). Since this showed that Parafibromin can indeed functionally substitute Hyx, larval tissues for the HA epitope of the transgene was monitored by immunofluorescence to analyze its sub-cellular localization. As expected, HA-Parafibromin was found to be exclusively nuclear in all cells of all tissues examined, independent of cellular Wg pathway activity (Fig. 17 E-J). These results strongly suggest that nuclear Parafibromin can fully replace Hyx in Drosophila, and that Hyx is presumably also a nuclear protein. Supporting this, the inventors observed HA- and EGPF- tagged Hyx, like Parafibromin fusion proteins, to localize to the nuclei of HEK 293T embryonic kidney cells (data not show), in agreement with the localization prediction based on Cdc73p homology, protein sequence analysis, RNAi epistasis and data from other reports (Cronshaw et al., 2002; Rozenblatt-Rosen et al., 2005; Tan et al., 2004; Woodard et al., 2005). These findings further support the idea that Parafibromin/Hyx is a nuclear component involved in Wnt/Wg target gene activation.

In summary, these results indicate that Hyx can substitute for Parafibromin, and further suggests that these proteins are true orthologs. Moreover, the results indicate a conserved role of metazoan Cdc73 homology proteins in nuclear Wnt/Wg signaling.

- 21 -

The Cdc73p similarity and the results so far confirm that Parafibromin/Hyx is a pleiotropic factor which assists in Wnt/Wg target gene activation, potentially by acting in a nuclear, yeast Pafl complex-like entity. It was therefore reasoned that Parafibromin/Hyx could represent a linker between a Pafl complex and known nuclear Wnt/Wg signaling components. To test this assumption, the inventors used the yeast-2-hybrid technique to look for interactions between a HRPT2 bait and prey constructs containing different known nuclear Wnt signalling effectors and the unrelated Spl/egr-like zinc finger transcription factor Huckebein (Hkb). Intriguingly, a specific interaction between both Parafibromin and Hyx to β-catenin was found (Fig. 18 A). The inventors confirmed the interaction using GST-Parafibromin and GST- Hyx coupled sepharose beads, which were able to efficiently pull-down in vitro translated full-length β-catenin and N-terminally truncated Arm (ΔN-Arm), respectively, as well as β- catenin from lysates of LiCl-treated HEK 293T cells (Fig. 18 D and data not shown). In an analogous set of pull-downs, GST-ΔN-β-catenin and GST-ΔN-Arm showed interactions with in vitro translated Parafibromin and Hyx as well as HA-Parafibromin from transiently transfected HEK 293T cells (Fig. 18 E and data not shown).

To verify the interaction in vivo, co-immunoprecipitation experiments were performed. For this task, the inventors transiently expressed HA-Parafibromin and FLAG-tagged β-catenin which contained the stabilizing S33Y mutation (β-catenin ^S33Y -FLAG) in HEK 293T cells, and immunoprecipitated lysates with α-FLAG or a control antibody. α-FLAG specifically precipitated HA-Parafibromin when β-catenin ^S33Y -FLAG was present in the lysates (Fig. 18 B and data not shown). More directly, the inventors succeeded in co-precipitating endogenous β-catenin together with Parafibromin from lysates of LiCl-treated 293T cells by using the α- Parafibromin antibody BL-649 (Fig. 18 C). Additionally, α-HA pulldowns from LiCl-treated, with HA-Hyx transiently transfected S2 cells specifically co-precipitated endogenous Arm (data not shown). This data strongly suggests that Parafibromin/Hyx and β-catenin/Arm coexist in a complex in vivo.

β-catenin, and by homology Armadillo, is a modular protein featuring an N-terminal domain (NTD), followed by 12 Armadillo repeats and a C-terminal domain (CTD) (Fig. 18 D) (Cox et al., 1999; Daniels and Weis, 2002; Hecht et al., 1999; Huber et al., 1997; Orsulic and Peifer, 1996). While the central repeats 4-8 are necessary for coupling β-catenin to TCF/LEF factors in the nucleus, the surrounding structure has been shown to interact with various transcriptional modulators. To determine the β-catenin region necessary for Parafibromin

- 22 -

recruitment, the inventors performed a series of GST pull-downs using in vitro translated β- catenin fragments and analyzed their binding to GST-Parafibromin. From these assays, and taking into account the possible instability of terminal Arm repeats in peptides, the inventors conclude that the β-catenin region of repeat 12 to the proximal C-terminus (amino acids 637- 781) harbours a minimal Parafibromin binding motif with full binding capacity (Fig. 18 D). This conclusion is further supported by the observation that separating the CTD from the Arm repeats of β-catenin abolishes binding of either fragment to Parafibromin (Fig. 18 D). The inventors also found that Hyx interacts with the corresponding part of Armadillo in a similar set of experiments (data not shown), indicating that the interaction region is conserved. Intriguingly, the Parafibromin/Hyx binding area at the repeat 12 to CTD joint is embedded in the β-catenin interaction regions for the chromatin remodelling factors CBP/p300 (repeat 10- C) (Hecht et al, 2000; Takemaru and Moon, 2000) and Brg-1 (repeats 7-12) (Barker et al, 2001).

The inventors next determined the β-catenin/ Arm binding domain of Parafibromin/Hyx. Results to date indicate that the region of Parafibromin spanning amino acids 1-343 is required for the interaction (Fig 18 E and data not shown). Importantly this region lies outside the Cdc73 domain and within the extended N-terminal portion of Hyx/Parafibromin that is conserved in metazoans (Fig. 13A). The inventors postulate that the N-terminal region of Hyx/Parafibromin may contain motifs responsible for the binding to β-catenin and potentially other pathway- specific transcription units.

Subsequently, the inventors analyzed if hTCF-4 and Parafibromin can simultaneously interact with β-catenin. For these experiments, a GST-coupled hTCF-4 fragment was used comprising amino acids 1-130 (GST-hTCF-4 ^1"130 ), which includes the N-terminal β-catenin binding motif. Although GST-hTCF-4 ^1'130 strongly interacts with β-catenin, it does not bind in vitro translated Parafibromin, even if provided in great excess (data not shown). In preliminary experiments, the inventors attempted to precipitate Parafibromin indirectly via β-catenin from lysates of HEK 293T cells transfected with HA-Parafibromin and β-catenin ^S33Y -FLAG, using GST-hTCF-4 ^1'130 or GST alone. They found that HA-Parafibromin is significantly enriched by GST-hTCF-4 ^1'130 in the presence of β-catenin ^S33Y -FLAG compared to the control (data not shown). This suggests that TCF/LEF factors and Parafibromin can interact with β-catenin simultaneously.

- 23 -

In summary, the inventors have presented several complementary lines of evidence that Parafibromin/Hyx is a novel nuclear component of the Wnt/Wg signaling cascade both in mammals and in Drosophila. In particular, they have demonstrated that Parafibromin is a novel β-catenin interacting protein, an interaction that is conserved in Drosophila, In addition, they have also identified minimal regions within β-catenin and Hyx/Parafibromin that are required for their interaction. The delineation of this minimal interaction region provides an avenue to modulate Wnt/Wg signaling for therapeutic purposes.

In addition, the observation of a minimal interaction domain within the extended N-terminal region of metazoan Hyx/Parafibromin homologs lends support to the hypothesis that additional transcriptional units may bind to the other conserved motifs within the N-terminal region. It is important to note that the evidence presented here and the data available to date do not imply that Wnt/Wg signaling is involved directly in the etiology of

Hyperparathyroidism, where Parafibromin has a tumor suppressor role. The novel implication from the results is that Hyx/Parafibromin can act as a direct link to pathway-specific transcriptional units and thereby serves as a therapeutic target to control those pathways.

- 24 -

EXPERIMENTAL PART

Example 1

EP-lines, hyx mutants and flystocks:

The UAS-lgs ^17E construct was generated using the site-directed mutagenesis kit (Stratagene). The EP-insertions EF '-hyx (P9) and PlO have been mapped by plasmid rescue and PCR and balanced with TM6b. UAS-hyx was created by cloning the full-length EST LD47989 into pUAS-T. The recessive lethal alleles hyx ¹ , hyx ² and hyx ^EY (EY6898, Drosophila Gene Disruption Project) were analyzed by PCR and balanced over TM6b. EP -hyx, PlO and all hyx alleles reported in this study fully complement neur ⁴¹⁰¹ , new ^j6cn and neur ¹ (Bloomington Stock Center). Transgenics carrying UAS-HRPT2 and tub Ia-EA-HRPTl were created by standard transformation. UAS-CG6220 was created using the full-length EST AT09112; EY3881 was obtained from the Drosophila Gene Disruption Project. The following additional flystocks were used: salE-Gal4/TM6b (also combined with UAS-lgs ^17E to generate yw; UAS- lgs ^17E ; salE-Gal4) and salE-lacZ (D. Nellen and K. B.); act5c-FRT-CD2-FRT-Gal4, with and without recombined UAS-GFP ^nls ; C765-Gal4 (also combined with UAS-lgs ^17E to generate^; UAS-lgs ^17E ; C765-Gal4)\ GMR-GaU; act5c-Gal4; dpp-lacZ; ptc-lacZ; saϊm-lacZ; yw, hs-flp; FRT82, ubi-GFPITMόb; yw, hs-flp, f; FRT82, Min, flTM2; TM6b, ubi-GFP; UAS-GFP ^nh (Bloomington Stock Center) and UAS-lacZ. All flystocks and crosses were kept at 25 °C if not otherwise indicated.

Example 2

EMS mutagenesis screen:

EP-element P9 reverses the wing phenotype of salE>lgs ^17E by hyx overexpression (see text).

To generate hyx mutant alleles, adult P9 males of an age between 1-2 days were collected and starved for 8h in empty bottles. They were subsequently transferred into bottles containing

- -

filter papers moistened with a 1% saccharose-water solution containing 0.4ml/100ml Ethyl methanesulfonate (EMS purum, Fluka). After 24h, the males were put into bottles containing regular food to recover for 24h, before they were mated in bulk to salE> lgs ^17E virginal females. Fl males carrying the mutagenized P9* chromosome marked with y ⁺ were then selected for re-occurence of the salE>lgs ^17E wing phenotype. Positive individuals were crossed to a salE-Gal4 stock to re-screen, and positive lines out of this selection were selected for the y ⁺ marker and balanced.

After genetic selection for candidates, 25-50 males with the 3rd chromosome genotype P9*/salE-Gal4 were collected and frozen for 20min. DNA preparations were then performed following standard protocols. A 2119bp fragment, containing the start and stop codon of the

CGl 1990 ORF was amplified by PCR (Expand High Fidelity PCR System, Roche) using primers with SEQ ID. Nos 15 and 20 out of the genomic DNA preparation, purified using the

QIAquick ^® PCR purification kit (Qiagen) after quality control on a 1-1.5% agarose gel and sequenced using primers with SEQ ID. Nos 15 to 20.

Example 3

Clonal analysis and immunohistochemistry: yw, hs-flp; FRT82, hyx ^EY /TM6b males were crossed to yw, hs-flp; FRT82, ubi-GFP virgins that carried lac-Z insertions of interest on the second chromosome. Larvae of these crosses were heat-shocked 48-72h after egg laying at 37.5°C for 45 min. 3rd instar larval tissue was

stained with mouse monoclonal anti-β-Gal (1:2000, Promega), mouse monoclonal anti-Dll

(1:2000), mouse polyclonal anti-CD2 (1:2000), rabbit monoclonal anti-Haemagglutinin (HA) (1:500, Promega) or mouse monoclonal anti-HA (HAI l, 1 :1000) (BAbCO). Goat secondary antibodies used were Alexa488 and Alexa594 (1 :400) (Molecular Probes). Minute

- 26 -

background clones were created as above but by crossing the males to yw, hs-flp; FRT82, Mm, ubi-GFP/TM6b virgins.

Vectors and Constructs: hyx constructs were PCR generated using the EST LD47989. HRPT2 constructs used the full- length EST V-1314 (Genecopoeia) as basis for PCR. CTNNBl and armadillo fragments for IVT production were PCR amplified and cloned into pcDNA3. β-catenin protein regions were taken as defined by GeneBank entry PRO2286). GST fusion constructs were cloned using pGEX-KG as backbone (Guan and Dixon, 1991). pTOP FLASH has been described previously (van de Wetering et al., 1997). The β-cateninS33Y-FLAG expression construct is as described previously (Kolligs et al., 1999). CMV-RL was obtained from Promega, and CMV-EGFP-Cl was obtained from Clontech.

Example 4

Mammalian cell culture experiments:

HEK 293T cells were seeded at a density of 2-2.5 x 10 ⁵ cells/ml in DMEM with 10% FCS and transfected 24h later with a total of 1.25 μg plasmid DNA per ml of medium (0.5 μg TOP/FOP-Flash, 0.25 μg CMV-RL and 0.5 μg cargo or empty vector, plus 1% of CMV- EGFP-Cl (Clontech) as transfection control) using CaC12. In siRNA experiments, 3 μg si RNA per ml of medium was additionally co-transfected. After 16h, the medium was changed and transfection efficiency verified by fluorescence microscopy for EGFP. If necessary, cells were treated by addition of 25mM LiCl (Sigma) 24h before harvesting. Cells where harvested 48h or 72h after transfection. Dual-Luciferase assays were performed as described below. siRNAs with SEQ ID. Nos 21 and 22 were used for HRPT2, siRNA with SEQ ID. No 23 were used for hPYGO2 and standard GFP-22 control (Quiagen).

- 27 -

Example 5

Protein interaction studies:

The yeast-2-hybrid system was used as described in Bartel and Fields, 1997. Radioactively labelled proteins for GST pulldowns were generated with the T7 TNT kit (Promega), GST pulldowns were done according to standard methods (Sambrook and Russell, 2001). For immunoprecipitation (IP) experiments, petri dishes seeded with HEK 293T cells (2x10 ⁶ ) were transfected as described above. Cells were lysed in NP-40 lysis buffer (Rozenblatt-Rosen et al., 2005), the lysates cleared by centrifugation and subsequently used for precipitation experiments (275 μl lysate, 30 μl Protein-A Sepharose (Amersham Biosciences), 2 μl antibody or the equivalent of 2 μg GST-fusion protein-coupled Sepharose plus Glutathione Sepharose (Amersham Biosciences) instead of Protein-A Sepharose). After 2h incubation at 4°C, pulldowns were analyzed on NuPAGE gels (Invitrogen). Antibodies used for IP were mouse α-FLAG (M2, Sigma), mouse α-Myc (9E10, Drosophila Hybridoma Bank), mouse HA-I l (BAbCO), rabbit α-HA (ICL) and rabbit α-Parafibromin (BL649, Bethyl Labs). Antibodies for western blotting also included rabbit α-Parafibromin (BL648, Bethyl Labs, 1:10.000) and mouse α-β-catenin (BD Biosciences, 1:2.000).

Example 6

Insect cell culture:

S2 cells were propagated in 1 x Schneider's Drosophila media (Invitrogen) supplemented with 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin in 75-cm2 T-flasks (Sarstedt) at room temperature. S2 cells stable lines were propagated in the same media supplemented with 200 μg/ml hygromycin (Roche Molecular Biochemicals) and/or 100 μg/ml blasticidin (Invitrogen).

" 28 "

Example 7

Transient transfection and generation of stable lines:

Standard protocols for transfection and the generation of stable cell lines were utilised, as detailed in the Drosophila Expression System manual (Invitrogen). Briefly, S2 cells were routinely transfected with Cellfectin (Invitrogen Life Technologies) using the following amounts for one well of a 6 well plate: 4 μg DNA, 16 μl Cellfectin per 3x106 cells in 1 ml of Drosophila expression system (DES) serum-free medium (Invitrogen). Three hours after addition of the DNA/Cellfectin mix 1 ml of Ix Schneider's media containing 20% FBS. For transient transfections the cells were harvested 48 hr post transfection. For the generation of the stable line the cells were allowed to grow for 24 hr post transfection before the selection medium was added. 0.2 μg of resistance plasmid were used (pCoHygro and or pCoBlast) per 4.0 μg total DNA. Polyclonal lines stably expressing the transfected constructs were allowed to grow out subsequently expanded.

For the generation of the Wg stable line (referred to as 197.10 2/3) the following plasmids were used: 0.1 μg LEF-luciferase reporter, 0.1 μg mouse LEF-I, 0.05 μg Renilla luciferase and 0.1 μgpTub_Dfz2.

For the Wg reporter system (transient) the following plasmids were used: 1.0 μg LEF- luciferase reporter, 1.0 μg mouse LEF-I, 0.2 μg Renilla luciferase and 0.5 μgpTub_Dfz2.

For the brinker reporter system the following plasmids were transfected (no stable lines used for this reporter): 1.0 μg briήker-lucif erase reporter and 0.2 μg Renilla luciferase.

For the metallothionien reporter system the following plasmids were transfected (no stable lines used for this reporter): 1.0 μg pMT-lucij ^" erase reporter and 0.2 μg Renilla luciferase,

Renilla luciferase was included in each transfection as a control for the efficiency of transfection. The final amount of DNA was adjusted to 4.0 μg using pBluescript vector DNA.

The published Drosophila expression constructs were cloned in the vectors as described for the LEF-luc reporter (Schweizer et al, 2003), the brinker reporter (Mueller et al, 2003). The tubulin driven constructs were generated by inserting the relevant coding sequences, with

Kozak sequence, downstream of the tubulin promoter (pTub_DFz2). The metallothionein inducible constructs were generated by inserting the relevant coding sequences with Kozak consensus downstream of the metallothionein promoter (pMT-Luc) in the pMT/V5-His B vector (Invitrogen).

Example 8

dsRNA production:

The method was adapted from the procedure published by the Dixon laboratory Worby et al., 2001). Individual DNA fragments approximately 700 bp in length, containing coding sequences for the proteins to be "knocked out" were amplified by using PCR. Each primer used in the PCR contained a 5' T7 RNA polymerase binding site (SEQ ID. No 24) followed by sequences specific for the targeted genes. The PCR products were purified by using the High Pure PCR Purification Kit (Roche Molecular Biochemicals). The purified PCR products were used as templates by using a MEGAS CRIPT T7 transcription kit (Ambion, Austin, TX) to produce dsRNA. The dsRNA products were ethanol-precipitated and resuspended in water. The dsRNAs were annealed by incubation at 65 °C for 30 min followed by slow cooling to

room temperature. The dsRNA were analyzed by 1% agarose gel electrophoresis to ensure that the majority of the dsRNA existed as a single band of approximately 700 bp. The dsRNA were stored at -20°C.

Primer sequences used to generate specific dsRNAs were obtained as follows:

Hyx SEQ ID. Nos 25 and 26 hyx b SEQ ID. Nos 27 and 28

APC SEQ ID. Nos 29 and 30 dAPC-2 SEQ ID. Nos 31 and 32

Arm SEQ ID. Nos 33 and 34 dAxin SEQID. Nos 35 and 36 lgs SEQ ID. Nos 37 and 38

CG6220 SEQ ID. Nos 39 and 40

GFP SEQ ID. Nos 41 and 42

Example 9

Conditions for RNAi in Drosophila cell culture: Drosophila cell culture cells were diluted to a final concentration of 1 x 106 cells/ml in Drosophila expression system (DES) serum-free medium (Invitrogen). One milliliter of cells was plated per well of a six- well cell culture dish (Corning). dsRNA was added directly to the media to a final concentration of approximately 15 μg of dsRNA per ml. This was followed immediately by vigorous agitation. The cells were incubated for 30 min at room temperature followed by addition of 1 ml of Ix Schneider's media containing 20% FBS. The cells were incubated for 24 hr to allow for turnover of the target protein and then the pathway was induced. The Wg pathway was activated by co-culture with Wg-producing cells generated by the Nusse laboratory (van Leeuwen et al., 1994). The Hh pathway was activated by inducing the expression of HhN by the addition of CuSO ₄ to a final concentration of 200 μM to the culture media.

_ _n _

Example IQ

Luciferase assays:

Approximately 48 hours after induction of the pathway, the cells were lysed in a passive lysis buffer (Promega) by shaking at room temperature for 30 minutes. The luciferase activities of the lysates were then analyzed by a luminometer (Wallac 1420 Victor 2 multilabel, multitask plate reader, Perkin Elmer) to measure the dual luciferase activities of LEF-luciferase reporter and Renilla luciferase using the Promega Dual-Luciferase reporter assay system. AU the experiments were performed at least three times, and each time in triplicates.

Example 11

Mapping of the β-catenin binding domain in Parafibromin: A GST-fusion comprising the N-terminus (amino acids 1-343) of Parafibromin interacted with β-catenin in vitro, while the C-terminal half of Parafibromin (amino acids 344-531) failed to do so (Figure 19). Further truncations allowed the inventors to define a stretch of amino acids (200-250) essential for β-catenin binding (Figure 19). This stretch corresponds to an evolutionarily conserved block in Parafibromin and its homologs centered on amino acids 218-263, which itself turned out to be sufficient for potent β-catenin binding (Figure 19). Therefore evidence exists that amino acids 218-263 of Parafibromin constitute a discrete β- catenin interaction domain (CID).

However, this minimal binding site is presumably only the core of the real interaction interface. Thus, referring to the experimental data, the Parafibromin fragment spanning amino acids 200-343 seems the smallest protein fragment with full binding capacity. Interestingly, amino acids 200-343 of Parafibromin are 60% identical with the corresponding fragment in

Drosophila Hyrax, which is functionally equivalent to Parafibromin and hence also bind β-

catenin.

REFERENCES

Baker, N. E. (1988). "Transcription of the segment-polarity gene wingless in the imaginal discs of Drosophila, and the phenotype of a pupal-lethal wg mutation." Development 102(3): 489-97.

Barker, N., Hurlstone, A., Musisi, H., Miles, A., Bienz, M., and Clevers, H. (2001). The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation. Embo J 20, 4935-4943.

Bauer, A., Chauvet, S., Huber, O., Usseglio, F., Rothbacher, U., Aragnol, D., Kemler, R., and Pradel, J. (2000). Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. Embo J 19, 6121-6130.

Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W., Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M., et al. (2004). The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761-781.

Boutros, M., Kiger, A. A., Armknecht, S., Kerr, K., HiId, M., Koch, B., Haas, S. A.,

Consortium, H. F. A., Paro, R., and Perrimon, N. (2004). Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells. Science 303 http://www.sciencemag.org/cgi/content/abstract/303/5659/832, 832-835.

Brand, A., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118 http://dev.biologists.Org/cgi/content/abstract/l 18/2/401, 401-415.

Brunner, E., Peter, O., Schweizer, L., and Basler, K. (1997). pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila. Nature 385, 829-833.

Cabrera, C. V., M. C. Alonso, et al. (1987). "Phenocopies induced with antisense RNA identify the wingless gene." Cell 50(4): 659-63.

Caplen, N. J., Fleenor, J., Fire, A., and Morgan, R. A. (2000). dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252, 95-105.

Carpten, J. D., Robbins, C. M., Villablanca, A., Forsberg, L., Presciuttini, S., Bailey- Wilson, J., Simonds, W. F., Gillanders, E. M., Kennedy, A. M., Chen, J. D., et al. (2002). HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-] aw tumor syndrome. Nat Genet 32, 676-680.

Chang, M., French-Cornay, D., Fan, H. Y., Klein, H., Denis, C. L., and Jaehning, J. A.

(1999). A complex containing RNA polymerase II, Paflp, Cdc73p, Hprlp, and Ccr4p plays a role in protein kinase C signaling. MoI Cell Biol 19, 1056-1067.

Chen, J. D., Morrison, C, Zhang, C, Kahnoski, K., Carpten, J. D., and Teh, B. T. (2003). Hyperparathyroidism-jaw tumour syndrome. J Intern Med 253, 634-642.

Chen, C. H., von Kessler, D. P., Park, W., Wang, B., Ma, Y., and Beachy, P. A. (1999). Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 98, 305-316.

Clemens, J. C, Worby, C. A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B. A., and Dixon, J. E. (2000). Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A 97, 6499-6503.

Collins, R. T., and Treisman, J. E. (2000). Osa-containing Brahma chromatin remodeling complexes are required for the repression of wingless target genes. Genes Dev 14, 3140-3152.

Cox, R. T., Pai, L. M., Kirkpatrick, C, Stein, J., and Peifer, M. (1999). Roles of the C terminus of Armadillo in Wingless signaling in Drosophila. Genetics 153, 319-332.

Cronshaw, J. M., Krutchinsky, A. N., Zhang, W., Chait, B. T., and Matunis, M. J. (2002). Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol 158 http://www.jcb.org/cgi/content/abstract/! 58/5/915, 915-927.

₃₅

Daniels, D. L., and Weis, W. I. (2002). ICAT inhibits beta-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural modules. MoI Cell 10, 573-584.

Gallet, A., Angelats, C, Erkner, A., Charroux, B., Fasano, L., and Kerridge, S. (1999). The C- terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila. Embo J 18, 2208-2217.

Gallet, A., Erkner, A., Charroux, B., Fasano, L., and Kerridge, S. (1998). Trunk-specific modulation of wingless signalling in Drosophila by teashirt binding to armadillo. Curr Biol 8, 893-902.

Gerlitz, O., and Basler, K. (2002). Wingful, an extracellular feedback inhibitor of Wingless. Genes Dev 16, 1055-1059.

Grosschedl R ₅ E. Q. (1999). "Regulation of LEF-ITCF transcription factors by Wnt and other signals." Current Opinion in Cell Biology 11: 233-240.

Guan, K.L. and Dixon, J.E. (1991). "Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase." Anal. Biochem., 192, 262-267.

Heberlein, U., E. R. Borod, et al. (1998). "Dorsoventral patterning in the Drosophila retina by wingless." Development 125(4): 567-77.

Hecht, A., Litterst, C. M., Huber, O., and Kemler, R. (1999). Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA- binding protein in vitro. J Biol Chem 274, 18017-18025.

Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F., and Kemler, R. (2000). The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. Embo J 19, 1839-1850.

Howell, V. M., Haven, C. J., Kahnoski, K., Khoo, S. K., Petillo, D., Chen, J., Fleuren, G. J., Robinson, B. G., Delbridge, L. W., Philips, J., et al. (2003). HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. J Med Genet 40, 657-663.

Huber, A. H., Nelson, W. J., and Weis, W. I. (1997). Three-dimensional structure of the armadillo repeat region of beta-catenin. Cell 90, 871-882.

Jho, E. H., Zhang, T., Domon, C, Joo, C. K., Freund, J. N., and Costantini, F. (2002). Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. MoI Cell Biol 22, 1172-1183.

Kiger, A., Baum, B., Jones, S., Jones, M., Coulson, A., Echeverri, C, and Perrimon, N. (2003). A functional genomic analysis of cell morphology using RNA interference. Journal of Biology http://jbiol.eom/content/2/4/27 2, 27.

Kolligs, F. T., Hu, G., Dang, C. V., and Fearon, E. R. (1999). Neoplastic transformation of RK3E by mutant beta-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression. MoI Cell Biol 19, 5696-5706.

Kramps, T., Peter, O., Brunner, E., Nellen, D., Froesch, B., Chatterjee, S., Murone, M., Zullig, S., and Basler, K. (2002). Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 109, 47-60. Krogan, N. J., Dover, J., Wood, A., Schneider, J., Heidt, J., Boateng, M. A., Dean, K., Ryan, O. W., Golshani, A., Johnston, M., et al. (2003). The Pafl Complex Is Required for Histone H3 Methylation by COMPASS and Dotlp. Linking Transcriptional Elongation to Histone Methylation. MoI Cell 11, 721-729.

Krogan, N. J., Kim, M., Ahn, S. H., Zhong, G., Kobor, M. S., Cagney, G., Emili, A., Shilatifard, A., Buratowski, S., and Greenblatt, J. F. (2002). RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. MoI Cell Biol 22, 6979- 6992.

Kuznicki, K. A., Smith, P. A., Leung-Chiu, W. M., Estevez, A. O., Scott, H. C, and Bennett, K. L. (2000). Combinatorial RNA interference indicates GLH-4 can compensate for GLH-I; these two P granule components are critical for fertility in C. elegans. Development 127, 2907-2916.

Lum, L., Yao, S., Mozer, B., Rovescalli, A., Von Kessler, D., Nirenberg, M., and Beachy, P. A. (2003). Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells, Science 299, 2039-2045.

Morin, P. J. (1999). "β-catenin signaling and cancer." Bioessays 21(12): 1021-30.

Mueller, C. L., and Jaehning, J. A. (2002). Ctr9, Rtfl, and Leol are components of the Pafl/RNA polymerase II complex. MoI Cell Biol 22, 1971-1980.

Mueller, C. L., Porter, S. E., Hoffman, M. G., and Jaehning, J. A. (2004). The Pafl complex has functions independent of actively transcribing RNA polymerase II. MoI Cell 14, 447-456.

Mύller, B., Hartmann, B., Pyrowolakis, G., Affolter, M., and Basler, K. (2003). Conversion of an Extracellular Dpp/BMP Morphogen Gradient into an Inverse Transcriptional Gradient. Cell 113, 221-233.

Neumann, C. J., and Cohen, S. M. (1997). Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124, 871-880.

Nusse, R. and H. E. Varmus (1982). "Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome." Cell 31(1): 99- 109.

Orsulic, S., and Peifer, M. (1996). An in vivo structure-function study of armadillo, the beta- catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for wingless signaling. J Cell Biol 134, 1283-1300.

Parisi, M., Nuttall, R., Edwards, P., Minor, J., Naiman, D., Lu, J., Doctolero, M., Vainer, M., Chan, C, Malley, J., et al. (2004). A survey of ovary-, testis-, and soma-biased gene expression in Drosophila melanogaster adults. Genome Biol 5, R40.

Parker, D. S., Jemison, J., and Cadigan, K. M. (2002). Pygopus, a nuclear PHD-fmger protein required for Wingless signaling in Drosophila. Development 129, 2565-2576.

Peifer, M. and P. Polakis (2000). "Wnt signaling in oncogenesis and embryogenesis~a look outside the nucleus." Science 287(5458): 1606-9.

Perrimon, N. and A. P. Mahowald (1987). "Multiple functions of segment polarity genes in Drosophila." Dev Biol 119(2): 587-600.

Phillips, R. G., and Whittle, J. R. (1993). wingless expression mediates determination of peripheral nervous system elements in late stages of Drosophila wing disc development. Development 118, 427-438.

Polakis, P., M. Hart, et al. (1999). "Defects in the regulation of β-catenin in colorectal cancer." Adv Exp Med Biol 470: 23-32.

Porter, S. E., Washburn, T. M., Chang, M., and Jaehning, J. A. (2002). The yeast pafl-rNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes. Eukaryot Cell 1, 830-842.

Potter, J. D. (1999). "Colorectal cancer: molecules and populations." Journal of the National Cancer Institute 91(11): 916-32.

Rijsewijk, F., M. Schuermann, et al. (1987). "The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless." Cell 50(4): 649- 57.

Rondon, A. G., Gallardo, M., Garcia-Rubio, M., and Aguilera, A. (2004). Molecular evidence indicating that the yeast PAF complex is required for transcription elongation. EMBO Rep 5,

47-53.

. .

Roose, J. and H. Clevers (1999). "TCF transcription factors: molecular switches in carcinogenesis." Biochimica et Biophysica Acta 1424 (2-3): M23-37.

Rozenblatt-Rosen, O., Hughes, C. M., Nannepaga, S. J., Shanmugara, K. S., Copeland, T. D., Guszczynski, T., Resau, J. H., and Meyerson, M. (2005). The parafibromin tumor suppressor protein is part of a human Pafl complex. MoI Cell Biol 25, 612-620.

Sambrook, J. and Russell, D.W. (2001). "Molecular Cloning — A Laboratory Manual.", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Schmid, A., Schindelholz, B., and Zinn, K. (2002). Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila. Trends Neurosci 25, 71-74. Schweizer, L., and Varmus, H. (2003). Wnt/Wingless signaling through beta-catenin requires the function of both LRP/Arrow and frizzled classes of receptors. BMC Cell Biol 4, 4.

Sharma, R. P. and V. L. Chopra (1976). "Effect of the Wingless (wgl) mutation on wing and haltere development in Drosophila melanogaster." Dev Biol 48(2): 461-5.

Shattuck, T. M., Valimaki, S., Obara, T., Gaz, R. D., Clark, O. H., Shoback, D., Wierman, M. E., Tojo, K., Robbins, C. M., Carpten, J. D., et al. (2003). Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. N Engl J Med 349, 1722-1729.

Shi, X., Chang, M., Wolf, A. J., Chang, C. H., Frazer-Abel, A. A., Wade, P. A., Burton, Z. F., and Jaehning, J. A. (1997). Cdc73p and Paflp are found in a novel RNA polymerase II- containing complex distinct from the Srbp-containing holoenzyme. MoI Cell Biol 17, 1160- 1169.

Squazzo, S. L., Costa, P. J., Lindstrom, D. L., Kumer, K. E., Simic, R., Jennings, J. L., Link, A. J., Arndt, K. M., and Hartzog, G. A. (2002). The Pafl complex physically and functionally associates with transcription elongation factors in vivo. Embo J 21, 1764-1774.

Struhl, G. and K. Basler (1993). "Organizing activity of wingless protein in Drosophila." Cell 72(4): 527-40.

Takemaru, K. L, and Moon, R. T. (2000). The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression. J Cell Biol 149, 249-254.

Tan, M. H., Morrison, C, Wang, P., Yang, X., Haven, C. J., Zhang, C, Zhao, P., Tretiakova, M. S., Korpi-Hyovalti, E., Burgess, J. R., et al, (2004). Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clin Cancer Res 10, 6629-6637.

Tatusova TA, M. T. (1999). "Blast 2 sequences— a new tool for comparing protein and nucleotide sequences." FEMS Microbiol Lett. 174: 247-250.

Thompson, B., Townsley, F., Rosin-Arbesfeld, R., Musisi, H., and Bienz, M. (2002). A new nuclear component of the Wnt signalling pathway. Nat Cell Biol 4, 367-373

van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A., et al. (1997). Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88, 789-799.

van Leeuwen F, Samos CH, Nusse R. (1994). Biological activity of soluble wingless protein in cultured Drosophila imaginal disc cells. Nature. 1994 Mar 24;368(6469):342-4.

Waltzer, L. and M. Bienz (1999). "The control of β-catenin and TCF during embryonic development and cancer." Cancer & Metastasis Reviews 18(2): 231-46.

Waltzer, L., Vandel, L., and Bienz, M. (2001). Teashirt is required for transcriptional repression mediated by high Wingless levels. Embo J 20, 137-145.

Wodarz, A. and R. Nusse (1998). "Mechanisms of Wnt signaling in development." Annual Review of Cell & Developmental Biology 14: 59-88.

Wood, A., Schneider, J., Dover, J., Johnston, M., and Shilatifard, A. (2003). The Pafl complex is essential for histone monoubiquitination by the Rad6-Brel complex, which signals for histone methylation by COMPASS and Dotlp. J Biol Chem 278, 34739-34742.

_ _

Woodard, G. E., Lin, L., Zhang, J. H., Agarwal, S. K., Marx, S. J., and Simonds, W. F. (2005). Parafibromin, product of the hyperparathyroidism-j aw tumor syndrome gene HRPT2, regulates cyclin Dl /PRADl expression, Oncogene 24, 1272-1276.

Worby, C. A., Simonson-Leff, N., and Dixon, J. E. (2001). RNA interference of gene expression (RNAi) in cultured Drosophila cells. Sci STKE 2001, PLl.

Xu, T., and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223-1237.

Zecca, M., Basler, K., and Struhl, G. (1996). Direct and long-range action of a wingless morphogen gradient. Cell 87, 833-844.