AU5873199A - Genes encoding for the human and murine death inducer-obliterator-1 - Google Patents

Description

WO 00/15787 PCT/GB99/03019 GENES ENCODING FOR THE HUMAN AND MURINE DEATH INDUCER-OBLITERATOR-1 The present invention relates to a novel DNA sequence that codes for expression of a human Death Inducer-Obliterator I (DIO-1)gene and the polypeptide derived from the DNA sequence. Expression vectors containing such sequences and 10 host cells transformed with such expression vectors are also disclosed. as are methods for the expression of the novel DIO-1 polypeptide of the invention. and uses thereof. Background The binding of FasL or TNF to their specific receptors triggers oligomerization 15 and activation of a series of events that results in apoptosis (Nagata. 1997). Dissection of the signal-transducing machinery for Fas-mediated apoptosis has revealed the presence of a set of molecules, FADD/Mortl, which is recruited by and associates with Fas following its activation. In a similar fashion. experiments with cells in which apoptosis was triggered by TNF-a show the existence of another protein. TRADD, 20 which associates to TNFR1 and triggers cell death. TRADD binds to FADD through its death domain such that both stimuli, Fas and TNF-a activate the same downstream caspase pathway. TNF-a activates still another apoptosis pathway through the recruitment of RIP, a serine/threonine kinase that activates the apoptotic pathway by yet unknown mechanisms. More recently, it has been clearly established that STATI 25 is required for TNF-a -triggered cell death (Kumar et al., 1997; Hoey. 1997). Three death factors, namely TNF, FasL and TRAIL (Pan el al., 1997; Gura, 1997), as well as four death receptors, Fas, TNFR1. DR3/Wsl-1 and CAR I (Walczak et al., 1997; Chinnaiyan et al., 1996), have to date been shown to play a role in apoptosis triggering. Loss-of-function mutations in the Fas system illustrate the 30 relevance of this death factor system in maintaining lymphocyte homeostasis. The binding of these factors to their specific receptors triggers a cascade of specific cysteil proteases, the caspases, which cleave various cellular components and lead to the morphological changes characteristic of apoptosis in cells and nuclei. The known signaling pathway initiates at the cell surface and operates in the cytoplasm. the main 35 location of the caspases as well as their inhibitors (Vucic et al.. 1997: Irmler ei al., 1997; Ghayur et al., 1997; Vaux. 1997; Chinnaivan ei al.. 1997). Very little is known SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 as to how these signals are transmitted to the nucleus. A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have recently been identified in the cytoplasmic fraction of a mouse lymphoma cell line. Caspase pathway activation by different stimuli cleaves ICAD. allowing CAD to enter the nucleus and degrade 5 chromosomal DNA (Enari et al.. 1998; Sakahira et al.. 1998). The invention The invention relates to the DNA sequence. amino acid sequences and compounds and methods as defined in the claims. 10 It also relates to the use of the new gene as defined in the claims. The new gene has been called DIO-1 gene by the inventors. The terms "variants" and "alleles" mean that they are derived from the sequences given in the figures and have the same function as those. 15 For the purpose of the invention. gene means both genomic DNA. cDNA. and synthetic DNA. The claimed nucleotide and amino acid sequences are new. They have been found to be useful for control of apoptosis and thereby useful not only for the treatment of diseases which are characterized in the alteration in cell death or by hyperproliferation, 20 but also for the treatment of metabolic, proliferative or inflammatory conditions. As examples cancer, autoimmune diseases, diabetes. rheumatoid artritis. benign and malignant tumors and hyperproliferative skin disorders can be mentioned. Figure legends 25 Figures 1 A, B, C and D and E. Nucleotide and predicted amino acid sequences of DIO-1. The bipartite NLS sequence is boxed and the zinc finger motifs are underlined. Figure 1 E. Schematic representation of the predicted murine DIO-1 ORF. The starting and ending positions of the amino acids defining the motifs are numbered on top. 30 Figures 2 a - 2e. Northern blots and Western blot analysis. cell death and DIO-1 expression analysis. Figures 3 a - 3c. DIO-1 expression pattern during murine limb development Figures 4A- 4F. Overexpression of DIO-I in chick limb. Figure 5A - 5D. Expression pattern of several transcription factors in DID-1-infected 35 chick limb bud. SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 Detailed description of the invention Methods 5 Cloning of DIG-i. Differential display experiments were carried out using an RNAmap kit (GenHunter Corp.,) according to the manufacturer's specifications. Briefly, 200 ng of total cytoplasmic RNA (after DNase treatment with the MessageClean Kit; GenHunter) 10 isolated from WOL-1 cells at 0, 2, 4 and 8 h after IL-7 withdrawal were reverse transcribed with oligo(dT) primers (T 12 MN) in the presence of Moloney murine leukemia virus reverse transcriptase. They were then amplified with several combinations of 5' decamer arbitrary primers and the T 12 MN used for RT in the presence of 3 5 S-dATP (1200 Ci/mmol). The amplified products were resolved in an 8 15 M urea, 6% polyacrylamide DNA sequencing gel and analyzed by autoradiography. Several bands of interest were isolated, reamplified, cloned in the pCR-Script SK(+) vector (Stratagene, La Jolla, CA) and further used for Northern analysis and sequencing. The full-length DIO-1 cDNA was obtained from WOL-1 cDNA by 5' RACE using a Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA), with a 3' 20 primer called L282 (5'-AGGTGTACCTTGTACAGCAGTGAAAC-3'). The resulting 2.6 Kbp band was excised from the gel and cloned in the TA-type vector pGEM-T (Promega, Madison, WI). The resulting clones were sequence-analyzed for orientation, and the oriented sense with respect to the T7 promoter was called DIG IpGEM-T. 25 To confirm the ORF sequence obtained, a cDNA library from mouse brain cloned in ?,ZAPII (Stratagene) was screened by probing with the RACE clone: the same probe was used to screen a human fetal kidney cDNA library (Clontech) from which the human DIG-I homologue was cloned. 30 Cells and transfections. WOL-1 cells were derived from the bone marrow of adult BALB/c mice. They are an untransformed, IL-7-dependent, stroma cell- independent pre-BI cell line, capable of reconstituting irradiated SCID mice. WOL-1 cells grow in Iscove's modified Dulbecco's medium (IMDM) supplemented with penicillin (100 U/ml), streptomycin 35 (100 ptg/ml), 1 mM sodium pyruvate, non-essential amino acids, 50 kM 2 mercaptoethanol, 2 mM L-glutamine, 10% fetal calf serum (FCS) and IL-7 (3% supernatant from a murine IL-7-producing cell line). A20, BAF/3. and FL5.12 cell SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 4 lines were maintained in RPMI 1640 with 10% FCS as described, and the FLS.l2hBcl 2 stable cell line was grown in the presence of I mg/ml G418. MEF(10.1)Val5MycER cells were cultured in phenol red-free Dulbecco's modified Eagle medium (DMEM) containing 10% FCS at 39 0 C. Where indicated. I ptM E2 (17b-estradiol) was added to 5 the medium to activate the MycER fusion protein after 24 h FCS starvation (Wagneer et al.. 1994). WOL-1, A20, BAF/3. and FL5.12 cell lines were cultured at 37"C, and all cell lines were kept in a humidified atmosphere with 5% CO. Transient DNA transfection was performed by electroporation. For each transfection, 2 x 106 log phase cells were collected by centrifugation. resuspended in 10 200 pil of complete RPMI 1640 medium without FCS. After addition of 10 pg of plasmid DNA (1 mg/ml). samples were gently shaken and electroporated in a 0.4 cm electrode gap gene pulser cuvette at 960 [pF and 320 V with a GenePulser apparatus (Bio-Rad, Hercules. CA). Samples were then diluted with 6 ml of the samc medium supplemented with 10% FCS and incubated at 370C in a humidified atmosphere with 15 5% CO. Cells were analyzed for cell cycle staining by FACS at 48 h post electroporation. Northern blot analysis. Total cytoplasmic RNA was prepared as described (Sambrook). RNA (10 Lg) was 20 Northern blotted using a QP-labeled DIO-1 riboprobe made by DIO-ipGEM-T digestion with Bgl II and in vitro transcribed from SP6 using the Riboprohe in vitro Transcription System (Promega). Hybridization was performed in 50% formamide at 65 0 C; washes were in 0.lX SSC + 0.1% SDS at 80 0 C. Blots were exposed on Kodak X-OMAT AR films at -80'C with two intensifying screens. 25 Antibodies and Western blotting. A peptide was synthesized corresponding to amino acids 58-72 of murine DIO-1 with an additional N terminal cysteine (CSLRRSGRQPKRTERV): it was then coupled to maleimide-activated keyhole limpet hemocyanin and the purified conjugate injected 30 into New Zealand White rabbits. Polyclonal antibody was affinity purified against the peptide coupled to a column. WOL-1 were IL-7 starved by washing our times in complete IMDM without FCS. then resuspended in the same volume of medium plus 10% FCS. SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 5 In situ hybridization and histology Whole mount in situ hybridization was carried out as described (Wilkinson, 1993) with some minor modifications (Izpisna Belmonte et al., 1993). The DIO-1 5 digoxigenin probe was made by BglII digestion of the DIO-lpGEM-T and transcription from the SP6 promoter. The probe used for Lhx-2 (700 bp) encompasses the homeobox and the second LIM domain. The remaining probes have been described elsewhere, and include Msx-1 (Robert et al., 1991), Fgf-8 (Vogel et al., 1995), NF-KB (Kanegae et al., 1998). To visualize the cartilage, embryos were fixed 10 in trichloroacetic acid after viral infection, stained with 0.1% alcian green and dehydrated/cleared in methyl salicylate. Production of virus and injection protocols Chicken embryos (either from MacIntyre Poultry, San Diego. CA, or SPAFAS, 15 Norwich, CT) were infected with a virus containing the full length cDNA of DIO-1. Virus preparation and injections were as previously described (Morgan et al., 1992). After injection, the embryos were returned to the incubator at 37'C and fixed at different time points either for in situ hybridization or for phenotypic analysis. 20 Example 1 To search for genes implicated in apoptosis, the differential display PCR technique was used (Liang and Pardee, 1992) with mRNA obtained from the WOL-1 pre-BI cell line as target. WOL-1 was derived from BALB/c adult bone marrow; it grows exponentially in the presence of IL-7 and undergoes apoptosis upon IL-7 withdrawal. 25 Using a set of oligonucleotide primers, one specific for the polyadenylated tail and the other arbitrary in sequence, mRNA was amplified from cells in exponential growth or 2 h after IL-7 deprivation; this was followed by RNA reverse transcription and resolution on a denaturing sequencing gel. Of 82 positive bands, 10 were initially identified as undergoing either upregulation or downregulation during apoptotic death 30 and were therefore considered candidates for further analysis. They were further amplified, sequenced and compared with known gene sequences using the NCBI BLAST program. Of these, one band (Death Inducer- Obliterator 1, DIO-1) revealed that the nucleotide sequence was a novel gene in that it showed no significant identity to any known gene or translated products in the data bases. Close inspection of the 35 DIO-1 gene showed a stretch of nucleotides with homology to Zn finger domains, nuclear localization signals, and acidic transcriptional activating domains in the amino terminal domain. Northern blot analysis of mouse tissue using a labeled DIO-1 probe SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 6 identified a major mRNA species of 9.5 Kb (see below). The cDNA clone encoded an open reading frame of 614 amino acids (Fig. IA). In the 3 untranslated region both TA tracts and a poly(A) tail were identified. DNA searches confirmed that this nucleotide sequence was that of a novel gene. 5 Nucleotide and predicted amino acid sequences of human and urine DIO-1 are shown in Figures lA, B C and D respectively. The bipartite NLS sequence is boxed and the zinc finger motifs are underlined. The DIO-1 protein does not belong to any typical family presently identified, and comprises an N-terminal domain. a central non-canonical Zn finger domain. and a C 10 terminus domain containing a K-rich region. See Figure lF which is a schematic representation of the predicted DIO-1 ORF. The starting and ending positions of the amino acids defining the motifs are numbered on top. Example 2 15 To further characterize DIG-I. its expression pattern was examined by Northern blot analysis using a DIG-1 probe of RNA samples isolated from several cell lines in exponential growth or undergoing apoptosis following triggering in different conditions. Northern blots containing 10 mg per lane of total cytoplasmic RNA from the indicated cell lines, treated with several apoptotic stimuli at different time points, 20 were hybridized to the DIG-1 riboprobe. The blots were reprobed with an actin probe for normalization of the amounts loaded. Figure 2a shows that WOL- 1 in exponential growth phase expresses low levels of DIG-1, which increase upon induction of apoptosis . Conclusion: DIO-1 is upregulated in cells deprived of IL-7. or treated with IFN-y or with dexamethasone. but not in cells treated with etoposide. UV irradiation or 25 in those undergoing Fas-mediated cell death Figure 2b shows Western blot analysis of WOL-1 cells driven to apoptosis by IL-7 starvation. The cells were collected at different time points after removal of IL-7 from the culture medium; 5 x 10' cells were lysed with RIPA buffer and the total extract 30 electrophoresed on an 8% PAGE-SDS gel, blotted and incubated with a affinity purified polyclonal antibody against amino acids 58-72 that specifically recognizes DIG-1 (1:100 dilution in TBS-I% dry milk). Equivalence of protein loading was confirmed by Ponceau S staining. The position of the DIG-1 gene product is indicated in Figure 2b. 35 The upregulation of DIG-1 mRNA levels in cells undergoing apoptosis was thus confirmed in Western blot, using the polyclonal antibody anti-DIG-l. Figure 2b shows that in cell extracts derived from WOL-1 cells undergoing IL-7 deprivation-induced SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 7 apoptosis, but not after etoposide-induced cell death. a 67 kDa band is upregulated two hours after induction. In all cases, there is a correlation between the kinetics of cell death and the upregulation of the mRNA encoding DIO-1 or of the DIO-1 protein itself. 5 Example 3 The effect on cell death was initially examined following transfection of DIO-I into 10 several cell lines using a transient transfection assay. The DIO-1 ORF was cloned into the pcDNA3 mammalian expression vector (Invitrogen, Inc., San Diego, CA). Both empty vector and the DIO-1 construct were transiently transfected by electroporation into A20 and BAF/3 cell lines. After 48 h expression, the cells were permeabilized and stained with propidium iodide, and cell 15 cycle analyzed by FACS. Under the same conditions. both FL5.12 wild type and stably transfected hBcl-2 cells were transiently transfected. In Figure 2c it is shown that transfection of a DIO-i expression plasmid into BAF/3 cells results in a dramatic loss of cell viability at 48 h post-transfection. and cells displayed morphological alterations characteristic of apoptosis. becoming rounded, 20 condensed and finally, dying. This effect was specific in that transfection of BAF/3 with an empty vector had no effect on cell survival. To verify these results, DIO-i plasmids were transfected into A20 (Fig. 2c) or FL5.12 cells (Fig. 2d) In Figure 2d it is shown that hBcl-2 suppresses DIO-I-induced cell death in FL5.12 cells Conclusion: Transient transfection with plasmids containing DIO-1 induced cell death 25 in both cases, with kinetics similar to those observed for BAF/3. When DIO-I was transfected in FL5.12 cells overexpressing human Bcl-2, cells were resistant to cell death, showing that Bcl-2 coexpression inhibits DIO-i death-promoting activity. In MEF(10.1)Va5MycER cells, when plasmids encoding full-length DIO-I were transfected, cells exhibited apoptotic morphology as assessed by 4'6'-diamidino-2 30 phenylindole (DAPI) staining (not shown). Furthermore, using the DIO-1-specific antibody, we find that DIO-i is located in the cytoplasm of MEF(10.1)Val5MycER cells in exponential growth. When apoptosis is triggered by addition of E2 (17b estradiol) (Wagneer et al., 1994), DIO-I is translocated to the nucleus in apoptotic cells (not shown). Extensive efforts to derive stable DIG-1 transfectants in these three 35 cell lines were unsuccessful, suggesting the lethality of DIO-1 expression in these cells (not shown). SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 8 DIO-I is thus differentially expressed under several apoptotic conditions and induces apoptosis when overexpressed. Example 4 5 DIO-I expression was analyzed in murine tissues by hybridization with the DIO-1 riboprobe of a mouse MTN Blot (Clontech). To determine DIO-1 transcript distribution, various tissues were analyzed in Northern blot. Two mRNA transcript bands corresponding to 9.5 and 5.4 kb were detected in most tissues tested. including thymus, spleen, heart, brain, lung, liver, skeletal muscle, kidney and testis. This 10 expression pattern was confirmed with the anti-DIO-1 antibody in Western blot. DIO I expression was also upregulated in vitro in various cell lines, derived from different tissues, when undergoing apoptosis The result is shown in Figure 2e. Molecular size markers are indicated on the left. Lanes: 1. heart. 2, brain. 3. spleen. 4. lung, 5, liver. 6. skeletal muscle. 7. kidney, 8. 15 testis. Example 5 The development of the vertebrate limb is an amenable system for the study of signaling pathways leading to tissue patterning, proliferation and cell death (Izpisna 20 Belmonte et al., 1993; Schwabe et al.. 1998). Limbs originate as a consequence of the differential growth of cells from the lateral plate mesoderm at specific axial levels (Summerbell et al., 1973). At the tip of the limb primordium. a morphologically homogenous and rapidly proliferating group of mesenchymal cells. called the progress zone, induces the overlying ectoderm to differentiate and form a specialized structure 25 termed the apical ectodermal ridge (AER). Subsequent limb outgrowth and maintenance of the AER requires reciprocal signaling between the ridge and the underlying mesodermal cells of the progress zone (Todt and Fallon. 1984: Morgan et al., 1992; Wilkinson, 1993: Vogel et al., 1996). A process that involves programmed death of mesenchymal cells is also required, and a specific gene. BMP-4. has been 30 implicated in this process. DIO-i expression during mouse fetal development was tested and it was found that it is expressed during limb development. DIO-i expression pattern during murine limb development by whole mount in situ hybridization is shown in Figure 3a-c. BALB/c embryos from days 10.5 (a). 11.5 (b) and 12.5 (c) were hybridized to DIO-1 digoxigenin probe, showing expression in the 35 postero-distal zone (a) of the limb. In (b), the pattern is clearly distal. with remarkable indentation in the nascent interdigitating spaces, while in (c), the expression is in the SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 9 interdigitating spaces, with no DIO-1 in the apical zone of the fingers. exhibiting another clear pattern in two areas of the antero-posterior axis. As shown in Figure 3a-c, DIO-1 is highly expressed at gestation day 12.5 in the interdigitating membranes, where programmed cell death is known to occur. 5 Example 6 Cell growth, cell differentiation and cell death are intimately linked during the development of the vertebrate embryo; the developing limb is perhaps one of the best model systems in which to study this process (for a review, see Schwabe et al., 1998). 10 The AER is a pseudostratified epithelium located at the distal part of the developing limb bud shown to be required for limb outgrowth (Summerbell el al., 1973; Todt and Fallon, 1984). Subsequent to the alteration in AER formation, limb outgrowth is arrested. To better understand the role of DIO-1, retroviral technology was used to misexpress it in the chick limb. A retroviral vector containing the RCAS-Dio-] 15 construct, the DIO-1-ORF, was injected into the limb primordia of chick embryos at stages 8-12. At 60-72 h after injection, infected limb buds failed to develop a normal AER. Embryos were examined at different stages following infection. The result is shown in Figures 4A-F in which A is whole mount preparation showing the leg of a wild type embryo. B is Alcian green staining of the same limb to visualize 20 the normal cartilage pattern. C shows an infected embryo 6 days after injection showing extensive truncation of the distal elements of the leg. D shows same embryo after cartilage staining. Note the complete absence of elements distal to the tibia-fibula joint. E and F show the whole mount and cartilage staining of an embryo 8 days after infection with the RCAS-Dio-] construct. 25 In most cases, the truncations occur in the most distal elements (showing an absence of digits, carpals and metacarpals (Figure 4C and 4D). In others, reduction in size and malformations of the tibia and fibula are observed (Figure 4E and 4F and data not shown). The infected limb is distorted and reduced in size. exhibiting an absence. reduction or 30 malformation of phalanges, tarsals and metatarsals (dot). In a few cases, the fibula was reduced in size (asterisk). Conclusion: Overexpression of DIO-I inhibits chick limb outgrowth. Example 7 35 Since misexpression of DIO-1 can perturb AER formation, it could be expected that it is preceded by changes in gene expression, both in the ectoderm and in the underlying limb bud mesoderm. In situ hybridization of embryos were infected with the RCAS SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 10 Dio-1 construct using riboprobes for mesodermal genes involved in limb outgrowth such as Msx-1, Lhx-2 , and NF-,cB (Kanegae el al., 1998). Msx-1 Lhx-2 - and NF-dB (show downregulation in their transcript levels, see Figure 5A. 5C and 5D, respectively. Furthermore, transcripts for ectodermal genes involved in limb 5 outgrowth, such as Fgf-8 , are also absent or downregulated, see Figure 5B). Note the reduced size of the infected limb buds (left limb buds in all cases). Transcripts for Msx-] (A), Fgf-8 (B), Lhx-2 (C) and NF-xB(D) are strongly downregulated (arrows) in the injected limb buds (compare with the normal expression pattern in the uninjected limb bud, right limb bud in all cases). Misexpression of the RCAS-Dio-I construct thus 10 leads to arrest in limb outgrowth that is preceded by changes in the expression of genes involved in outgrowth of the limb. It is not known whether the misexpression of DIO 1 is directly responsible for the downregulation of ectodermal gene markers (i.e., Fgf 8) or if this is a consequence of the previously altered mesodermal gene expression. The combination of these results indicates that DIO-1 may regulate cell death and 15 proliferation during limb development. Conclusion: DIO-1 overexpression alters gene expression in the developing chick limb bud. Discussion 20 Cell growth, cell differentiation and cell death signals are regulated through triggering of specific receptors. which leads to the activation of specific mediators, giving rise in turn to gene transcription activation and/or postranslational modification. Nonetheless, the mechanism through which this activation takes places has not yet been identified. Much work has been done on the mechanism activated by Fas and 25 TNFR ligation in the triggering of apoptosis, but we know very little of the mechanism implicated in transcriptional regulation during the apoptotic process. Few of the molecules presently associated with apoptosis regulation are transcriptional regulators, among which p53 (Wagneer el al., 1994), Nur77 (Chong et al., 1997), the glucocorticoid receptor, STATI (Kumar et al., 1997) and NF-KB (Kanegae et al., 30 1998) are probably the only ones identified so far. Neither Fas- nor TNF-a-mediated apoptosis require gene transcription for induction; it may nonetheless play a role in these cases. We have recently shown that Nur77 is upregulated during Fas-mediated apoptosis, and that constitutive expression of Nur77 renders cells more susceptible to Fas-induced death. On the contrary, nuclear translocation of NF-KB prevents Fas- and 35 TNF-a -triggered cell death., and constitutive expression of IKB favors cell death. SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 11 The structure and features of DIO-l makes this gene useful for control of apoptosis. 5 References Chinnaiyan AM, K et al. 1996. Science 274: 990-992. Chinnaiyan AM, D et al.. 1997. Nature 388: 728-729. Chong LE-C et al.. 1997.. EMBO J 16: 1865-1875. 10 Enari M, et al. 1998. Nature 391: 43-50. Ghayur T, S et al.. Nature 386: 619-623. Gura T. 1997.. Science 277: 768. Nature 388: 714-715. Hoey T. 1997. Science 278: 1578-1579. Irmler M, M et al.. 1997. Nature 388: 190-195. 15 Izpisia Belmonte JC et al., 1993.. Cell 74: 645-659. Kanegae, Y et al.. 1998Nature 392: 611-614. Kumar A, M Commane, et al.. 1997. Science278: 1630-1632. Liang P and AB Pardee. 1992. Science 257: 967-971. Morgan, BA, et al.. 1992.. Nature 358: 236. 20 Nagata S. 1997.. Cell 88: 355-365. Pan G et al. 1997. Science 277: 815-821. Robert, B, et al.. 1991.. Genes Dev. 5: 2363-2374. Sakahira H et al. 1998. Nature 391: 96-99. Sambrook et al. 1989. Molcular Cloning: A laboratory Manual. Cold Spring Harbor 25 Laboratory Press. Schwabe, J et al.. 1998. Trends Genet. 14:229-235. Summerbell, D., J. et al. 1973 Nature 244: 492-496. Todt, W and JF Fallon. 1984.. J. Embryol. Exp. Morph. 80: 21-41. VauxDL. 1997.. Cell 90:389-390. 30 Vogel, A, et al.. 1996. Development 122: 1737-1750. Vucic D et al.. 1997. Proc. Nat. Acad. Sci. USA 94: 10183-10188. Wagneer AJ et al.. 1994.. Genes Dev. 8: 2817-2830. Walczak H et al. 1997. EMBO J. 16: 5386-5397. Wilkinson, D.G. 1993. Whole mount in situ hybridisation of vertebrate embryos. In In 35 Situ Hybridisation (ed. D.G. Wilkinson). Oxford University Press, Oxford. SUBSTITUTE SHEET (RULE 26)

Claims (30)

1. An isolated DNA sequence according to Figure IA and variants and alleles thereof that codes for expression of the human Death Inducer-Obliterator I (DIO-I)Lene. 10

2. A DNA sequence according to claim 1, wherein the DNA sequence is that given in Figure IA.

3. An isolated DNA sequence according to Figure 1B and variants and alleles thereof 15 that codes for expression of the murine Death Inducer-Obliterator I (DIO-1 Igene.

4. A DNA sequence according to claim 3, wherein the DNA sequence is that given in Figure 1B. 20

5. A fragment of an isolated DNA sequence according to any of claims I to 4, which comprises an N-terminal domain, a central non-canonical Zn finger domain, and a C-terminus domain containing a K-rich region.

6. An isolated DIO-1 polypeptide derived from the DNA sequence according to any of 25 claims 1 to 2 comprising the mature human amino acid sequence shown in Figure 1 C and variants thereof.

7. A polypeptide according to claim 6 comprising the mature human amino acid sequence shown in Figure IC. 30

8. An isolated DIG-1 polypeptide derived from the DNA sequence according to any of claims 3 to 4 comprising the mature murine amino acid sequence shown in Figure 1 D and variants thereof. 35

9. A polypeptide according to claim 8 comprising the mature murine amino acid sequence shown in Figure 1 D. SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 13

10. A nucleic acid probe for the detection of a nucleic acid sequence encoding a polypeptide according to any of claims 6-9 in a sample.

11. A nucleic acid probe according to claim 10 wherein said probe comprises at least 5 14 contiguous nucleotides of the sequence given in Figure 1A or lB.

12. A DNA sequence of any of claims 1 to 5 wherein the isolated DNA comprises a cDNA sequence. 10

13. An expression vector containing a DNA sequence of any of claims 1-5.

14. A cell transformed with a DNA sequence of any of claims 1-5, such that it allows the direct replication and expression of said DNA sequence.

15 15. A cell according to claim 14 wherein said cell is a mammalian or a bacterial cell

16. A process for producing a protein according to any of claims 6 to 9 which process comprises the culture of a cell of any of claims 14 to 15 in a suitable culture medium and the isolation of the protein therefrom. 20

17. A method for identifying clones encoding a DIO-1 polypeptide according to any of claims 6-9, said method comprising screening a genomic or cDNA library with a nucleic acid probe according to any of claims 10 to I1 under low stringency hybridization conditions, and identifying those clones which display a substantial 25 degree of hybridization to said probe.

18. A method of identifying agonists and antagonists of the protein according to any of claims 6-9 comprising transduction or transfection of a mammalian cell line with an expression vector comprising nucleic acid sequences lacking the nuclear 30 localization sequences or lacking the Zn finger domain or lacking the acidic domain or lacking the lysine-rich domain and thereafter identifying the agonist or antagonist interacting with the DIG-1 gene according to claims 6-9.

19. An agonists or antagonists according to claim 18. 35 SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 14

20. A method of identifying ligands with which the polypeptide according to any of claims 6-9, interacts, following cloning into and expression in appropriate vectors and using the two-hybrid method. 5

21. A method to produce specific monoclonal and polyclonal antibodies against the polypeptide according to any of claims 6 to 9 comprising the injection of the polypeptide to a mammalian.

22. Method for treatment of diseases which are characterized by the alteration in cell 10 death or by hyperproliferation, characterized by the administration of compounds according to any of claims 6 to 9 or 19 .

23. Method according to claim 22 by administration of a therapeutically effective amount of the compound. 15

24. Method according to claim 22 in which the disease is cancer. an autoimmune disease and/or diabetes.

25. Method according to claim 22 in which the disease is rheumatoid artritis. benign 20 and malignant tumors or hyperproliferative skin disorders.

26. Method for treatment of diseases which are characterized in the alteration in cell death or by hyperproliferation, comprising introducing into a mammal a nucleic acid vector according to claim 13 and wherein said nucleic acid vector is capable of 25 transforming a cell in vivo and expressing said polypeptide in said transformed cell.

27. A pharmaceutical formulation comprising compounds according to any of claims 6 to 9 or 19 and one or more therapeutically acceptable excipients. 30

28. A method for identifying a ligand to the compound according to any of claims 6 to 9 or 19, by a cell-based reporter assay, transgenic-animal reporter assay or in vitro binding assay.

29. A method for identifying a substance for treatment of a condition afTected by a 35 polypeptide according to any of claims 6 to 9, comprising screening for an agonist or an antagonist of the polypeptide signal transduction to be used for treating metabolic, proliferative or inflammatory conditions. SUBSTITUTE SHEET (RULE 26) WO 00/15787 PCT/GB99/03019 15

30. A compound according to any of claims 6 to 9 or 19 for use as a medicament. SUBSTITUTE SHEET (RULE 26)