GB2364051A - SKAT-2, a zinc finger protein - Google Patents

Abstract

There is described the nucleic and protein sequence of a kruppel-like zinc finger protein called SKAT-2 isolated from murine T cells. SKAT-2 may induce apoptosis and cytokine production. It is anticipated that SKAT-2 may be useful in identifying agents which modulate Th-2 responses, or in inflammatory disease therapy.

Description

2364051 PROTEIN

Field of the invention

This invention relates to a novel transcription factor which is preferentially expressed during production of Th 2 helper cells and mediates apoptosis and cytokine production.

Background of the Invention

The ability of naive CD 4 ' T cells to differentiate into distinct effector populations is critical in generating an appropriate immune response to any given infective agent The major driving force behind the establishment of the correct immune response is the pattern of cytokines expressed by the differentiating CD 4 ' T cells The two best characterised effector T cell subsets are Thi and Th 2 cells which are defined by the cytokines they secrete namely IL-2, TNFP and interferon y (IFN y) by Thi cells and IL-4, IL-5, IL-10 and IL-13 by Th 2 cells The cytokines produced by both Thi and Th 2 cells are mutually inhibitory to the differentiation of the other phenotype This probably explains the strong bias towards Thi or Th 2 subtypes of effector cell seen during different infections as predominant production of one or other type at an early stage of infection results in the rapid skewing of the effector cell profile by both positive and negative regulatory mechanisms Polarised Thi and Th 2 responses have also been found in a number of auto-immune and allergic diseases where the secretion of cytokines is thought to be fundamental to the pathology of the disease Thus Thl cells secreting TNF and IF Ny predominate in synovial tissue from rheumatoid arthritis patients and Th 2 cells are thought to promote the damage to the bronchial epithelium characteristic of allergic asthma via direct or indirect recruitment and activation of mast cells and eosinophils.

Regulation of cytokine expression at the level of transcription is likely to play a major role in determining the profile of cytokines expressed in differentiating T cells Given that negative regulation of cytokines and their receptors seems to be an integral part of the mechanism by which polarised T helper cell populations are generated, it could be expected that transcription factors capable of exerting repressive effects on target genes will be involved in regulating the early stages of 2 differentiation.

One of the best characterised and most common repressive domains found in transcription factors is the Kruppel-associated box (KRAB) found in association with approximately one third of all C 2 H 2-type zinc finger proteins When linked to heterologous DNA binding domains, KRAB domains have been shown to shut off transcription of target genes containing appropriate DNA binding sites Commonly composed of two parts (A and B) KRAB domains are approximately 75 amino acids in length and mediate protein-protein interactions which may influence chromatin condensation to mediate their repressive effect Since individual members of KRAB zinc finger sub-families can show tissue-specific expression profiles then their strong repressor activity may indicate a potential role for some of these proteins in stabilising expression profiles in cells during the differentiation process.

Other modular sequence motifs have been described in association with zinc finger transcription factors including the poxvirus and zinc finger (POZ) domain, the finger-associated box (FAX) and the SCAN box or domain None of these sequences have been characterised to the same extent as the KRAB domain SCAN boxes have recently been associated with an oligomerisation function where they mediate the association of one or more SCAN domain containing proteins No positive or negative transcriptional regulatory function has as yet been attributed to the SCAN domain and it may be that its role is primarily to recruit other functional elements to the promoter such as the KRAB domain with which it is often associated.

Alzheimers disease is a neurodegenerative disorder characterised by the loss of neurons and the generation of neuritic plaques and neurofibrillary tangles and glial cell activation FAC 1 is a protein that has been found to be associated with degenerating neurons and activated microglial cells during the early stages of disease progression FAC 1 is probably a DNA binding protein and potential binding sites have been found in regulatory regions of a number of genes associated with the neurodegenerative process Thus FAC 1 may participate in the regulation of gene expression during Alzheimers in cells intimately associated with the degenerative phenotype of the disease, including those undergoing targetted cell death (apoptosis).

Recently a c DNA encoding a protein called BPTF has been identified which is almost identical to FAC 1 but which contains an additional 6 Kb of sequence at the 3 ' 3 end of the c DNA (Ref: Jones et al Genomics 63, 35-39 2000) This may represent an alternatively spliced form of FACI or FAC 1 may be a transcribed pseudogene or represent a partial BPTF clone.

Summary of the Invention

We have now identified a new gene referred to herein as SKAT-2 This gene is selectively expressed in differentiating Th 2 cells but not Thl cells in vitro and expressed in inflammatory cells in the lungs of mice primed and airway challenged with ovalbumin SKAT-2 enhances IL-4 and IL-13 secretion from T cells SKAT- 2 may also induce apoptosis SKAT-2 provides a novel target to identify agents which may be useful in allergic inflammatory disease such as asthma SKAT-2 or variants thereof may also be -useful in targeting expression to Th 2 cells or other antigen presenting cells, for enhancing or repressing Th 2 or humoral responses generated to an antigen, or for reducing the development of a Thl phenotype.

In one aspect, the present invention provides an isolated SKAT-2 polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which maintains or impairs a function of SKAT-2 selected from transcription factor activity, biasing a Th 2 phenotype, inducing apoptosis or inducing cytokine production; or (iii) a fragment of (i) or (ii) which maintains the same function as (i) or (ii).

In another aspect the invention provides a polynucleotide encoding a SKAT2 polypeptide which is capable of at least one function selected from transcription factor activity, biasing production of Th 2 phenotype, inducing apoptosis or inducing cytokine production which polynucleotide comprises:

(a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a 4 sequence as defined in (a) or (b); or (d) a sequence having at least 60 % identity to a sequence as defined in (a), (b) or (c).

In further aspects, the invention relates to an expression vector comprising a polynucleotide of the invention, a cell line comprising the expression vector; and an antibody specific for a polypeptide of the invention.

Description of the Figures

Figure 1 I-4/IL-5 promoter activity in EL-4 cells Expression vectors ( 10 Og) encoding GATA 3, SKAT-2 and ZFP 141 were transfected into EL-4 cells along with 1 O l Og of a reporter construct encoding luciferase driven by either the IL-4 or IL-5 promoters 24 hours after electroporation of plasmids cells were treated with PMA ( 1 Ong/ml) and lonomycin ( 1 pg/ml) or were left untreated After a further 24 hours cells were harvested, lysed and luciferase activity measured The amount of luciferase measured after transfection of an empty expression vector in place of those encoding transcription factors was taken as a baseline and this activity was deducted from that measured in all other transfections The resulting activity is plotted as shown.

Figure 2 Cytokine secretion from stable cell lines expressing SKAT-2/GATA 3.

Plasmids encoding SKAT-2 or GATA 3 cloned in frame with the modified ligand binding domain of the estrogen receptor were used to generate stable cell lines in EL- 4 cells Clones were cultured in 24 well plates for 48 hours before addition of the ligand tamoxifen at various concentrations to induce activation of the fusion proteins.

Cells were stimulated with PMA ( 1 Ong/ml) and Ionomycin ( lpg/ml) and supernatants were measured after 24 hours for IL-4, IL-5 and IL 13 secretion by ELISA using specific antibodies Since the SKAT-2 induction caused some cell death, results are expressed as ng cytokine per 100000 viable cells.

Figure 3 Effect of caspase inhibitors on SKAT-2 dependent cell death One of the SKAT-2 inducible cell lines (clone Bl 1) was grown in 24 well plates and treated with PMA and lonomycin (PI) to activate the cells and tamoxifen to induce SKAT-2 expression Cells were cultured in the presence of 100,10 or 1 pi M inhibitors to caspase 1 and caspase 8 The % viability of the cultures was measured by Trypan Blue staining 24 hours after cell induction 50 u 1 of cells were diluted 1:1 with 0 4 % trypan blue in saline and live/dead cells counted in a haemocytometer The % of viable cells was compared to controls of unstimulated cells and also cells stimulated with Pl and inhibitor but no tamoxifen.

Figure 4 Taqman PCR on Dendritic cell c DNA e DNA was prepared from dendritic cells derived from bone marrow by culture in GMCSF An aliquot was amplified using Taqman PCR machine with primers specific for SKAT-2 A duplicate PCR with water instead of c DNA was also performaed as a negative control.

Brief Description of the Sequences

SEQ ID No 1 is the amino acid sequence of human protein SKAT-2 and its encoding DNA.

SEQ ID No 2 is the amino acid sequence alone of human SKAT-2.

SEQ ID No 3 is the amino acid sequence of murine protein SKAT-2 and its encoding DNA.

SEQ ID No 4 is the amino acid sequence alone of murine SKAT-2.

SEQ ID No 5 is the amino acid sequence of a peptide termed 7 A 1 and its encoding DNA.

Detailed Description of the Invention

Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises", "comprising",

"includes" and "including" are to be interpreted inclusively That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The present invention relates to a polypeptide, referred to herein as SKAT-2, and variants thereof Sequence information for human SKAT-2 is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2 The polypeptides of the invention consist essentially of the amino acid sequence of SEQ ID NO: 2 or of a variant of that sequence SKAT-2 is found to be upregulated by IL-4 SKAT-2 may 6 operate as part of the IL-4 pathway SKAT-2 is preferentially expressed in Th 2 cells and assists in differentiation of T cells into Th 2 cells Expression of SKAT-2 leads to upregulation of the expression of IL-4 and IL 13 SKAT-2 has a KRAB domain which may operate by repressing transcription SKAT-2 may be capable of transcription factor activity and may operate for example by repressing a repressor.

SKAT-2 may act as a transcriptional activator via recruitment of cofactors such as BPTF Expression of SKAT-2 is associated with induction of apoptosis in cells.

SKAT-2 is also expressed in antigen presenting cells SKAT-2 has also been shown to bind to a peptide referred to herein as 7 A 1 which shows homology to a protein named BPTF (Genbank accession No AB 032251) which comprises a full length extension of FACI (U 05237) FAC 1 has been associated with Alzheimers disease and myoglial degenerative processes.

The polypeptides are provided in isolated form The term "isolated" is intended to convey that the polypeptide is not in its native state, insofar as it has been purified at least to some extent or has been synthetically produced, for example by recombinant methods The polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated The term "isolated" therefore includes the possibility of the polypeptide being in combination with other biological or non- biological material, such as cells, suspensions of cells or cell fragments, proteins, peptides, expression vectors, organic or inorganic solvents, or other materials where appropriate, but excludes the situation where the polypeptide is in a state as found in nature.

A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50 %, e g more than 80 %, 90 %, 95 % or 99 %, by weight of the polypeptide in the preparation is a polypeptide of the invention Routine methods, can be employed to purify and/or synthesise the proteins according to the invention Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Edition, CSH Laboratory Press ( 1989), the disclosure of which is included herein in its entirety by way of reference.

7 The term "variants" refers to polypeptides which have a same essential character or basic biological functionality as SKAT-2 In an alternative aspect, a polypeptide of the invention is one which impairs or inhibits an essential character or basic biological functionality of SKAT-2 Preferably, a variant polypeptide is one which has at least one of the following functions: transcription factor activity, induction of the Th 2 phenotype in naive T lymphocytes, induction of apoptosis, ability to bias the immune response to an antigen, induction of cytokine production, and binding to the polypeptide 7 A 1 or a fragment or variant thereof SKAT-2 preferentially controls expression certain cytokines in T-lymphocytes which may assist in inducing the Th 2 phenotype In particular, SKAT-2 induces expression of IL-4 and IL-13 in T-lymphocytes A polypeptide having the same essential character as SKAT-2 may be identified, for example, by expressing the polypeptide in T- lymphocytes or antigen presenting cells and monitoring for one of the functions outlined above, as discussed in more detail below.

Preferably, a full length protein is one which includes at least one zinc finger.

Preferably, multiple zinc fingers are present and are tandemly arranged, for example, the polypeptide has tandemly arranged kruppel-type (C 2 H 2) C terminal zinc fingers.

Preferably, a full length polypeptide also includes a SCAN box and a KRAB domain, preferably towards the N terminus Preferably, the polypeptide is a transcription factor with the potential to repress expression of target genes.

Typically, polypeptides with more than about 65 % identity preferably at least % or at least 90 % and particularly preferably at least 95 % at least 97 % or at least 99 % identity, with the amino acid sequences of SEQ ID NO: 2, are considered as variants of the proteins Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains a basic biological functionality of the SKAT-2 polypeptide, or alternatively inhibits a function of SKAT-2.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions The modified polypeptide generally retains activity as a SKAT-2 polypeptide Conservative substitutions may be made, for example according to the following Table Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar G A P ILV Polar-uncharged C S T M NQ Polar-charged D E KR AROMATIC H F W Y Shorter polypeptide sequences are within the scope of the invention For example, a peptide of at least 20 amino acids or up to 50, 60, 70, 80, 100, 150 or 200 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates at least one biological functionality of SKAT-2 In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a zinc finger or a number of tandemly arranged zinc fingers, for example, C-terminal domain or may represent a KRAB domain or a SCAN domain, which may confer transcriptional repression (N-terminal domain) Such fragments can be used to construct chimeric proteins, for example, incorporating zinc fingers or other KRAB or SCAN domains from other transcription factor proteins Such fragments can also be used to raise anti-SKAT-2 antibodies In this embodiment the fragment may comprise an epitope of the SKAT-2 polypeptide and may otherwise not demonstrate the binding or other properties of SKAT-2.

Polypeptides of the invention may be chemically modified, e g posttranslationally modified For example, they may be glycosylated or comprise modified amino acid residues They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane Such modified polypeptides fall within the scope 9 of the term "polypeptide" of the invention.

The invention also includes nucleotide sequences that encode for SKAT-2 or variant thereof as well as nucleotide sequences which are complementary thereto.

The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or c DNA Preferably the nucleotide sequence is a DNA sequence and most preferably, a c DNA sequence Nucleotide sequence information is provided in SEQ ID NO: 1 Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background Background hybridization may occur, for example, because of other c DN As present in a c DNA library The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1 The intensity of interaction may be measured, for example, by radiolabelling the probe, e g with 32 P Selective hybridisation may typically be achieved using conditions of low stringency ( 0 3 M sodium chloride and 0.03 M sodium citrate at about 40 C), medium stringency (for example, 0 3 M sodium chloride and 0 03 M sodium citrate at about 50 C) or high stringency (for example, 0 03 M sodium chloride and 0 003 M sodium citrate at about 60 C).

The coding sequence of SEQ ID No: 1 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions The polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

The modified polynucleotide generally encodes a polypeptide which has a SKAT-2 activity or inhibits a SKAT-2 activity Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1.

Sequence identity of polynucleotide or peptide sequences of the invention can be calculated using the CGC (University of Wisconsin) suite of programs and in particular using Clustal W The default matrix for all protein-protein comparisons is preferably BLOSUM 62 For nucleotide sequences, the blast 2 0 program may be used Altschul et al 1997 "Gapped BLAST and PSI-BLAST".

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i e higher sequence identity over longer lengths) being preferred Thus, for example a polynucleotide which has at least 90 % sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95 % sequence identity over 40 nucleotides.

The nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo.

The nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques Nucleotides complementary to those encoding SKAT-2, or antisense sequences, may also be used in gene therapy, such as in strategies for down regulation of expression of the proteins of the invention.

Polynucleotides of the invention may be used as a primer, e g a PCR primer, a primer for an alternative amplification reaction, a probe e g labelled with a revealing label by conventional means using radioactive or non- radioactive labels, or the polynucleotides may be cloned into vectors.

Such primers, probes and other fragments will preferably be at least 10, 11 preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO: 1.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention Such expression vectors are routinely constructed in the art of molecular biology and may o for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression Other suitable vectors would be apparent to persons skilled in the art By way of further example in this regard we refer to Sambrook et al.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA Antisense RNA or other antisense polynucleotides may also be produced by synthetic means Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i e the vector is an expression vector The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter The vectors may contain 12 one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed For example, yeast promoters include S cerevisiae GAL 4 and ADH promoters, S pombe nmtl and adh promoter Mammalian promoters include the metallothionein promoter which can be I 0 induced in response to heavy metals such as cadmium Viral promoters such as the SV 40 large T antigen promoter or adenovirus promoters may also be used All these promoters are readily available in the art.

Mammalian promoters, such as P-actin promoters, may be used Tissuespecific promoters are especially preferred Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) , the rous sarcoma virus (RSV) LTR promoter, the SV 40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR) Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses Gene transfer techniques using these viruses are known to those skilled in the art Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome Replication-defective adenovirus vectors 13 by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the SKAT-2 polypeptide or a variant thereof Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK 293 T, CHO, He La and COS cells, T-lymphocytes and antigen presenting cells Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide Expression may be achieved in transformed oocytes A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably a mouse A transgenic non-human animal expressing a polypeptide of the invention is included within the scope of the invention A polypeptide of the invention may also be expressed in Xenopus laevis oocytes or melanophores, in particular for use in an assay of the invention.

It is also possible for the proteins of the invention to be transientlyexpressed in a cell line or on a membrane, such as for example in a baculovirus expression system Such systems, which are adapted to express the proteins according to the invention, are also included within the scope of the present invention.

According to another aspect, the present invention also relates to antibodies (either polyclonal or preferably monoclonal antibodies, chimeric, single chain and fab fragments) which have been raised by standard techniques and are specific for a polypeptide of the invention Such antibodies could for example, be useful in purification, isolation or screening methods involving immunoprecipitation techniques and may be used as tools to further elucidate the function of SKAT-2 or a variant thereof, or indeed as therapeutic agents in their own right Antibodies may also be raised against specific epitopes of the polypeptides according to the invention Such antibodies may be used to block activity of the polypeptide An antibody, or other compounds, "specifically binds" to a protein when it binds with high affinity to the protein for which it is specific but does not bind or binds with only low affinity to other proteins A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an 14 antibody are well known in the art (see for example Maddox et al 1993) Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods to identify compounds that may act as modulators of SKAT-2 activity Any suitable form may be used for the assay to identify a modulator of SKAT-2 activity Agents which bind to the proteins of the present invention can also be identified by binding assays.

The binding of a test substance to a polypeptide of the invention can be determined directly For example, a radio labeled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored Typically, the radiolabeled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached The membranes can then be separated from a non-bound test substance and 1 5 dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting Non-specific binding of the test substance may also be determined by repeating the experiment in the presence of a saturating concentration of a non-radioactive ligand.

Assays may also be carried out to monitor for the ability of SKAT-2 or fragments thereof or variants thereof to bind to DNA In order to identify the preferred or consensus binding site for SKAT-2 on DNA a variety of techniques can be used A SKAT-2 fusion protein over-expressed and purified from E Coli could be coupled to a column and used to bind oligonucleotides from within a random pool.

After washing, elution and re-binding, oligonucleotides containing specific SKAT-2 binding sequences could be identified by sequencing A similar process can be carried out using fragmented genomic DNA This has the advantage of identifying binding sites on potentially functional sites within the genome A third even more powerful technique involves cross-linking proteins to DNA in vivo, fragmenting the chromatin and precipitating with an anti-SKAT-2 antibody DNA associated with specifically precipitated protein will represent the in vivo sites of SKAT-2 binding, possibly with some coding sequences of regulated genes being identified as well.

Once the binding site preference for SKAT-2 has been determined, gel shift assays can be carried out using labelled oligonucleotides in order to monitor the binding of SKAT-2 in vitro.

SKAT-2 has also been identified as binding to a polypeptide referred to herein as 7 A 1 and having the amino acid sequence set out in SEQ ID No 5 7 A 1 has been shown to be identical to the 3 'end of a protein termed BPTF The 5 'region of the BPTF aligns to the protein FAC-1 which is associated with Alzheimer's disease and in particular with myoglial degenerative processes Thus, other assays may involve monitoring the binding of SKAT-2 or a variant thereof with 7 A 1 or other related polypeptides.

Assays may also be carried out to identify a modulator of SKAT-2 mediated activity A modulator may effect the binding characteristics of SKAT-2, may enhance or inhibit SKAT-2 binding to DNA or a polypeptide A modulator may be one which modulates the activity of SKAT-2, for example, by interfering or enhancing the transcription factor activity, inducement of cytokine production, apoptosis or cell expression pattern of SKAT-2.

Typically, a cell is provided The cell may comprise a polynucleotide construct or expression vector encoding SKAT-2 or a variant thereof or may comprise a cell which has the ability to express SKAT-2 A test substance may be added to the extracellular medium under conditions in which expression SKAT-2 is induced in the absence of the test substance and SKAT-2 activity is then monitored.

Alternatively, the conditions may be selected such that no expression of SKAT-2 is expected to identify test substances which induce such expression The activity to be monitored may comprise expression of SKAT-2, for example, to identify agents which upregulate or down regulate SKAT-2 expression Alternatively, expression of other polypeptides mediated by SKAT-2 transcription factor activity may be monitored For example, where the cell comprises a T-lympocyte, expression of cytokines which are upregulated on SKAT-2 expression such as IL-4 or IL- 13 may be monitored Alternatively, a cell may be provided with a polynucleotide construct encoding a reporter gene under the control of a promoter that is responsive to SKAT- 2 mediated transcription factor activity For example, a reporter gene comprising the IL-4 promoter operably linked to a suitable reporter gene may be provided Normal expression of SKAT-2 in the cell would lead to expression of the reporter gene 16 through the IL-4 promoter.

In an alternative aspect of the invention, T-lymphocytes are used for the assay SKAT-2 activity may be monitored by reference to the induction of Th 2 phenotype in the T-lymphocyte An inhibitor of SKAT-2 activity may have the effect of inducing a Thl phenotype Polynucleotide constructs encoding SKAT-2 or reporter genes may be recombinantly expressed in an cell The cell may be transiently or stabily transfected or transformed Cells can be transfected by methods well known in the art, for example, by electroporation, calcium phosphate precipitation, lipofection or heat shock The proteins may be expressed in mammalian cells such as human cells or non-mammalian cells such as yeast or bacteria In an alternative aspect, T-lymphocytes are used in accordance with the assay and SKAT-2 expression may be induced, for example, by incubation of the cells with IL-4.

In accordance with an assay of the invention, the test substance may comprise a polypeptide The test substance may be provided into the extracellular medium or may be provided as a further polynucleotide construct Expression of the test polypeptide may be induced during the assay.

In an alternative aspect of the invention, the SKAT-2 activity to be monitored may be the induction of apoptosis.

Control experiments may be carried out in which the substance to be tested in omitted or agents which are known to effect SKAT-2 activity are included.

Examples of reporter polypeptides which may be used include 13glucoronidase, green fluorescent protein, luciferase, chloramphenicol transferase or 3-galactasidase.

Suitable test substances which can be tested in the above assays include combinatorial libraries, defined chemical entities, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e g phage display libraries) and antibody products.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or activation tested individually Test substances may be used at a concentration of 17 from 1 n M to 1 OOOM, preferably from 1 p M to 1 Op M, more preferably from 1 M to 10 LM.

Another aspect of the present invention is the use of polynucleotides encoding the SKAT-2 polypeptides of the invention to identify mutations in SKAT-2 genes which may be implicated in human disorders Identification of such mutations may be used to assist in diagnosis or susceptibility to such disorders and in assessing the physiology of such disorders Alternatively, such polynucleotides may be used to monitor SKAT-2 expression and in particular to monitor for up or down regulation of SKAT-2 and the association of such up and down regulation with a disease condition, or in the presence of a compound under test.

Another aspect of the present invention is the use of the compounds that have been identified by screening techniques referred to above in the treatment or prophylaxis of disorders which are responsive to regulation of SKAT-2 activity.

Alternatively, SKAT-2 polypeptides, polynucleotides encoding SKAT-2 or agents which up regulate SKAT-2 expression may be used in a method of treatment or prophylaxis of such disorders Alternatively, variants of SKAT-2 which inhibit SKAT-2 activity, polynucleotides, antisense polynucleotides or agents which down regulate SKAT-2 expression may be used in the treatment or prophylaxis of such SKAT-2 associated disorders.

Modulators of SKAT-2 activity may be useful in the treatment of asthma, allergic disorders such as hayfever, atopic dermatitis, allergic rhinitus etc, Alzheimer's and other neuronal disorders and in particular neuronal disorders associated with apoptosis SKAT-2 may also be useful for manipulation of the immune response produced, for example, to an antigen in a vaccine formulation, auto immune conditions, infections and cancer.

SKAT-2 has been shown to be involved in the induction of a Th 2 phenotype.

Accordingly, agents which stimulate up regulation of SKAT-2 expression or agents which enhance the activity of SKAT-2 such as enhancing SKAT-2 mediated expression of IL-4 or IL 13 may be used to bias the immune response to a Th 2 type response Such agents include polynucleotides encoding SKAT-2 which may be used to transform cells to induce the Th 2 phenotype as well as compounds 18 identifiable by an assay in accordance with the invention which are shown to activate SKAT-2 activity.

In an alternative aspect of the invention, agents and compounds which down regulate SKAT-2 activity may be used, for example, to reduce the Th 2 cell response that is generated Such agents include compounds which have been shown to down regulate SKAT-2 expression, compounds which inhibit SKAT-2 activity such as inhibiting SKAT-2 induced transcription and cytokine production and antisense polynucleotides which inhibit SKAT-2 expression in vivo Such agents may be useful, in particular, in the treatment of inflammatory disease such as asthma o associated with a Th 2 cell response Alternatively and/or in addition, such agents may be useful to prevent induction of a Th 2 phenotype and may have utility in inducing Thi type responses, for example, where cytotoxic T-cell helper responses are required.

In preferred embodiments, SKAT-2 may be used in a vaccine composition.

SKAT-2 may be provided as a polypeptide for administration with an antigen to enhance the immune response produced to the antigen and in particular to assist in biasing the immune response generated to a Th 2 type response Alternatively, SKAT-2 may be administered as a polynucleotide, for example, in a nucleic acid vaccine Expression of SKAT-2 in antigen presenting cells and/or T cells may be effective to enhance production of Th 2 type responses to the antigen Alternatively, regulatory sequences of SKAT-2 such as the SKAT-2 promoter may be used to drive expression of the antigen of interest and in particular to obtain preferential expression of the antigen in antigen presenting cells and other cells of the immune system to enhance the immune response generated to the antigen e g selective up or down- regulation of Th 2 response In an alternative aspect of the invention, an inhibitor of SKAT-2 activity or one which down regulates SKAT-2 expression is co- administered with an antigen to assist in modulating the Thl/Th 2 balance and/or promoting a cytotoxic T-cell helper response where such a response is desired to the antigen being administered.

Antigens such as those allergens derived from house dustmite and cockroach or grass pollens can be used to induce a response in T cells after processing and presentation by dendritic cells or other antigen presenting cells Similarly viral and 19 bacterial antigens such as LPS tend to lead to a predominantly Thi-like population of T cells It is thought that the AP Cs involved undergo quite different differentiation processes depending on the antigen and environment which they encounter such that when T cells are stimulated there is an APC-derived bias towards Thi or Th 2 development Since SKAT-2 has been shown to be associated with AP Cs which are being used to drive T cells to a Th 2 but not Thi phenotype, it is possible that SKAT- 2 or enhancers of SKAT-2 might be useful in biasing an immune response towards or away from (eg SKAT-2 might promote death of the APC in context of Th 2 response and thus serve to down-regulate rather than promote) the Th 2 direction Similarly, inhibitors of SKAT-2 might be able to modulate the APC drive of the Thl/2 balance.

SKAT-2 has been shown to play a role in apoptosis Accordingly, agents which induce expression of SKAT-2, polynucleotides encoding SKAT-2 or agents which are shown to enhance SKAT-2 activity and in particular SKAT-2 mediated apoptosis may be used in the treatment of disorders for which apoptosis is required.

Agents which inhibit expression of SKAT-2, antisense SKAT-2 polynucleotides or agents which are shown to inhibit SKAT-2 activity may be used in particular where it is desired to inhibit apoptosis In this embodiment, such agents may be particularly useful to inhibit apoptosis in neuronal tissue and in particular disorders such as Alzheimer's.

Disorders in which it might be desirable to induce apoptosis include cancer (unregulated cell division) and some inflammatory and auto-immune diseases such as asthma and rheumatoid arthritis (RA) In the latter two cases cells such as eosinophils and macrophages secrete products which directly and indirectly cause many of the deleterious pathology associated with the disease It has been suggested that the disease itself may be caused by the failure of such cells to die by apoptosis By promoting cell death in these cases it may be possible to treat the inflammation successfully.

The compounds identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art, and as fully described in Remmington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania 17th Ed 1985, the disclosure of which is included herein of its entirety by way of reference.

The compounds may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen A physician will be able to determine the required route of administration and dosage for any particular patient A typical daily dose is from about 0 1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration Preferably, daily dosage levels are from 5 mg to 2 g.

1 5 Nucleic acid encoding SKAT-2 or a variant thereof which maintains or inhibits SKAT-2 activity may be administered to the mammal Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal In particular, such nucleic acid may be administered as a vaccine composition with a nucleic acid encoding an antigen to which it is desired to generate an immune response.

Nucleic acid encoding the peptide(s) may be administered to the animal by any available technique For example, the nucleic acid may be introduced by injection, preferably intradermally, subcutaneously or intramuscularly Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery The nucleic acid may be administered topically to the skin, or to the mucosal surfaces for example by intranasal, oral, intravaginal, intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.

Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam The 21 dosage of the nucleic acid to be administered can be altered Typically the nucleic acid is administered in the range of 1 pg to 1 ming, preferably to 1 pg to 1 Oig nucleic acid for particle mediated gene delivery and 1 Otg to 1 mg for other routes.

The following Examples illustrate the invention.

Identification of SKAT-2, a Novel Murine Kruppel Zinc Finger Gene.

In an attempt to identify transcription factors which are differentially expressed between developing T helper cell subsets, we have carried out low stringency PCR using redundant primers designed to highly conserved regions of the Kruppel-type zinc finger motif namely the HC linker region and the sequence around o the two cysteine residues PCR amplification of c DNA using these primers generates a ladder of fragments when resolved on agarose gels corresponding to an 81 bp zinc finger repeat We have performed such a PCR on naive CD 4 ' spleen cells from DO 10 TCR transgenic mice in addition to cells showing both Thi and Th 2 type cytokine expression profiles derived in culture from naive cells Bands corresponding to 1 5 multiple zinc finger repeats were purified, cloned and sequenced and the data used to identify clones which showed a potential bias in expression towards the Thi or Th 2 populations Such clones were used as probes in Southern blots against total excised inserts from Th 1 and Th 2 c DNA libraries as a preliminary confirmation of their differential expression (not shown).

One such zinc finger insert which appeared to be significantly biased in representation in the Th 2 library was used to obtain a partial c DNA clone by PCR between the zinc finger region and the flanking vector sequences in the library The complete open reading frame was completed by extending the sequence at the 5 ' end by cloning genomic DNA with a Promoter Finder kit (Clontech) The deduced 5 ' end of the clone was verified by PCR analysis on c DNA DNA sequence from several independent PCR amplified clones was used to compile the final sequence of the c DNA in order to minimise the chances of PCR-derived sequence errors.

The single open reading frame of 2277 nucleotides encodes a protein of 759 amino acids with a deduced relative molecular mass of 86 7 Kda The predicted protein encoded by this clone contains 14 tandemly arrayed Kruppel-type (C 2 H 2) C terminal zinc fingers and an N terminal SCAN box and KRAB domain Both of these 22 domains are typically found in association with kruppel type zinc fingers within transcription factors Although no function has as yet been ascribed to SCAN boxes, KRAB domains have been shown to confer potent transcriptional repression when tethered to reporter constructs Thus the predicted protein SKAT-2 (SCAN/KRAB protein associated with a Th 2 phenotype) is a putative transcription factor with the potential to repress expression of target genes.

Searching of public databases with the mouse c DNA sequence has led us to identify the human homologue of SKAT-2 from within a large 169 9 Kb genomic DNA sequence in public databases (accession number AC 005822) The SKAT-2 1 o gene is contained within a 15 5 Kb region of this clone and is split into 5 exons covering the whole gene We have designed PCR primers corresponding to the 5 ' and 3 ' ends of the coding sequence of the human DNA based on our knowledge of the mouse c DNA and have confirmed the existence of the corresponding message by successfully amplifying the human c DNA from a library The sequence of the human clone and its protein translation are given in SEQ ID No 1.

The open reading frame has 2283 nucleotides encoding a protein of 761 amino acids.

Expression profile of SKA T-2.

In order to confirm the differential expression of SKAT-2 in Thl/Th 2 cells we performed a Northern blot analysis on total RNA isolated from Thi and Th 2 cells 4 hours after re-stimulation with anti-CD 3 and anti-CD 28 A single m RNA species of 6 5 Kb corresponding to SKAT-2 was clearly present at much higher levels in the Th 2 cells and was indeed only just detectable in the Thi cells In order to verify that the SKAT-2 protein was also differentially expressed we prepared protein extracts from Thi and Th 2 cells and carried out a Western blot using an anti-SKAT- 2 peptide antibody.

No SKAT-2 protein was detectable in this way at any stage of Thl development whereas a band with an apparent molecular weight of-1 OOK Da was evident in the extracts made from cells cultured under Th 2 biasing conditions at both 2 and 3 days after primary stimulation The apparent discrepancy between the theoretical and observed molecular mass of SKAT-2 could potentially reflect post- transcriptional modification of the protein.

23 The tissue distribution of SKAT-2 was also determined by Northern blot analysis A band was detected in mouse brain and there were also weak signals in the kidney and spleen whereas none of the other adult mouse tissues tested contained measurable levels of SKAT-2 The absence of any detectable transcript in the thymus RNA could suggest that SKAT-2 may be restricted in its expression to either differentiating or fully committed Th 2 cells and is probably absent or present at only low levels in naive T cells When tested on a number of haematopoietic cell lines SKAT-2 was found to be expressed in only 1 of 6 T cell lines and more weakly in 3 of 5 late myeloid and 2 of 3 mastocytic cell lines None of the erythroid, l O megakaryocytic, early myeloid and plasma lines contained any detectable message.

Interestingly 2 of the 5 pre-B cell lines expressed a transcript of -4 5 kb We have recently identified a clone corresponding to the human homologue of SKAT- 2 which was derived from B cell germinal centres This clone corresponds to the 5 ' end of SKAT-2 and lacks the zinc fingers which are replaced by a poly A tail and could potentially correspond to the message seen on the Northern blot in the B cell lines.

SKAT-2 and GATA 3 show similar expression profiles.

Since m RNA encoding the zinc finger transcription factor GATA 3 has been shown to be expressed at high levels during commitment to the Th 2 phenotype, we wished to see how the temporal expression of SKAT-2 compared to that of GATA 3 in our cells at the level of m RNA In order to determine at what stage in T cell development SKAT-2 is expressed we carried out in situ hybridisation of a DIG- labelled SKAT-2 riboprobe on cytospins of fixed CD 4 + spleen cells and Thl and Th 2 cells from days 1-5 after primary stimulation with antigen Naive CD 4 + T cells derived from spleens of DO 10 TCR transgenic mice were stimulated with ovalbumen peptide and APC's and cultured under conditions favouring Thl (anti-IL-4, IL-12) or Th 2 (anti IF Ny, anti-IL-12 and IL-4) development Cells were harvested every 24 hours for 5 days and fixed in paraformaldehyde before collecting on glass slides using a cytospin centrifuge SKAT-2 m RNA was detected using a DIG- labelled riboprobe corresponding to the linker region between the KRAB domain and the zinc fingers The presence of bound probe was detected after high stringency washing using an anti-DIG antibody and an anti-rabbit Ig G HRP secondary antibody.

Purified CD 4 + spleen cells appear to contain very low levels of SKAT-2 24 m RNA but when cells are driven towards a Th 2 phenotype by culturing in IL-4 and anti-IL-12 there is a strong and sharp peak of expression of SKAT-2 at about 48 hours after the primary stimulation This level is down-regulated fairly rapidly thereafter and is returned to the basal level seen in the unstimulated cells by day 5.

No SKAT-2 m RNA was detectable at any stage in cells driven towards the Thl phenotype above the background level In situ hybridisation of a GATA 3 riboprobe to cytospins of developing Thl and Th 2 cells revealed a remarkably similar profile of expression to that of SKAT-2 Message for GATA 3 appears to peak strongly 48 hours after stimulation and subsides to the basal level by day 5 The basal level of l 0 m RNA seen in the CD 4 + unstimulated cells is slightly higher than seen for SKAT-2.

SKA T-2 upregulates the IL-4 but not the IL-5 promoter in T cells.

GATA 3 has been shown by others to upregulate the IL-4 and IL-5 promoters in a number of different cell lines in transient transfections We wished to see if over- expression of SKAT-2 in a similar system would have any effect on transcription driven by the IL-4 and IL-5 promoters Luciferase reporter constructs under the control of the IL-4 and IL-5 promoters were transfected into EL-4 cells either with an expression vector alone or the same vector coding GATA 3, SKAT-2 or zfp 141, a control protein containing kruppel-type zinc fingers and a KRAB domain.

Luciferase activity was measured in unstimulated cells 24 hours after transfection and also 24 hours after stimulation of transfected cells with PMA/Ionomycin (Fig 1).

The previously described effect of GATA 3 on the two promoters was clearly observed in these cells with significant promoter activity measured on over- expression of GATA 3 in the presence of PMA and lonomycin However whereas zfp 141 was unable to stimulate any activity above basal levels from these promoters, the other KRAB domain containing protein SKAT-2 was able to transactivate the IL- 4 but not IL-5 promoter Although the levels of activation over the background level observed with the vector alone were relatively small ( 2-3 fold) they were consistently observed over a number of experiments and presumably reflect a significant level of activation Since we do not know the DNA recognition sequence for SKAT-2 it is possible that the IL-5 promoter used in this construct was not sufficiently large to contain any possible sites for this factor Thus SKAT-2 could potentially be able to activate the IL-5 promoter under different conditions.

In order to overcome this limitation we have generated stable EL-4 cell lines expressing inducible forms of GATA 3 or SKAT-2 and have measured the effect of inducing protein expression on the production of endogenous cytokines This should enable us to monitor the effect of GATA 3 and SKAT-2 on intact cytokine promoters in their normal chromatin configuration The inducible system used is based on the oestrogen receptor (ER) which is held in an inactive complex with heat shock proteins in the absence of ligand On ligand binding the protein is released and can move into the nucleus and regulate transcription of target genes In this work we have made fusion proteins between a modified ligand binding domain of the ERand either GATA 3 or SKAT-2 The modified ER has a high affinity for the oestrogen antagonist 4-hydroxytamoxifen (OHT) and no binding affinity for the 17 P- oestradiol ligand for the unmodified LBD Thus we can measure the effect on cytokine expression of releasing increasing amounts of our fusion proteins by titrating in OHT to cell cultures.

As can be seen in Figure 2 GATA 3 is able to upregulate IL-4 and IL-5 secretion in a tamoxifen-dependent manner confirming the results from the transient transfections using reporter constructs driven by these promoters In addition production of another Th 2-type cytokine IL-13 is also increased in the same way For each cytokine the level of induction is at least 8 fold although a precise measure is not possible since the amount of IL-5 and IL-13 made by the untreated cells is too low to measure accurately The results obtained using the SKAT-2 fusion protein again confirm those obtained using the promoter /reporter constructs as IL-4 but not IL-5 secretion is increased in a dose-dependent manner by tamoxifen Interestingly, like GATA 3 SKAT-2 is able to upregulate IL-13 production in EL-4 cells although the magnitude of the increase is much lower Since SKAT-2 is selectively expressed during the process of Th 2 cell differentiation at similar times to GATA 3 and is capable of upregulating some Th 2-type cytokines in T cell lines, it is possible that this protein may like GATA 3 play some role in establishing the Th 2 phenotype in vivo.

Apoptosis Since we had noticed that induction of SKAT-2 expression in EL-4 cells caused significant cell death we looked to see if the cells were undergoing apoptosis.

26 In each of the stable SKAT-2 cell lines tested there was a significant degree of apoptosis occurring as measured by sub-GI peak analysis of PI-labelled cells and annexin staining of phosphatidylserine surface expression No such apoptosis was detected in any of the stable GATA 3 expressing cell lines In order to demonstrate further that SKAT-2 was causing apoptosis in these cells we measured the ability of two caspase inhibitors to block the observed cell death As can be seen in Figure 3 one of the inhibitors (caspase 1 inhibitor) was able to block apoptosis in a dose dependent manner with virtually no cell death observed in the presence of 1 00 Ou M inhibitor Since caspases are an intrinsic part of certain apoptotic processes then the l O ability to observe a block in cell death with a specific caspase inhibitor implies that the SKAT-2 dependent cell death is caused by apoptosis.

Expression of SKAT-2 in antigen presenting cells In situ hybridisation of SKAT-2 riboprobes to cultures of naive T cells/AP Cs in the presence of antigen under conditions which will drive either Thl or Th 2 differentiation were carried out No hybridisation was observed in any cell type unrder Thi-biasing conditions, whereas in the Th 2 cultures, a distinct signal corresponding to SKAT-2 m RNA was detected in the AP Cs within 24 hours of the initiation of the differentiation process This signal occurred in those AP Cs which had T cells closely associated with them which may imply a relationship between expression of SKAT-2 in AP Cs and the process of T cell stimulation We have confirmed the presence of SKAT-2 m RNA in these dendritic cells by Taqman PCR using SKAT-2 specific primers (see Figure 4).

Binding of SKA T-2 with 7 A 1 In order to identify potential binding partners for SKAT-2, we have carried out yeast two-hybrid screening assays using various domains from SKAT-2 as bait.

One particular clone was identified ( 7 A 1) using the zinc finger region of SKAT-2 as bait This clone has not been identified as a binding partner in any of the other two- hybrid screens carried out using this library and thus it is unlikely to represent a promiscuous binding protein Furthermore the specificity checks carried out for binding to 7 A 1 using 7 different non-SKAT-2 baits confirms the specificity of the SKAT-2/7 A 1 interaction (see Table).

P-Gal Activity in Yeast BAIT PREY (chemiluminescence units) SKAT-2 (ZF) 7 A 1 44,000 No 1 7 A 1 12 No.2 7 A 1 16 No.3 7 A 1 28 No.4 7 A 1 16 No 5 7 A 1 32 No.6 7 A 1 12 No.7 7 A 1 28 Clone 7 A 1 was sequenced and found to correspond to the 3 ' end of a c DNA encoding BPTF (Bromodomain/PHD finger Transcription Factor) This clone in turn is almost identical at the DNA level with a shorter c DNA clone FAC 1 which is also present in public databases Both BPTF and FAC 1 apparently share the same initiation methionine and the entire FAC 1 sequence is contained within the N terminal part of BPTF BPTF however contains an extra 1971 amino acids C terminal to this and so FAC 1 may be a partial clone or expressed pseudogene Since SKAT-2 appears to bind to 7 A 1 (BPTF) in the two-hybrid assay, then it may well interact with FAC 1/BPTF in vivo Thus SKAT-2 may play some functional role in the regulation of cellular processes involved with the progression of Alzheimers disease with which FAC 1 is associated.

28 SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > PROTEIN < 130 > P 78720 GCW SER < 140 > < 141 > < 160 > 5 < 170 > Patent In Ver 2 1 < 210 > 1 < 211 > 2286 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > ( 1) ( 2286) < 400 > 1 atg tta gcc tca agc aag agg atg aac agt tct tca cgt tct caa atc 48 Met Leu Ala Ser Ser Lys Arg Met Asn Ser Ser Ser Arg Ser Gin Ile 1 5 10 15 ctt cta agg tgg aag tca gac aag gct cag agt gga ccc tac aat gtt 96 Leu Leu Arg Trp Lys Ser Asp Lys Ala Gin Ser Gly Pro Tyr Asn Val 25 30 gag aag gaa atc ctt act tca aga ttc ttg cgt gac act gag acc tgt 144 Glu Lys Glu Ile Leu Thr Ser Arg Phe Leu Arg Asp Thr Glu Thr Cys 40 45 cga cag aat ttt agg aat ttt cca tac cca gac ctg gct ggt ctt cga 192 Arg Gin Asn Phe Arg Asn Phe Pro Tyr Pro Asp Leu Ala Gly Leu Arg 55 60 aag gca ttg agt caa ctc cga gag ctc tgc ctt aag tgg ctg aga cct 240 Lys Ala Leu Ser Gin Leu Arg Glu Leu Cys Leu Lys Trp Leu Arg Pro 65 70 75 80 gag att cac tca aag gaa caa att ttg gag ctg ctg gtg ctg gag caa 288 Glu Ile His Ser Lys Glu Gin Ile Leu Glu Leu Leu Val Leu Glu Gln 90 95 ttc ctg acc atc ctg cct ggt gag gtt agg act tgg gta aag tcc cag 336 Phe Leu Thr Ile Leu Pro Gly Glu Val Arg Thr Trp Val Lys Ser Gin 105 110 tat cca gag agc agc gag gaa gca gtg act ctg gtg gag gat ttg act 384 Tyr Pro Glu Ser Ser Glu Glu Ala Val Thr Leu Val Glu Asp Leu Thr 120 125 cag att cta gaa gag gaa gct cct caa aac tct acc ctt tcc caa gat 432 Gin Ile Leu Glu Glu Glu Ala Pro Gin Asn Ser Thr Leu Ser Gin Asp 135 140 acc cca gag gaa gac ccc aga gga aaa cat gct ttc cag aca gga tgg 480 Thr Pro Glu Glu Asp Pro Arg Gly Lys His Ala Phe Gin Thr Gly Trp 150 155 160 29 cta aat gac ttg gtg acc aaa gaa tcg atg aca ttc aaa gat gtg gct 528 Leu Asn Asp Leu Val Thr Lys Glu Ser Met Thr Phe Lys Asp Val Ala 170 175 gta gac atc acc cag gag gac tgg gag tta atg cgt cct gtg cag aag 576 Val Asp Ile Thr Gin Glu Asp Trp Glu Leu Met Arg Pro Val Gin Lys 185 190 gaa tta tac aag act gtg acg tta cag aac tat tgg aac atg gtt tct 624 Glu Leu Tyr Lys Thr Val Thr Leu Gin Asn Tyr Trp Asn Met Val Ser 200 205 ctg gga ctt aca gtg tac aga cca act gtg att ccc ata ttg gaa gaa 672 Leu Gly Leu Thr Val Tyr Arg Pro Thr Val Ile Pro Ile Leu Glu Glu 210 215 220 cca tgg atg gtg ata aaa gaa att tta gaa ggc cct agt cca gaa tgg 720 Pro Trp Met Val Ile Lys Glu Ile Leu Glu Gly Pro Ser Pro Glu Trp 225 230 235 240 gaa act aaa gcc caa gca tgt act cca gtg gag gat atg tct aaa ctc 768 Glu Thr Lys Ala Gin Ala Cys Thr Pro Val Glu Asp Met Ser Lys Leu 245 250 255 aca aag gaa gaa acc cat acc atc aaa tta gaa gac tca tat gac tac 816 Thr Lys Glu Glu Thr His Thr Ile Lys Leu Glu Asp Ser Tyr Asp Tyr 260 265 270 gat gat aga cta gag agg cga gga aaa ggt ggc ttc tgg aaa att cac 864 Asp Asp Arg Leu Glu Arg Arg Gly Lys Gly Gly Phe Trp Lys Ile His 275 280 285 act gat gaa aga ggt ttc agt ttg aag tca gtc ctt tca caa gaa tat 912 Thr Asp Glu Arg Gly Phe Ser Leu Lys Ser Val Leu Ser Gin Glu Tyr 290 295 300 gat cct aca gaa gaa tgt ctt agt aaa tat gat ata tat aga aat aat 960 Asp Pro Thr Glu Glu Cys Leu Ser Lys Tyr Asp Ile Tyr Arg Asn Asn 305 310 315 320 ttt gaa aag cat tca aac cta att gta cag ttt gat acc caa tta gat 1008 Phe Glu Lys His Ser Asn Leu Ile Val Gin Phe Asp Thr Gin Leu Asp 325 330 335 aat aaa act tct gtg tat aat gaa ggc agg gca acc ttc aat cat gtc 1056 Asn Lys Thr Ser Val Tyr Asn Glu Gly Arg Ala Thr Phe Asn His Val 340 345 350 tca tat ggt att gta cat agg aaa ata ctt cct gga gag aag cct tac 1104 Ser Tyr Gly Ile Val His Arg Lys Ile Leu Pro Gly Glu Lys Pro Tyr 355 360 365 aag tgt aat gtg tgt ggg aaa aaa ttt agg aaa tac cca tcc ctc ctg 1152 Lys Cys Asn Val Cys Gly Lys Lys Phe Arg Lys Tyr Pro Ser Leu Leu 370 375 380 aaa cac caa agt acc cat gcc aaa gag aaa tcg tat gaa tgt gaa gaa 1200 Lys His Gin Ser Thr His Ala Lys Glu Lys Ser Tyr Glu Cys Glu Glu 385 390 395 400 tgt ggg aaa gag ttt agg cat atc tca tcc ctt att gca cat cag aga 1248 Cys Gly Lys Glu Phe Arg His Ile Ser Ser Leu Ile Ala His Gin Arg 405 410 415 atg cac act gga gaa aaa cca tat gaa tgc cac cag tgt ggt aaa gcc 1296 Met His Thr Gly Glu Lys Pro Tyr Glu Cys His Gin Cys Gly Lys Ala 420 425 430 ttc agc cag cgt gca cac ctt act ata cat cag aga att cat act gga 1344 Phe Ser Gin Arg Ala His Leu Thr Ile His Gin Arg Ile His Thr Gly 435 440 445 gag aaa ccc tat aag tgt gat gac tgt ggg aaa gac ttc agt cag cgt 1392 Glu Lys Pro Tyr Lys Cys Asp Asp Cys Gly Lys Asp Phe Ser Gin Arg 450 455 460 gca cac ctt acc atc cat caa agg aca cat act gga gag aaa cca tat 1440 Ala His Leu Thr Ile His Gin Arg Thr His Thr Gly Glu Lys Pro Tyr 465 470 475 480 aaa tgc ttg gaa tgt ggt aaa acc ttc agt cat agt tca tca ctg att 1488 Lys Cys Leu Glu Cys Gly Lys Thr Phe Ser His Ser Ser Ser Leu Ile 485 490 495 aat cat cag aga gtt cat act gga gaa aaa cct tat ata tgc aat gaa 1536 Asn His Gin Arg Val His Thr Gly Glu Lys Pro Tyr Ile Cys Asn Glu 500 505 510 tgt ggg aag act ttc agt cag agt aca cac ctt ctt cag cat caa aaa 1584 Cys Gly Lys Thr Phe Ser Gin Ser Thr His Leu Leu Gin His Gin Lys 515 520 525 ata cat act ggg aag aaa cca tat aaa tgc aat gaa tgt tgg aaa gtg 1632 Ile His Thr Gly Lys Lys Pro Tyr Lys Cys Asn Glu Cys Trp Lys Val 530 535 540 ttt agt cag agt act tac ctt att cga cat cag aga att cat tct gga 1680 Phe Ser Gin Ser Thr Tyr Leu Ile Arg His Gin Arg Ile His Ser Gly 545 550 555 560 gag aag tgt tat aaa tgt aat gaa tgt gga aaa gcc ttt gct cat tcc 1728 Glu Lys Cys Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ala His Ser 565 570 575 tca acc ctt att caa cat caa acc act cac act gga gag aaa tcc tat 1776 Ser Thr Leu Ile Gin His Gin Thr Thr His Thr Gly Glu Lys Ser Tyr 580 585 590 ata tgt aat ata tgt ggg aaa gcc ttc agc cag agt gca aat ctt act 1824 Ile Cys Asn Ile Cys Gly Lys Ala Phe Ser Gin Ser Ala Asn Leu Thr 595 600 605 caa cat cat aga aca cat act gga gag aaa cca tat aaa tgc agt gtg 1872 Gin His His Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Ser Val 610 615 620 tgt ggg aaa gca ttc agc cag agt gtg cac ctt act caa cat cag agg 1920 Cys Gly Lys Ala Phe Ser Gin Ser Val His Leu Thr Gin His Gin Arg 625 630 635 640 att cat aat gga gaa aaa ccc ttt aaa tgc aat ata tgt ggg aaa gca 1968 Ile His Asn Gly Glu Lys Pro Phe Lys Cys Asn Ile Cys Gly Lys Ala 645 650 655 tat aga caa ggc gca aat'ctt act cag cat caa agg att cat act gga 2016 Tyr Arg Gin Gly Ala Asn Leu Thr Gin His Gin Arg Ile His Thr Gly 660 665 670 gaa aaa ccc tat aaa tgt aat gaa tgt ggg aaa gct ttc att tat tcc 2064 Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ile Tyr Ser 675 680 685 tca tca ctt aat caa cat cag aga act cat act gga gag aga ccc tat 2112 Ser Ser Leu Asn Gin His Gin Arg Thr His Thr Gly Glu Arg Pro Tyr 690 695 700 aaa tgt aat gaa tgt gat aag gat ttt agc cag aga aca tgc ctt att 2160 Lys Cys Asn Glu Cys Asp Lys Asp Phe Ser Gin Arg Thr Cys Leu Ile 705 710 715 720 caa cac cag aga att cac aca gga gag aaa ccc tat gca tgt cgt ata 2208 Gin His Gin Arg Ile His Thr Gly Glu Lys Pro Tyr Ala Cys Arg Ile 725 730 735 tgt ggt aaa acc ttc acc cag agt aca aac ctt att cag cat caa cgt 2256 Cys Gly Lys Thr Phe Thr Gin Ser Thr Asn Leu Ile Gin His Gin Arg 740 745 750 gtt cat aca ggt gcc aaa cat cgt aat taa 2286 Val His Thr Gly Ala Lys His Arg Asn 755 760 < 210 > 2 < 211 > 761 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Leu Ala Ser Ser Lys Arg Met Asn Ser Ser Ser Arg Ser Gin Ile 1 5 10 15 Leu Leu Arg Trp Lys Ser Asp Lys Ala Gin Ser Gly Pro Tyr Asn Val 25 30 Glu Lys Glu Ile Leu Thr Ser Arg Phe Leu Arg Asp Thr Glu Thr Cys 40 45 Arg Gin Asn Phe Arg Asn Phe Pro Tyr Pro Asp Leu Ala Gly Leu Arg 55 60 Lys Ala Leu Ser Gin Leu Arg Glu Leu Cys Leu Lys Trp Leu Arg Pro 70 75 80 Glu Ile His Ser Lys Glu Gin Ile Leu Glu Leu Leu Val Leu Glu Gin 85 90 95 Phe Leu Thr Ile Leu Pro Gly Glu Val Arg Thr Trp Val Lys Ser Gin 105 110 Tyr Pro Glu Ser Ser Glu Glu Ala Val Thr Leu Val Glu Asp Leu Thr 120 125 Gin Ile Leu Glu Glu Glu Ala Pro Gin Asn Ser Thr Leu Ser Gin Asp 135 140 Thr Pro Glu Glu Asp Pro Arg Gly Lys His Ala Phe Gin Thr Gly Trp 150 155 160 Leu Asn Asp Leu Val Thr Lys Glu Ser Met Thr Phe Lys Asp Val Ala 165 170 175 Val Asp Ile Thr Gin Glu Asp Trp Glu Leu Met Arg Pro Val Gin Lys 185 190 Glu Leu Tyr Lys Thr Val Thr Leu Gin Asn Tyr Trp Asn Met Val Ser 200 205 Leu Gly Leu Thr Val Tyr Arg Pro Thr Val Ile Pro Ile Leu Glu Glu 210 215 220 Pro Trp Met Val Ile Lys Glu Ile Leu Glu Gly Pro Ser Pro Glu Trp 225 230 235 240 Glu Thr Lys Ala Gin Ala Cys Thr Pro Val Glu Asp Met Ser Lys Leu 245 250 255 Thr Lys Glu Glu Thr His Thr Ile Lys Leu Glu Asp Ser Tyr Asp Tyr 260 265 270 Asp Asp Arg Leu Glu Arg Arg Gly Lys Gly Gly Phe Trp Lys Ile His 275 280 285 Thr Asp Glu Arg Gly Phe Ser Leu Lys Ser Val Leu Ser Gin Glu Tyr 290 295 300 Asp Pro Thr Glu Glu Cys Leu Ser Lys Tyr Asp Ile Tyr Arg Asn Asn 305 310 315 320 Phe Glu Lys His Ser Asn Leu Ile Val Gin Phe Asp Thr Gin Leu Asp 325 330 335 Asn Lys Thr Ser Val Tyr Asn Glu Gly Arg Ala Thr Phe Asn His Val 340 345 350 Ser Tyr Gly Ile Val His Arg Lys Ile Leu Pro Gly Glu Lys Pro Tyr 355 360 365 Lys Cys Asn Val Cys Gly Lys Lys Phe Arg Lys Tyr Pro Ser Leu Leu 370 375 380 32 Lys His Gin Ser Thr His Ala Lys Glu Lys Ser Tyr Glu Cys Glu Glu 385 390 395 400 Cys Gly Lys Glu Phe Arg His Ile Ser Ser Leu Ile Ala His Gin Arg 405 410 415 Met His Thr Gly Glu Lys Pro Tyr Glu Cys His Gin Cys Gly Lys Ala 420 425 430 Phe Ser Gin Arg Ala His Leu Thr Ile His Gin Arg Ile His Thr Gly 435 440 445 Glu Lys Pro Tyr Lys Cys Asp Asp Cys Gly Lys Asp Phe Ser Gin Arg 450 455 460 Ala His Leu Thr Ile His Gin Arg Thr His Thr Gly Glu Lys Pro Tyr 465 470 475 480 Lys Cys Leu Glu Cys Gly Lys Thr Phe Ser His Ser Ser Ser Leu Ile 485 490 495 Asn His Gin Arg Val His Thr Gly Glu Lys Pro Tyr Ile Cys Asn Glu 500 505 510 Cys Gly Lys Thr Phe Ser Gin Ser Thr His Leu Leu Gin His Gin Lys 515 520 525 Ile His Thr Gly Lys Lys Pro Tyr Lys Cys Asn Glu Cys Trp Lys Val 530 535 540 Phe Ser Gin Ser Thr Tyr Leu Ile Arg His Gin Arg Ile His Ser Gly 545 550 555 560 Glu Lys Cys Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ala His Ser 565 570 575 Ser Thr Leu Ile Gin His Gin Thr Thr His Thr Gly Glu Lys Ser Tyr 580 585 590 Ile Cys Asn Ile Cys Gly Lys Ala Phe Ser Gin Ser Ala Asn Leu Thr 595 600 605 Gin His His Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Ser Val 610 615 620 Cys Gly Lys Ala Phe Ser Gin Ser Val His Leu Thr Gin His Gin Arg 625 630 635 640 Ile His Asn Gly Glu Lys Pro Phe Lys Cys Asn Ile Cys Gly Lys Ala 645 650 655 Tyr Arg Gin Gly Ala Asn Leu Thr Gin His Gin Arg Ile His Thr Gly 660 665 670 Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ile Tyr Ser 675 680 685 Ser Ser Leu Asn Gin His Gin Arg Thr His Thr Gly Glu Arg Pro Tyr 690 695 700 Lys Cys Asn Glu Cys Asp Lys Asp Phe Ser Gin Arg Thr Cys Leu Ile 705 710 715 720 Gin His Gin Arg Ile His Thr Gly Glu Lys Pro Tyr Ala Cys Arg Ile 725 730 735 Cys Gly Lys Thr Phe Thr Gin Ser Thr Asn Leu Ile Gin His Gin Arg 740 745 750 Val His Thr Gly Ala Lys His Arg Asn 755 760 < 210 > 3 < 211 > 2280 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > ( 1) ( 2280) < 400 > 3 atg gca aat tct tca ctt tcc caa gtc ctt cta atg tgg aag cca ggc 48 Met Ala Asn Ser Ser Leu Ser Gin Val Leu Leu Met Trp Lys Pro Gly 1 5 10 15 aag atc cag aag gga ccc tgc agt gct gag cag cgg aca ctc acc tca 96 Lys Ile Gin Lys Gly Pro Cys Ser Ala Glu Gin Arg Thr Leu Thr Ser 25 30 aga ctc ttg cgt gac act gaa acc tgt cga cgg aat ttt aga aat ttc 144 Arg Leu Leu Arg Asp Thr Glu Thr Cys Arg Arg Asn Phe Arg Asn Phe 40 45 cca tac ccg gat gtg gca ggc cct cgg aaa gcg ttg tgt cag ctc cga 192 Pro Tyr Pro Asp Val Ala Gly Pro Arg Lys Ala Leu Cys Gin Leu Arg 55 60 gag ctc tgc ctg aag tgg ctg aga cct gag gtt cat tcc aag gaa caa 240 Glu Leu Cys Leu Lys Trp Leu Arg Pro Glu Val His Ser Lys Glu Gin 70 75 80 att ctg gag ttg ctg gtg ctg gag caa ttt ctg agc atc ttg cct ggg 288 Ile Leu Glu Leu Leu Val Leu Glu Gin Phe Leu Ser Ile Leu Pro Gly 85 90 95 gag gtt agg act tgg gta aat tct cag tac cca gag agc agc gaa gaa 336 Glu Val Arg Thr Trp Val Asn Ser Gin Tyr Pro Glu Ser Ser Glu Glu 105 110 gtg gtg gct ttg gtg gag gat ttg act cag atc cta gaa gaa gaa gaa 384 Val Val Ala Leu Val Glu Asp Leu Thr Gin Ile Leu Glu Glu Glu Glu 120 125 gct cct cag agt tct gcc ctc ccc caa gac acc cca gag gat gac ccc 432 Ala Pro Gin Ser Ser Ala Leu Pro Gin Asp Thr Pro Glu Asp Asp Pro 135 140 aac cat gac ccc aac cct gct tcc cag gca ggg tgg ctc agt gac gtg 480 Asn His Asp Pro Asn Pro Ala Ser Gin Ala Gly Trp Leu Ser Asp Val 150 155 160 gtg acc aaa gac ttg gtg aca ttc aat gac gtg gct gtg gac atc acc 528 Val Thr Lys Asp Leu Val Thr Phe Asn Asp Val Ala Val Asp Ile Thr 165 170 175 caa gaa gac tgg gaa ctg atg ccc cct gtt cag aag gaa ttg tat aag 576 Gin Glu Asp Trp Glu Leu Met Pro Pro Val Gin Lys Glu Leu Tyr Lys 185 190 act gtg act tta cag aac tat tgg aac atg gtt tct cta gga ctg act 624 Thr Val Thr Leu Gin Asn Tyr Trp Asn Met Val Ser Leu Gly Leu Thr 200 205 gtg tac agg cca act gtg att ccc gtc tta gaa gag ccg tgg atg gtg 672 Val Tyr Arg Pro Thr Val Ile Pro Val Leu Glu Glu Pro Trp Met Val 210 215 220 ata aaa gaa att gta gaa ggc cct aat cca gga tgg gaa cct aaa gct 720 Ile Lys Glu Ile Val Glu Gly Pro Asn Pro Gly Trp Glu Pro Lys Ala 225 230 235 240 cag gca cag tgc cca gca aag cac ctc cct gaa ctc aag cag gac gga 768 Gln Ala Gin Cys Pro Ala Lys His Leu Pro Glu Leu Lys Gin Asp Gly 245 250 255 acc caa acc gta aaa ctg gaa gat tcc tat gac gac gac aac gat gac 816 Thr Gin Thr Val Lys Leu Glu Asp Ser Tyr Asp Asp Asp Asn Asp Asp 260 265 270 agc gta gag agt ccg cca gtg tgt gcc ttc ggg atg atc cac ata gat 864 Ser Val Glu Ser Pro Pro Val Cys Ala Phe Gly Met Ile His Ile Asp 275 280 285 gag gaa ggc ttc agt gtg aag tca gag ctt tca caa gaa gac cct aca 912 34 Glu Glu Gly Phe Ser Val Lys Ser Glu Leu Ser Gin Glu Asp Pro Thr 290 295 300 gaa gaa tac ctt agc aaa tgt gac ata tat aga gtg act ttt gaa aag 960 Glu Glu Tyr Leu Ser Lys Cys Asp Ile Tyr Arg Val Thr Phe Glu Lys 305 310 315 320 cac aca aac cta ggg gtc cag ttt gat acc cag tcg gat gat aaa act 1008 His Thr Asn Leu Gly Val Gin Phe Asp Thr Gin Ser Asp Asp Lys Thr 325 330 335 gct ctg cat aat gaa agc aag cca ccg ttt agc aat gcc tcg tct ggt 1056 Ala Leu His Asn Glu Ser Lys Pro Pro Phe Ser Asn Ala Ser Ser Gly 340 345 350 ggt gcc gtg cgt ggg aaa ata ctt cct gga gat aag cct tat tcc tgt 1104 Gly Ala Val Arg Gly Lys Ile Leu Pro Gly Asp Lys Pro Tyr Ser Cys 355 360 365 aat gtc tgt ggg aaa cag ttt aga aag tac cct tcc ctc ctg gcg cac 1152 Asn Val Cys Gly Lys Gin Phe Arg Lys Tyr Pro Ser Leu Leu Ala His 370 375 380 cga gag aac cac gcc aaa gag aaa gct tat gag tgt gaa gaa tgc ggc 1200 Arg Glu Asn His Ala Lys Glu Lys Ala Tyr Glu Cys Glu Glu Cys Gly 385 390 395 400 aaa gaa ttt aag cat ctc tcc tcc ctc atc gca cat cag aga atg cac 1248 Lys Glu Phe Lys His Leu Ser Ser Leu Ile Ala His Gin Arg Met His 405 410 415 acc gga gaa aaa ccg tac gaa tgc cac cag tgt ggg aaa gcc ttc agt 1296 Thr Gly Glu Lys Pro Tyr Glu Cys His Gin Cys Gly Lys Ala Phe Ser 420 425 430 cag cga gca cac ctg act ata cac cag aga att cac act gga gag aaa 1344 Gln Arg Ala His Leu Thr Ile His Gin Arg Ile His Thr Gly Glu Lys 435 440 445 ccc tac aaa tgt gag gac tgt ggg aaa gac ttc agt cag cgc gcg cac 1392 Pro Tyr Lys Cys Glu Asp Cys Gly Lys Asp Phe Ser Gin Arg Ala His 450 455 460 ctc acc atc cat caa agg aca cac act ggg gag aag ccg tat aaa tgt 1440 Leu Thr Ile His Gin Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys 465 470 475 '480 ctg gag tgc agt aaa acc ttt agc cat agt tca tca ctg att aat cat 1488 Leu Glu Cys Ser Lys Thr Phe Ser His Ser Ser Ser Leu Ile Asn His 485 490 495 cag aga gtt cat act gga gaa aaa cct tat ata tgc aac gaa tgt ggg 1536 Gin Arg Val His Thr Gly Glu Lys Pro Tyr Ile Cys Asn Glu Cys Gly 500 505 510 aag act ttc agt cag agc acg cac ctt ctc cag cat cag aaa atc cac 1584 Lys Thr Phe Ser Gin Ser Thr His Leu Leu Gin His Gin Lys Ile His 515 520 525 act ggg aaa aag ccg tac aag tgc aac gag tgt tgg aaa gtg ttc agc 1632 Thr Gly Lys Lys Pro Tyr Lys Cys Asn Glu Cys Trp Lys Val Phe Ser 530 535 540 cag agc act tac ctc atc cga cac cag aga atc cat tct gga gag aag 1680 Gln Ser Thr Tyr Leu Ile Arg His Gin Arg Ile His Ser Gly Glu Lys 545 550 555 560 tgc tac aaa tgc act gcc tgt ggg aag gcc ttt gcc cac tcc tca act 1728 Cys Tyr Lys Cys Thr Ala Cys Gly Lys Ala Phe Ala His Ser Ser Thr 565 570 575 ctc att caa cat cag acc acc cac acc gga gag aag tcc tat ata tgc 1776 Leu Ile Gin His Gin Thr Thr His Thr Gly Glu Lys Ser Tyr Ile Cys 580 585 590 aac gtg tgt ggg aaa gcc ttc agc caa agc gcc aac ctt acc cag cat 1824 Asn Val Cys Gly Lys Ala Phe Ser Gin Ser Ala Asn Leu Thr Gin His 595 600 605 cat aga aca cac act gga gag aaa ccg tac aaa tgt agt gtg tgt gga 1872 His Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Ser Val Cys Gly 610 615 620 aaa gcc ttc agc cag agt gtg cac ctc acc cag cac cag agg att cac 1920 Lys Ala Phe Ser Gin Ser Val His Leu Thr Gln His Gln Arg Ile His 625 630 635 640 aat gga gaa aag ccc ttt aag tgt aat acc tgt ggg aaa gct tac aga 1968 Asn Gly Glu Lys Pro Phe Lys Cys Asn Thr Cys Gly Lys Ala Tyr Arg 645 650 655 cag ggt gca aac ctg act cag cac caa cgg gtc cac act gga gag agg 2016 Gin Gly Ala Asn Leu Thr Gin His Gin Arg Val His Thr Gly Glu Arg 660 665 670 ccc tac aaa tgt cac cac tgc ggg aaa gcc ttt atc tac tct tca tcc 2064 Pro Tyr Lys Cys His His Cys Gly Lys Ala Phe Ile Tyr Ser Ser Ser 675 680 685 ctt aac caa cat cga aga act cac act gga gag cga ccc tat aag tgt 2112 Leu Asn Gin His Arg Arg Thr His Thr Gly Glu Arg Pro Tyr Lys Cys 690 695 700 agt cac tgt aac aaa gat ttt agc cag aga aca tgc ctt att caa cac 2160 Ser His Cys Asn Lys Asp Phe Ser Gin Arg Thr Cys Leu Ile Gin His 705 710 715 720 cag agg att cac aca gga gaa aag ccc tac gga tgc cgt ata tgt gag 2208 Gin Arg Ile His Thr Gly Glu Lys Pro Tyr Gly Cys Arg Ile Cys Glu 725 730 735 aaa gcc ttc acc cag agt acc aat ctt att cag cat cag cgg gtt cat 2256 Lys Ala Phe Thr Gin Ser Thr Asn Leu Ile Gin His Gin Arg Val His 740 745 750 acg ggt gcc aga cat cgc aat tga 2280 Thr Gly Ala Arg His Arg Asn 755 760 < 210 > 4 < 211 > 759 < 212 > PRT < 213 > Mus musculus < 400 > 4 Met Ala Asn Ser Ser Leu Ser Gin Val Leu Leu Met Trp Lys Pro Gly 1 5 10 15 Lys Ile Gin Lys Gly Pro Cys Ser Ala Glu Gln Arg Thr Leu Thr Ser 25 30 Arg Leu Leu Arg Asp Thr Glu Thr Cys Arg Arg Asn Phe Arg Asn Phe 35 40 45 Pro Tyr Pro Asp Val Ala Gly Pro Arg Lys Ala Leu Cys Gin Leu Arg 55 60 Glu Leu Cys Leu Lys Trp Leu Arg Pro Glu Val His Ser Lys Glu Gin 70 75 80 Ile Leu Glu Leu Leu Val Leu Glu Gin Phe Leu Ser Ile Leu Pro Gly 90 95 Glu Val Arg Thr Trp Val Asn Ser Gin Tyr Pro Glu Ser Ser Glu Glu 105 110 Val Val Ala Leu Val Glu Asp Leu Thr Gin Ile Leu Glu Glu Glu Glu 115 120 125 Ala Pro Gin Ser Ser Ala Leu Pro Gin Asp Thr Pro Glu Asp Asp Pro 135 140 Asn His Asp Pro Asn Pro Ala Ser Gin Ala Gly Trp Leu Ser Asp Val 150 155 160 Val Thr Lys Asp Leu Val Thr Phe Asn Asp Val Ala Val Asp Ile Thr 165 170 175 Gin Glu Asp Trp Glu Leu Met Pro Pro Val Gln Lys Glu Leu Tyr Lys 185 190 Thr Val Thr Leu Gin Asn Tyr Trp Asn Met Val Ser Leu Gly Leu Thr 200 205 Val Tyr Arg Pro Thr Val Ile Pro Val Leu Glu Glu Pro Trp Met Val 210 215 220 Ile Lys Glu Ile Val Glu Gly Pro Asn Pro Gly Trp Glu Pro Lys Ala 225 230 235 240 Gln Ala Gin Cys Pro Ala Lys His Leu Pro Glu Leu Lys Gin Asp Gly 245 250 255 Thr Gin Thr Val Lys Leu Glu Asp Ser Tyr Asp Asp Asp Asn Asp Asp 260 265 270 Ser Val Glu Ser Pro Pro Val Cys Ala Phe Gly Met Ile His Ile Asp 275 280 285 Glu Glu Gly Phe Ser Val Lys Ser Glu Leu Ser Gin Glu Asp Pro Thr 290 295 300 Glu Glu Tyr Leu Ser Lys Cys Asp Ile Tyr Arg Val Thr Phe Glu Lys 305 310 315 320 His Thr Asn Leu Gly Val Gin Phe Asp Thr Gin Ser Asp Asp Lys Thr 325 330 335 Ala Leu His Asn Glu Ser Lys Pro Pro Phe Ser Asn Ala Ser Ser Gly 340 345 350 Gly Ala Val Arg Gly Lys Ile Leu Pro Gly Asp Lys Pro Tyr Ser Cys 355 360 365 Asn Val Cys Gly Lys Gin Phe Arg Lys Tyr Pro Ser Leu Leu Ala His 370 375 380 Arg Glu Asn His Ala Lys Glu Lys Ala Tyr Glu Cys Glu Glu Cys Gly 385 390 395 400 Lys Glu Phe Lys His Leu Ser Ser Leu Ile Ala His Gin Arg Met His 405 410 415 Thr Gly Glu Lys Pro Tyr Glu Cys His Gin Cys Gly Lys Ala Phe Ser 420 425 430 Gin Arg Ala His Leu Thr Ile His Gin Arg Ile His Thr Gly Glu Lys 435 440 445 Pro Tyr Lys Cys Glu Asp Cys Gly Lys Asp Phe Ser Gin Arg Ala His 450 455 460 Leu Thr Ile His Gin Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys 465 470 475 480 Leu Glu Cys Ser Lys Thr Phe Ser His Ser Ser Ser Leu Ile Asn His 485 490 495 Gin Arg Val His Thr Gly Glu Lys Pro Tyr Ile Cys Asn Glu Cys Gly 500 505 510 Lys Thr Phe Ser Gin Ser Thr His Leu Leu Gin His Gin Lys Ile His 515 520 525 Thr Gly Lys Lys Pro Tyr Lys Cys Asn Glu Cys Trp Lys Val Phe Ser 530 535 540 Gin Ser Thr Tyr Leu Ile Arg His Gin Arg Ile His Ser Gly Glu Lys 545 550 555 560 Cys Tyr Lys Cys Thr Ala Cys Gly Lys Ala Phe Ala His Ser Ser Thr 565 570 575 Leu Ile Gin His Gin Thr Thr His Thr Gly Glu Lys Ser Tyr Ile Cys 580 585 590 37 Asn Val Cys Gly Lys Ala Phe Ser Gin Ser Ala Asn Leu Thr Gin His 595 600 605 His Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Ser Val Cys Gly 610 615 620 Lys Ala Phe Ser Gin Ser Val His Leu Thr Gin His Gin Arg Ile His 625 630 635 640 Asn Gly Glu Lys Pro Phe Lys Cys Asn Thr Cys Gly Lys Ala Tyr Arg 645 650 655 Gln Gly Ala Asn Leu Thr Gin His Gin Arg Val His Thr Gly Glu Arg 660 665 670 Pro Tyr Lys Cys His His Cys Gly Lys Ala Phe Ile Tyr Ser Ser Ser 675 680 685 Leu Asn Gin His Arg Arg Thr His Thr Gly Glu Arg Pro Tyr Lys Cys 690 695 700 Ser His Cys Asn Lys Asp Phe Ser Gin Arg Thr Cys Leu Ile Gin His 705 710 715 720 Gin Arg Ile His Thr Gly Glu Lys Pro Tyr Gly Cys Arg Ile Cys Glu 725 730 735 Lys Ala Phe Thr Gin Ser Thr Asn Leu Ile Gin His Gin Arg Val His 740 745 750 Thr Gly Ala Arg His Arg Asn 755 < 210 > 5 < 211 > 735 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > ( 1) ( 447) < 400 > 51 ggc atc ttg caa agt gag gca gag ctc att gat gag tat gtc tgt cca 48 Gly Ile Leu Gin Ser Glu Ala Glu Leu Ile Asp Glu Tyr Val Cys Pro 1 5 10 15 cag tgc cag tca aca gag gat gcc atg aca gtg ctc acg cca cta aca 96 Gln Cys Gin Ser Thr Glu Asp Ala Met Thr Val Leu Thr Pro Leu Thr 25 30 gag aag gat tat gag ggg ttg aag agg gtg ctc cgt tcc tta cag gcc 144 Glu Lys Asp Tyr Glu Gly Leu Lys Arg Val Leu Arg Ser Leu Gin Ala 40 45 cat aag atg gcc tgg cct ttc ctt gaa cca gta gac cct aat gat gca 192 His Lys Met Ala Trp Pro Phe Leu Glu Pro Val Asp Pro Asn Asp Ala 50 55 60 cca gat tat tat ggt gtt att aag gaa cct atg gac ctt gcc acc atg 240 Pro Asp Tyr Tyr Gly Val Ile Lys Glu Pro Met Asp Leu Ala Thr Met 70 75 80 gaa gaa aga gta caa aga cga tat tat gaa aag ctg acg gaa ttt gtg 288 Glu Glu Arg Val Gin Arg Arg Tyr Tyr Glu Lys Leu Thr Glu Phe Val 90 95 gca gat atg acc aaa att ttt gat aac tgt cgt tac tac aat cca agt 336 Ala Asp Met Thr Lys Ile Phe Asp Asn Cys Arg Tyr Tyr Asn Pro Ser 105 110 gac tcc cca ttt tac cag tgt gca gaa gtt ctc gaa tca ttc ttt gta 384 Asp Ser Pro Phe Tyr Gin Cys Ala Glu Val Leu Glu Ser Phe Phe Val 120 125 38 cag aaa ttg aaa ggc ttc aaa gct agc agg tct cat aac aac aaa ctg 432 Gin Lys Leu Lys Gly Phe Lys Ala Ser Arg Ser His Asn Asn Lys Leu 135 140 cag tct aca gct tct taaagttcag cgtgttaacc taacataaaa cacagcaaga 487 Gin Ser Thr Ala Ser atctggttgt ctgaactatt ttaaattaag gagccagatg tttttagtca ggctatcctg 547 acaagacttg acctaaactt cgtttttatt ggtcataaca gtccaattat attcttggcc 607 aattttgtcc aacggacaag aaaaaagcaa agtcaacgac accattatct tgtcaagatc 667 agatggtttt actattgtgg cagaagcgag aaaactttgt ttattgaaaa aaaaagaaaa 727 agaaagca 735 39

Claims (19)

1 An isolated SKAT-2 polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (iv) a variant thereof which maintains or impairs a function of SKAT-2 selected from transcription factor activity, biasing a Th 2 phenotype, inducing apoptosis or inducing cytokine production; or (v) a fragment of (i) or (ii) which maintains the same function as (i) or (ii).

2 A polypeptide according to claim 1 wherein the variant (ii) has at least io 80 % identity to the amino acid sequence of SEQ ID NO: 2.

3 A polynucleotide encoding a polypeptide according to claim 1 or 2.

4 A polynucleotide according to claim 3 which is a c DNA sequence.

A polynucleotide encoding a SKAT-2 polypeptide which is capable of at least one function selected from transcription factor activity, biasing production of Th 2 phenotype, inducing apoptosis or inducing cytokine production which polynucleotide comprises:

(a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60 % identity to a sequence as defined in (a), (b) or (c).

6 An expression vector comprising a polynucleotide sequence according to any one of claims 3 to 5, which is capable of expressing a polypeptide according to claim 1 or 2.

7 A cell line comprising an expression vector according to claim 6.

8 An antibody specific for a polypeptide according to claim 1 or 2.

9 A method for identifying a compound that modulates SKAT-2 activity, which method comprises contacting a polypeptide according to claim 1 or 2 with a test compound and monitoring for SKAT-2 activity.

A method according to claim 9 wherein the polypeptide is expressed in a cell.

11 A method according to claim 10 wherein said cell further comprises a reporter gene under the control of a promoter which is responsive to SKAT2 and the method comprises monitoring for expression of the reporter gene to thereby determine whether the test substance modulates SKAT-2 transcription factor activity.

12 A method according to claim 10 wherein the method is for identification of a compound that modulates SKAT-2 mediated apoptosis and wherein the method comprises monitoring SKAT-2 mediates apoptosis to thereby o determine whether a test compound modulates such SKAT-2 mediated apoptosis.

13 A method according to claim 10 for identification of a compound that modulates SKAT-2 induced cytokine production which method comprises monitoring for SKAT-2 cytokine production in the presence of the test substance and thereby determining whether the test substance modulates SKAT-2 mediated cytokine production.

14 A method according to claim 10 for identifying a compound which modulates SKAT-2 differentiation of T-lymphocytes to Th 2 cells wherein the cell comprises a T-lymphocyte and the method comprises monitoring for Th 2 cell differentiation in the presence of the test substance to thereby determine whether the test substance modulates Th 2 cell differentiation.

A method according to any one of claims 10 to 13 wherein the cell is transformed with an expression vector encoding a polypeptide according to claim 1 or 2 and expression of the polypeptide is induced during the course of the method.

16 A compound which modulates SKAT-2 mediated activity and which is identifiable by a method according to any one of claims 9 to 15.

17 A method of treating a subject having a disorder that is responsive to modulation of SKAT-2 mediated activity, which method comprises administering to said subject an effective amount of a compound according to claim 16, a polypeptide according to claim 1 or 2 or a polynucleotide according to claim 3, 4 or 5.

18 A method according to claim 17 wherein the condition is selected from asthma, allergic disorders such as hay fever, atopic dermatitis, allergic rhinitis etc, Alzheimer's and other neuronal disorders, conditions involving an alteration of T 41 helper response or apoptosis, and in vaccination against an antigen.

19 Use of a compound as defined in claim 16 a polypeptide according to claim 1 or 2 or a polynucleotide according to claim 3, 4 or 5 in the manufacture of a medicament for treatment or prophylaxis of a condition that is responsive to stimulation or modulation of SKAT-2 condition activity.

A use according to claim 19 wherein the condition is a disorder selected from asthma, allergic disorders such as hay fever, atopic dermatitis, allergic rhinitis etc, Alzheimers disease and other neuronal disorders, conditions involving an alteration of T helper response or apoptosis, and in vaccination against an antigen.

l O 21 A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a cell line as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.