WO2004003004A1

WO2004003004A1 - Compositions and methods involving inhibiting or enhancing the deubiquitylation of an enzyme

Info

Publication number: WO2004003004A1
Application number: PCT/US2003/016238
Authority: WO
Inventors: Shelley L. Berger; Karl W. Henry, Jr.; Steven B. Mcmahon
Original assignee: The Wistar Institute Of Anatomy And Biology
Priority date: 2002-06-27
Filing date: 2003-06-27
Publication date: 2004-01-08
Also published as: AU2003247401A1

Abstract

The human homologs of the yeast ubp8 gene and polypeptide are required for chromatin development and gene regulation of the genes in the SAGA complex, including H2B. Compounds that inhibit the expression or activity of the human Ubp8 homolog polypeptide and the nucleic acid sequences encoding it are useful in rendering a cancer cell more sensitive to additional anti-tumor therapies, in diagnosing tumorigenic cells, or in treating or diagnosing other disorders dependent upon regulation of genes requiring deubiquitylation for transcription.

Description

COMPOSITIONS AND METHODS INVOLVING INHIBITING OR ENHANCING THE DEUBIQUITYLATION ACTIVITY OF AN ENZYME

This invention has received funding from National Institutes of Health Grant No. 5 R01 GM 55360-04. The United States government has an interest in this invention.

BACKGROUND OF THE INVENTION

Gene expression is regulated at the DNA template through covalent modifications of both histones and DNA-binding transcription factors. Certain posttranslational modifications of histones within chromatin are required for the transcriptional regulation of a group/subset of inducible genes. These modifications include acetylation, methylation, phosphorylation, ADP-ribosylation, and . ubiquitylation (Bradbury, 1992 Bioessays, 14:9; Strahl and Allis, 2000 Nature 403:41). In the model yeast Saccharomyces cerevisiae, many of the complexes responsible for these modifications, as well as their antagonistic counterparts responsible for removing the modification, are known. One of the better-understood histone modifying complexes is the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex, which is responsible for the acetylation of histone H3 and, to a lesser extent, H2B, and is required for the expression of approximately 5% of yeast genes (Grant et al, 1997

Genes Dev., ii:1640-1650; Holstege et β/, 1998 Cell, 95:717-728).

One of the more poorly understood modifications is the ubiquitylation of histones. hile ubiquitylation of H2A and H2B is conserved in eukaryotes and is associated with active DNA its specific role in gene regulation and chromatin architecture is unknown (see Chen et al, 1998; J. Biol. Chem). Recent evidence indicates that ubiquitylation of Lys-123 in the C-terminus of H2B occurs in S. cerevisiae. H2B ubiquitylation is important for cell cycle progression in both mitosis and meiosis, and thus gene regulation.

The yeast Ubp8, a ubiquitin-specific protease, has been reported to associate with components of the SAGA complex (Gavin et al, 2002 Nature, 415: 141-147; Ho et al, 2002 Nature, ¥75:180-183; and Sanders et al, 2002M / Cell Biol., 22:4723- 381). The yeast Ubp8 protease sequence is reported in the NCBI database at Locus and Accession No. NP_013950 (SEQ ID NO: 2) (see, A. Goffeau et al, 1996 Science, 274:546 and S. Bowman et al, 1997 Nature, 387:90-93). A nucleic acid sequence encoding yeast Ubp8 is found in SEQ ID NO: 4.

Only a few mammalian ubiquitin proteases have been characterized to date, and have been shown to be involved in growth control and/or oncogenesis (L. M. DeSalle et al, 2001 Oncogene, 20:5538-5542). Murine ubiquitin nuclear protein (Unp) has been determined to interact with the product of the retinoblastoma gene. A human homolog, Unph (also known as hUBP8), has been mapped to a chromosomal region frequently rearranged in human tumor cells and is proposed to be involved in the neoplastic process resulting in small cell tumors and adenocarcinomas of the lung (D. A. Gray et al, 1995 Oncogene, 70:2179-2183). Other mammalian enzymes have been identified but not characterized as to function. See, e.g., the human ubiquitin specific protease 22 (USP22), published in the NCBI database at Locus/ Accession No.

XP_042698 as a 420 a ino acid linear sequence on May 13, 2002 (amino acids 106- 525 of SEQ ID NO: 1). This protein was originally identified as part of a 593 amino acid linear protein encoded by an unidentified human gene from brain in the NCBI database at Locus/Accession No. BAA83015, published on August 4, 1999 (SEQ ID NO: 3). A subsequent submission to the database on April 28, 2003 (referred to herein as bUBP8) identifies the sequence as a 525 amino acid sequence (SEQ ID NO: 1). However, to date there has been no publication describing the specific activity or substrate of hUBP8.

Errors in gene regulation and chromatin architecture during cell growth and differentiation result in dramatically altered growth properties. Histone modifications have been shown to be directly linked to gene silencing in cancer cells. Thus, the enzymes that mediate histone modifications and thereby regulate gene activity have increasing interest as targets of drug evaluation.

There remains a need in the art for the identification of additional methods and compositions useful in the diagnosis and treatment of cancer, particularly the identification of additional enzymes that monitor and control genes that function as tumor suppressors and oncogenes, as well as methods and compositions that permit the screening of drugs useful for treatment of cancer. The present invention satisfies this need.

SUMMARY OF THE INVENTION

This invention relates generally to the identification of the human homolog of a yeast ubiquitin specific protease, compositions containing same and methods of use therefor. More specifically, this invention relates to the identification of a human ubiquitin specific protease 22 (SEQ ID NO: 1) as the human homolog of the protein encoded by the yeast gene ubp8, that controls histone deubiquitylation, particularly of histone H2B. Thus, human ubiquitin specific protease 22 or "hUBP8" as referred to herein, when over-expressed, can result in the over-production of certain genes, e.g., oncogenes. When the protease is defective or the gene encoding it is defective, the reduced level of protease activity may also reduce the production of a tumor suppressor gene, resulting in cancer. Similarly, other genes regulated by hUBP8 activity can result in disease if the enzymatic activity is abnormally increased or defective activity of hUBP8 can underexpress gene products necessary for prevention of disease, e.g., cytokines, etc. Thus, hUBP8 and/or one of its homologs such as yeast UBP8, can be used as a target for screening for compounds that inhibit its enzymatic activity in circumstances in which excessive enzymatic activity is related to disease. hUBP8 or a homolog thereof can be used as a target for screening for compounds that enhance the activity of hUBP8, where reduced enzymatic activity is related to disease. This enzyme can also be used in methods for diagnosis or screening of cancers and other diseases characterized by the over-production or under-production of a gene product, the transcription of which is controlled by hUBP8. Thus, this invention provides methods for identifying compositions for use in the diagnosis, treatment and prevention of cancers and other diseases identified herein. The invention also provides methods for diagnosis of disease.

In one aspect, the invention provides an assay for identifying a composition or compound that inhibits the activity or expression of hUBP8, and/or inhibits the activity or expression of one of its homologs, such as yeast Ubp8. The method involves contacting with a suitable amount of a test compound a cell capable of expressing enymatically active hUBP8 or a homolog thereof in the presence of a substrate that can be deubiquitylated. The level of deubiquitylating enzymatic activity of hUBP8 or a homolog thereof in the cell is then assessed. This can be done by measuring the level of ubiquitylated or de-ubiquitylated substrate after contact with the test compound. The level of activity of hUBP8 or a homolog in an otherwise identical cell and substrate, which has not been contacted with the test compound, is also determined and the two levels of hUBP8 or homolog expression and activity compared. A lower level of activity of the hUBP8 or homolog in the former cell compared with the level of activity of hUBP8 or homolog in the latter cell indicates that the test compound is a hUBP8 inhibitor. UBP8 inhibitors identified by this method are also provided.

In another aspect, the invention provides an assay for detecting and identifying compositions that enhance the enzymatic activity of MJBP8, by performing the above- noted steps. An increased level of activity of the hUBP8 or homolog in the former cell compared with the level of activity of hUBP8 or homolog in the latter cell indicates that the test compound enhances hUBP8 activity.

In another aspect, the invention provides a method of retarding the growth of a cancer cell, the method comprising administering to the cell a hUBP8 inhibitor that suppresses transcription of a gene, e.g., an oncogene, regulated by hUBP8 enzymatic activity. This method may be performed ex vivo by administration to cancer cells removed from a mammal's body, or in vivo by direct administration to the mammal. Such inhibitors include antisense sequences that hybridize to the hUBP8 sequences and prevent or retard transcription thereof or RNAi. In another aspect, the invention provides a method of retarding the growth of a cancer cell, the method comprising administering to the cell a hUBP8 enhancer that enhances transcription of a gene, e.g., a tumor suppressor, regulated by hUBP8 enzymatic activity. This method may be performed ex vivo by administration to cancer cells removed from a mammal's body, or in vivo by direct administration to the mammal. In another aspect, the invention provides a method of determining tumorigenic potential of a cell comprising examining the cell for the presence of a normal or defective hUBP8 nucleic acid sequence in the cell, and/or examining the cell for the presence of an increased or decreased level of hUBP8 enzymatic activity, wherein either a reduced level of hUBP8 expression or activity in a cell bearing a tumor suppressor gene or an increased level of hUBP8 expression or activity in a cell carrying an oncogene, indicates that the cell is predisposed to tumorigenesis.

In a further aspect, the invention provides a composition for diagnosis, treatment or prevention of disease comprising a nucleotide sequence that binds to the hUBP8 nucleic acid sequence or a fragment thereof, the reagent sequence associated with a detectable label. For example, such a sequence is an antisense sequence or an RNAi.

In still another aspect, the invention provides a composition for diagnosis, treatment or prevention of disease comprising a ligand that binds to hUBP8, the ligand being optionally associated with a detectable label.

Yet another aspect of this invention is a kit for detecting the tumorigenic potential of a cell or screening for hUBP8 inhibitors or enhancers, which comprises at least one of the above-mentioned compositions, and optional suitable components for detection of a label, among other components. In another aspect, the invention provides a composition that is an inhibitor or antagonist of the biological activity of hUBP8. In one embodiment, this composition is an antisense sequence or RNAi that inhibits transcription and thus expression of the gene.

In another aspect, the invention provides a composition that is an enhancer or agonist of the biological activity of MJBP8.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 is a bar graph showing the H2B ubiquitylation is required for transcription of the Gcn5-dependent genes, SUC2, INOl and GALl. In the S. cereviseae htbl K123R strain, considerably less expression of each gene is indicated by the dark bars. FIG. 2 is a bar graph showing the ChDIP analysis of ubH2B during galactose induction in S. cereviseae wildtype HTB1+ strain. The level of immunoprecipitated chromatin from the GALl promoter (white bars) and Int. V region (black bars) is shown as relative immunoprecipitation as a ratio of immunoprecipitated material to input chromatin. FIG. 3 is a graph showing GALl transcription during galactose induction in S. cereviseae htbl-KR strain, with RNA isolated and analyzed as in FIG. 2, using inducing conditions.

FIG. 4 is a graph comparing GALl RNA accumulation and promoter- associated ubiquitylation of H2B. Data from Figs. 2 and 3, as well as data from a GALl RNA SI nuclease assay (not shown) measuring GALl transcription during galactose induction in 2% galactose for 2.5 hours at OD₆₀o_nm 0.8 in the same strains, were graphed together.

FIG. 5 is a ChDIP analysis of ubH2B at GALl promoter in ubp8Δ, as described in Example 1. The strains were wild type (white bars) or ubp8Δ (black bars).

FIG. 6 is a bar graph shown SUC2 transcription in ubp8Δ strain. SUC2 expression as determined by an SI nuclease assay was performed as described in Example 1 in wild type (white bars), htbl-KR (black bar) and ubp8Δ strains. Values are the average of four independent experiments. FIG. 7 is a bar graph showing the ChIP analysis of Gcn5 and Ubp8. Gcn5-

3HA (white bars) and Ubp8-3HA (black bars) binding in wild type strains was analysied by ChIP at the GALl promoter in glucose (0 timepoint) and in galactose (60 and 120 minute timepoints). Association was compared to Int. V (Gen5-3HA sample (white bars, black stripes) or Ubp8-3HA (black bars, white stripes). FIG. 8 is a bar graph of HAT activity of SAGA derived from wildtype or ubp8 strains. Threefold serial dilutions of equivalent amounts of wildtype (white bars) or mutant (grey bars) SAGA were assayed for HAT activity on core histones (Sigma) (39). Background (black bars) was incorporation of 3H-acetate without added SAGA complex.

FIG. 9 is an H3 3meK4 ChIP analysis of the GALl promoter in wildtype and ubp8d strains. An antibody specific for 3mek$ of histone He (Abeam) was used in ChIP at the GALl promoter. Smaples were taken from glucose (Omin) and at the indicated times after the switch to glactose. Samples were analyzed from wildtype (white bars), ubp8<d (grey bars) and htbl-KR (black bars) strains.

FIG. 10 is a model of the role of ubH2B and 3meH2B on GALl expression. Schematic represenation of the GALl promoter under poised (see dashed lines around core nucleosome), activated (see lower left free core nucleosome) and repressed (see lower right) conditions in UBP8+ and ubp8<d backgrounds.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that the human gene, referred to as Ubp22 or hUBP8 (SEQ ID NO: 1), functions to control the chromatin modification, and regulation of histone, preferably the histone H2B, which is critical for gene transcription. Thus, the hUBP8 gene and the hUBP8 protein expressed therefrom, as well as other homologs, such as yeast Ubp8, are useful in diagnostic, therapeutic and drug-screening applications. In the following description, wherever the term hUBP8 is employed in the description of assays, kits, compositions and the like, unless otherwise specified, the term encompasses both the human protein as well as other homologs thereof.

A. Function of Yeast Ubp8

Characterization of the histone modifying complexes, as well as their antagonistic counterparts in the model yeast Saccharomyces cerevisae has led to a paradigm that modifications function as "on/off" switches for gene regulation. The inventors discovered a transcriptional role of histone ubiquitylation and deubiquitylation, preferably of histone H2B, in regulating activation of certain genes, among which are included oncogenes, tumor suppressors, cytokines, and others.

Modification of the histone was orchestrated by the yeast ubiquitin hydrolase, Ubp8 (SEQ ID NO: 3). As supported in the examples detailed below, histone H2B

5 ubiquitylation is required for, and enhances, transcription of several yeast Gcn5- dependent genes, namely SUC2 and GALl. It is also observed that H2B is ubiquitylated and subsequently de-ubiquitylated at the GALl promoter in wild-type S. cerevisiae during galactose induction. This modification is not present in a strain that lacks the ubiquitylation site (htbl-KR) or at a region of the DNA that lacks an open l o reading frame (Int. V) .

The yeast ubiquitin hydrolase Ubp8 was found to be a stable component of the histone acetylation complex SAGA and the related complex SALSA but not present in the ADA complex. The inventors determined that SAGA-associated Ubp8 targets H2B for deubiquitylation in vivo and in vitro. This is not a function of any yeast Ubp.

15 Either substitution of the ubiquitylation site in H2B (Lys-123 or K123), or loss of

Ubp8, lowered yeast GALl expression, indicating that ubiquitylation is an essential modification for GALl transcription but is also involved in SUC2 expression. Thus, the balance of H2B ubiquitylation during transcription is essential for full gene activation. Disruption of the yeast Ubp8 did not alter the integrity or activity of the

20 multicomponent SAGA complex. It also did not affect expression of the Gal4 activator protein, essential for GALl expression and cell growth. What was observed was that the methylation of Lys4 of histone H3 was altered in the ubp8- strain. H3 Lys4 methylation has been found to track with gene activation and this alteration may be responsible for the disruption in GALl gene activity (Santos-Rosa et al, 2002

25 Nature, 419:407). Deubiquitylation of histone H2B requires SAGA-associated yeast ubiquitin protease Ubp8. The removal of ubiquitin from H2B is required for transcription to occur. Both ubiquitylation of H2B and then deubiquitylation are necessary for transcriptional activation. The role of ubiquitylation of Lys-123 in histone H2B in gene regulation reveals that ordered ubiquitylation/deubiquitylation of

30 H2B is required for optimal GALl transcription and gene activation. As supported in the examples detailed below, yeast ubiquityl hydrolase Ubp8 was found to be a stable component of the histone acetylation complex SAGA and the related complex SALSA, but not present in the ADA complex. Disruption of the yeast Ubp8 did not alter the integrity of the multicomponent SAGA complex. Ubp8 targets H2B for deubiquitylation in vivo, an essential modification for GALl transcription.

Either substitution of the ubiquitylation site in H2B (Lys-123), or loss of Ubp8, lowered yeast GALl expression, indicating that the balance of H2B ubiquitylation during transcription is essential for full gene activation.

Thus, the inventors discovered a unique relationship for Ubp8 with respect to histone modifications. Unlike acetylation/deacetylation whose effects are in opposition, both ubiquitylation and deubiquitylation are required for full gene activation. These effects may be due to the importance of initially opening the chromatin using the large ubiquitin moiety, and then removing the ubiquitin to allow later steps in transcription. The inventors' results, reported in the examples below, provide evidence that ubiquitylation of histones has a unique role, i.e., to orchestrate an ordered pathway of chromatin alterations in certain genes. Inhibition of the UBP8 deubiquitylating function can turn transcription of certain genes on or off, as desired.

B. The Human Ubp8 Homolog

The unique function of Ubp8 in yeast as it relates to modification of histones, particularly H2B, lead to the discovery that the human Ubp22 (SEQ ID NO: 1), a protein with a previously unknown function and substrate, is the human homolog of yeast Ubp8 (SEQ ID NO: 3). Thus, hUBP8 controls the deubiquitylation of histone H2B in the human chromatin, and is essential for gene activation of a variety of genes in the human SAGA complex, among others. The transcriptional regulatory complex with which hUBP8/USP22 associates in human cells has been shown to be critical for transcriptional regulation by a number of protein families important in human cancer. This includes the MYC, E2F, p53 and nuclear hormone receptor families (Yanagisawa, J. et al, 2002 Mol . Cell, 9(3): 553-62; Park, J. et al, 2001 Genes Dev, 75(13): 1619-24; McMahon, S.B. et al, 2000 Mol. Cell. Biol, 20: 556-562; McMahon, S.B. et al., 1998 Cell, 94: 363-374; Lang, S.E. et al, 2001 J. Biol. Chem., 276(35): 32627-34; and Ard, P.G etal, 2002 Mol. Cell. Biol, 22: 5650-5661). In addition, these complexes are targeted by tumor suppressors (Vieyra, D., et al, 2002 J. Biol. Chem., 277: 29832-9). For example, hUBP8 is useful in effecting p53 pathways in tumorigenesis (see, e.g., Liu etal, 1999 Mol. Cell Biol, 19: 1202-9; Sakaguchi et al, 1998 Genes Dev., 72:2831-41).

Further, because hUBP8 is a component of the PCAF/Gcn5 complexes, it is likely to operate as an on/off switch to control inflammation (Bradney et al, 2003 J. Biol. Chem., 278:2370-6; Brockmann et al, 2001 Gene, 277: 111-20). Similarly, methods or agents that effect hUBP8 expression are likely to be useful as such "switches" to treat HIV infections (Kieman et al, 1999 EMBO J., 75:6106-18; Bres et al, 2002 EMBO J., 27:6811-9). Still another use of the hUBP8 assays and compositions of this invention is in the regulation of IFN-β (Merika and Thanos, 2001 Curr. Opin. Genet. Dev., 77:205-8; Lomvardas and Thanos, 2002 Cell, 770:261-71). Still another use of hUBP8 assays, compositions, and inhibitors/enhancers is in the treatment of Alzheimers (see, e.g., Cao and Sudhof, 2001 Science, 293:115-20) or fungal infections (see Example 7 below).

The protein Ubp22 or hUBP8 is therefor implicated in chromatin modification in humans and also tumorigenesis, inflammation, viral or fungal infection, when the gene encoding hUBP8 is absent or damaged. The gene encoding hUBP8 in humans and the enzyme expressed thereby can thus function as a regulator for transcription.

The ubp8 gene and encoded protein expressed therefrom can be used in assays for drug screening, and in diagnostic and therapeutic applications.

Thus, in one embodiment, the invention includes an isolated nucleic acid of a hUBP8 gene and the use thereof. The term "isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from sequences that flank it in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment, such as the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

The isolated nucleic acid of hUBP8 according to this invention should be construed to include any and all nucleotide sequences that encode the hUBP8 protein (SEQ ID NO: 1), or a fragment thereof of greater than 20 consecutive nucleic acids. In another embodiment a fragment of hUBP8 or its analogs or homologs includes greater than 40 nucleic acids. In another embodiment a fragment of hUBP8 or its analogs or homologs includes greater than 60 nucleic acids. In another embodiment a fragment of hUBP8 or its analogs or homologs includes greater than 100 nucleic acids. In another embodiment a fragment of hUBP8 or its analogs or homologs includes greater than 150 nucleic acids and up to the nucleic acid sequence encoding the entire protein. Any such isolated nucleic acid desirably encodes a polypeptide having the biological activity of the hUBP8 polypeptide disclosed herein.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3' ATTGCC 5' and 3' TATGGC 5' share 50% homology. As used herein, "homology" is used synonymously with "identity". Percent identity, percent similarity or percent homology of one polynucleotide or polypeptide with respect to another identified polynucleotide or polypeptide may be calculated using algorithms, such as the Smith- Waterman algorithm (J. F. Collins et al, 1988, Comput. Appl BioscL, 4:61-12; J. F. Collins etal, Molecular Sequence Comparison and Alignment, (M. J. Bishop et al, eds.) In Practical Approach Series:

Nucleic Acid and Protein Sequence Analysis XVIII, IRL Press: Oxford, England, UK (1987) pp.417), and the BLAST and FASTA programs (E. G. Shpaer etal, 1996, Genomics, 38: 179- 191). A preferred algorithm is the computer program BLAST, especially blastp or tblastn (Altschul et al, 1997 Nucl. Acids Res., 25(17):3389-3402). These references are incorporated herein by reference. Sequence homology for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. Unless otherwise specified, the parameters of each algorithm discussed above are the default parameters identified by the authors of such algorithms.

Among such homologous nucleotide sequences of this invention are allelic variants of the hUBP8 sequences within a species (i.e., sequences containing some individual nucleotide differences from a more commonly occurring sequence within a species, but which nevertheless encode the same polypeptide or a protein with the same function). Additionally anti-sense strands or biologically active fragments thereof are homologous sequences according to this invention. An example of a highly stringent hybridization condition is hybridization in 2XSSC at 65°C, followed by a washing in 0. IXSSC at 65 °C for an hour. Alternatively, an exemplary highly stringent hybridization condition is in 50% formamide, 4XSSC at 42°C. Moderately high stringency conditions may also prove useful, e.g., hybridization in 4XSSC at 55°C, followed by washing in 0. IXSSC at 37°C for an hour. An alternative exemplary moderately high stringency hybridization condition is in 50% formamide, 4XSSC at

30°C.

According to the invention, the hUBP8 nucleic acid sequence encoding the protein of SEQ ID NO: 1 or an analog thereof may be modified. Utilizing the sequence data of SEQ ID NO: 1, it is within the skill of the art to obtain or prepare synthetically or recombinantly polynucleotide sequences, or modified polynucleotide sequences, encoding the full-length hUBP8 protein or useful fragments of the invention. Such modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences that are silent or that change the amino acids, e.g. to improve expression. Also included are allelic variations, caused by the natural degeneracy of the genetic code. Additional homologous sequences can include mutants including 5' or 3' terminal or internal deletions, which truncated or deletion mutant sequence may be expressed for the purpose of affecting the activity of the full- length or wild-type hUBP8 polypeptide or fragments.

In still another embodiment, the invention provides a substantially pure polypeptide of hUBP8 (SEQ ID NO: 1). The term "substantially pure" describes a compound, e.g., a protein or polypeptide that has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

The substantially pure preparation of hUBP8 according to this invention should not be construed as being limited solely to the amino acid sequences presented herein, e.g., SEQ ID NO: 1, but rather should be construed to include any and all amino acid sequences that share homology (i.e., have sequence identity) with the amino acid sequences presented herein. Preferably, the invention includes a polypeptide having an amino acid sequence that is 70% identical, more preferably, 75% identical, even more preferably, 80% identical, yet more preferably, 85% identical, even more preferably, 90% identical, more preferably, 95% identical and most preferably, 99% or 100% identical to the amino acid sequence presented in SEQ ID NO: 1. This definition of the preparation of hUBP8 includes the definitions of 'homologous", "homology" and "percent identity" as discussed above, including the list of computer algorithms available to calculate these homologies. Any such preparation of a homologous polypeptide has the enzymatic activity of the hUBP8 polypeptide disclosed herein.

Also included in the invention are modified versions of the hUBP8 polypeptide. Typically, such polypeptides differ from the specifically identified hUBP8 polypeptide of SEQ ID NO: 1 by only one to four codon changes. Examples include polypeptides with minor amino acid variations from the illustrated amino acid sequence of hUBP8 SEQ ID NO: 1, in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. Further encompassed by this invention are additional fragments of the hUBP8 polypeptide. These fragments may be designed or obtained in any desired length. In one embodiment, a fragment includes at least 10 consecutive amino acids in length. In another embodiment, a fragment includes at least

20 amino acids in length. In another embodiment, a fragment includes at least 40 amino acids in length. In another embodiment, a fragment includes at least 60 amino acids in length. In another embodiment, a fragment includes at least 100 amino acids in length. In another embodiment, a fragment includes at least 420 amino acids in length. In another embodiment, a fragment includes up to 525 amino acids in length. These fragments may be useful as probes, primers, molecular weight markers, etc. Useful fragments of hUBP8 that are smaller than the full-length hUBP8 are desirably characterized by having a biological activity similar to that displayed by the complete hUBP8 polypeptide, including, e.g., the ability to deubiquitylate its substrate(s), such as H2B. hUBP8 polypeptides of this invention may be characterized by measurements including, without limitation, western blot, macromolecular mass determinations by biophysical determinations, such as SDS-PAGE/staining, HPLC and the like, and assays such as those in the examples below to identify the biological activity. By the term "biological activity of UBP8" as used herein, is meant the ability to deubiquitylate histones, particularly the H2B histone, wherein in the absence, reduction or inactivation of the hUBP8 activity, cells containing tumor suppressor genes regulated by hUBP8 are predisposed to tumorigenesis and wherein in a cell that over-expresses hUBP8 activity, cells containing oncogenes regulated by hUBP8 are predisposed to tumorigenesis.

C. Methods of Preparing Sequences of this Invention Methods for obtaining the nucleic acids and polypeptides of the invention should be apparent to those skilled in the art upon a reading of the present disclosure and by following any of the instructions in the art. For example, the nucleotide and polypeptide sequences of the invention may be prepared conventionally by resort to known chemical synthesis techniques, e.g., solid-phase chemical synthesis, such as described by Merrifield, J. Amer. Chem. Soc, 55:2149-2154 (1963), and J. Stuart and J. Young, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, IL (1984), or detailed in the examples below. Alternatively, the nucleotide and polypeptide sequences of this invention may be prepared by known recombinant DNA techniques and genetic engineering techniques, such as polymerase chain reaction, by cloning and expressing within a host microorganism or cell a DNA fragment carrying a nucleic acid sequence encoding the above-described polypeptides, etc. (See, e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d Edit., Cold Spring Harbor Laboratory, New York (1989); Ausubel et al (1997), Current Protocols in Molecular Biology, John Wiley & Sons, New York). The hUBP8 sequence may be obtained from gene banks derived from whole genomic DNA. These sequences, fragments thereof, modifications thereto and the full-length sequences may be constructed recombinantly using conventional molecular biology techniques, site-directed mutagenesis, genetic engineering or PCR, and the like by utilizing the information provided herein. For example, methods for producing the above-identified modifications of the sequences, include mutagenesis of certain nucleotides and/or insertion or deletion of nucleotides, or codons, thereby effecting the polypeptide sequence by insertion or deletion of, e.g., non-natural amino acids, are known and may be selected by one of skill in the art.

7. Expression In Vitro

To produce recombinant hUBP8 or other fragments of this invention in vitro, the appropriate DNA sequences are inserted into a suitable expression system. Desirably, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected protein is operably linked to a heterologous expression control sequence permitting expression of the protein. Numerous types of appropriate expression vectors are known in the art for protein expression, by standard molecular biology techniques. Such vectors are selected from among conventional vector types including insects, e.g., baculovirus expression, or yeast, fungal, bacterial or viral expression systems. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose. Methods for obtaining such expression vectors are well-known. See, Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory, New York (1989); Miller et al, Genetic Engineering, 5:277-298 (Plenum Press 1986) and references cited therein.

Suitable host cells or cell lines for transfection by this method include bacterial cells. For example, the various strains of E. coli (e.g., HB101, MC1061, and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, Streptomyces, and other bacilli and the like are also be employed in this method. Mammalian cells, such as human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NIH mice are used. Another suitable mammalian cell line is the CV-1 cell line. Still other suitable mammalian host cells, as well as methods for transfection, culture, amplification, screening, production, and purification are known in the art. (See, e.g., Gething and Sambrook, 1981 Nature,

293:620-625, or alternatively, Kaufrnan etal, 1985 Mol. Cell. Biol, 5(7): 1750-1759 or Howley et al, U. S. Patent 4,419,446). Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Other fungal cells may also be employed as expression systems. Alternatively, insect cells such as Spodoptera frugipedera (Sf9) cells may be used.

Thus, the present invention provides a method for producing a recombinant hUBP8 protein, which involves transfecting, e.g., by conventional means such as electroporation, a host cell with at least one expression vector containing a polynucleotide of the invention under the control of a transcriptional regulatory sequence. The transfected or transformed host cell is then cultured under conditions that allow expression of the protein. The expressed protein is recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art. For example, the proteins are isolated in soluble form following cell lysis, or extracted using known techniques, e.g., in guanidine chloride. If desired, the proteins or fragments of the invention are produced as a fusion protein to enhance expression of the protein in a selected host cell, to improve purification, or for use in monitoring the presence of the desired protein in tissues, cells or cell extracts. Suitable fusion partners for the proteins of the invention are well known to those of skill in the art and include, among others, beta-galactosidase, glutathione-S-transferase, and poly-histidine.

2. Expression In Vivo

Alternatively, where it is desired that the hUBP8 protein of the invention or proteinaceous inhibitors thereof (whether full-length or a desirable fragment) be expressed in vivo, e.g., to induce antibodies, or as a therapeutic, an appropriate vector for delivery is readily selected by one of skill in the art. Exemplary vectors for in vivo gene delivery are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus (International patent application No. PCT/US91/03440), adenovirus vectors (M. Kay et al, 1994 Proa Natl. Acad. Sci. USA, 97:2353; S. Ishibashi et al, 1993 J. Clin. Invest., 92:883) or other viral vectors, e.g., various poxviruses, vaccinia, etc. Methods for insertion of a desired gene, e.g., P7-1, and obtaining in vivo expression of the encoded protein, are well known to those of skill in the art.

The preparation or synthesis of the nucleotide and polypeptide sequences disclosed herein, whether in vitro or in vivo (including ex vivo) is well within the ability of the person having ordinary skill in the art using available material.

The synthetic methods are not a limitation of this invention.

D. Agonists (Enhancers) and Antagonists (Inhibitors) ofhUBP8 or I1UBP8 of the Invention and Compositions Containing Them

In still another embodiment, the invention provides inhibitors of the hUBP8 gene or hUBP8 polypeptide, or its homologs, such as yeast hubp8 and yUBP8. Such inhibitor compositions have utility as diagnostic reagents or as therapeutic reagents in the methods described below. By the use of the term "hUBP8 inhibitor" as used herein is meant a compound that is capable of inhibiting expression and/or biological activity of hUBP8. Inhibition of hUBP8 activity or expression may be assessed by following the procedures presented in the examples herein, which permit the progress (or the lack thereof) of histone modification or the progress of gene expression through transcription to be monitored.

1. Nucleotide sequence inhibitors

One such inhibitor is a nucleotide sequence that binds to the hUBP8 nucleic acid sequence or a fragment thereof. Such inhibitors when contacted with a cell expressing hUBP8 inhibit the expression of (or inactivate) hUBP8 in that cell. For example, an inhibitor of hUBP8 expression or function includes an oligonucleotide molecule that is preferably in an antisense orientation with respect to the nucleic acid sequence of hUBP8. As used herein, the term "antisense oligonucleotide" means a nucleic acid polymer, at least a portion of which is complementary to a hUBP8 nucleic acid. "Antisense" refers particularly to the nucleic acid sequence of the noncoding strand of a double stranded DNA molecule encoding a protein, or to a sequence that is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences. In one embodiment, the antisense oligonucleotides of the invention preferably comprise about 10 nucleotides. In another embodiment, the antisense oligonucleotides of the invention preferably comprise about 12 nucleotides. In still another embodiment, the antisense oligonucleotides of the invention preferably comprise about 16 nucleotides. In still another embodiment, the antisense oligonucleotides of the invention preferably comprise about 20 nucleotides. In still another embodiment, the antisense oligonucleotides of the invention preferably comprise about 30 nucleotides. In still another embodiment, the antisense oligonucleotides of the invention preferably comprise about 50 or more nucleotides. Such nucleotides may be complementary to a consecutive sequence of nucleotides in the nucleotide sequence encoding the selected UBP8 homolog. The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides. Methods for synthesizing oligonucleotides, phosphorothioate oligonucleotides, and otherwise modified oligonucleotides are well known in the art (U.S. Patent No. 5,034,506; Nielsen et al, 1991, Science 254: 1497). See also the general teachings regarding the production of antisense nucleotides in texts such as, H. M. Weintraub, 1990 Scient. Amer., pp.40- 46 and Milner et al, 1991 Nat. Biotech., 75:537-541, among others in the art.

For example, suitable antisense sequences generated to inhibit hubp8 expression include, without limitation, the following sequences that hybridize to portions of SEQ ID NO: 1. One embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 1-50 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 51-100 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 101-150 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 151-200 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 201-250 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 251-300 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 301-350 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 351-400 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 401-450 of SEQ ID

NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 451-500 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 501-525 of SEQ ID NO: 1. Other antisense sequences can complement portions of the sequence spanning the specifically-exemplified regions. For example, antisense sequences can be directed to the Cys Box at amino acid positions 138-167 of SEQ ID NO: 1, the His Box at positions 411-465 of SEQ ID NO: 1, or within the active sites at or around the amino acids at position 146, 419 and 427. Still other regions of SEQ ID NO: 1 may be targeted by antisense or other inhibitors.

Still another embodiment of nucleotide inhibitors of this invention are homologous double-stranded RNA formed by annealing sense and antisense strands of hubp8 and/or a homolog thereof, e.g., yeast ubp8. By introducing such ds RNA into cells the gene may be silenced post-transcriptionally. See, e.g., this process described in Hasuwa et al, 2002 FEBSLett., 532(l-2):221-30; Rubinson et al, 2003 Nat. Gnet, 33(3):40\-6; Song etal, 2003 Nat. Med, 9(3):347-51; McManus et al, 2002RNA, 5(6): 842-50; and Kennerdell and Carthew 1998 £>eve/., 95:1017-26, among others, incorporated by reference herein.

For example, suitable dsRΝA sequences are generated synthetically by the methods described in the references cited above to silence hubp8 expression include, without limitation, the following sequences based on portions of a nucleic acid sequence encoding SEQ ID NO: 1. One embodiment of an ds RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 1-50 of SEQ ID NO: 1. Another embodiment of an ds RNA sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 51-100 of SEQ ID NO: 1. Another embodiment of an antisense sequence is a nucleotide sequence complementary to a sequence within the nucleotide sequence encoding amino acids 101-150 of SEQ ID NO: 1. Another embodiment of an ds RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 151-200 of SEQ ID NO: 1. Another embodiment of an ds RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 201-250 of SEQ ID NO: 1. Another embodiment of an ds RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 251-300 of SEQ ID NO: 1. Another embodiment of an ds RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 301-350 of SEQ ID NO: 1. Another embodiment of an ds RNA sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 351-400 of SEQ ID NO: 1. Another embodiment of an ds

RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 401-450 of SEQ ID NO: 1. Another embodiment of an ds RNAi sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 451-500 of SEQ ID

NO: 1 Another embodiment of an ds RNA sequence is a ds ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding amino acids 501-525 of SEQ ID NO: 1. Other RNA or dsRNA sequences can complement portions of the sequence spanning the specifically- exemplified regions such as the active sites, His and Cys boxes identified above.

2. Polypeptide/protein inhibitors

In another embodiment, another inhibitor composition of the invention includes a ligand that binds to hUBP8 polypeptide. Such a ligand is desirably an antibody that binds to hUBP8, thereby inhibiting the function thereof. The term "antibody," as used herein, refers to an immunoglobulin molecule that is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, high affinity polyclonal antibodies, monoclonal antibodies, synthetic antibodies, chimeric antibodies, recombinant antibodies and humanized antibodies. Such antibodies may originate from immunoglobulin classes IgG, IgM, IgA, IgD and IgE. Such antibodies may include a Fab, Fab' or F(ab')2, or Fc antibody fragment thereof that binds hUBP8. Still another useful ligand is a single chain Fv antibody fragment that binds hUBP8. Another useful ligand is a recombinant construct comprising a complementarity determining region of an antibody, a synthetic antibody or a chimeric antibody construct or a humanized antibody construct that shares sufficient CDRs to retain functionally equivalent binding characteristics of an antibody that binds hUBP8. By the term "synthetic antibody" as used herein, is meant an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody that has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic

DNA or amino acid sequence technology that is available and well known in the art. The antibodies of this invention are generated by conventional means utilizing the isolated, recombinant or modified hUBP8 or fragments thereof as antigens of this invention. For example, polyclonal antibodies are generated by conventionally stimulating the immune system of a selected animal or human with a hUBP8 antigen, allowing the immune system to produce natural antibodies thereto, and collecting these antibodies from the animal or human's blood or other biological fluid. Preferably a recombinant version of hUBP8 is used as an immunogen. Monoclonal antibodies (MAbs) directed against hUBP8 are also generated conventionally. Hybridoma cell lines expressing desirable MAbs are generated by well-known conventional techniques, e.g. Kohler and Milstein and the many known modifications thereof. Similarly desirable high titer antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens (see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit et al, 1986 Science, 233:141-153; Queen et al,

1989 Proc. Nat'l. Acad. Sci. USA, 56:10029-10033; PCT Patent Application No. PCT/WO9007861; Riechmann et /., 1988 Nature, 332:323-321; Huse et α/, 1988 Science, 246:121 '5-1281).

Given the disclosure contained herein, one of skill in the art may generate ligands or antibodies directed against hUBP8 by resort to known techniques by manipulating the complementarity determining regions of animals or human antibodies to the antigen of this invention. See, e.g., E. Mark and Padlin, "Humanization of Monoclonal Antibodies", Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer- Verlag (June, 1994); Harlow et al, 1999, Using Antibodies: A Laboratory Manual,

Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 55:5879-5883; and Bird etal, 1988, Science 242:423-426.

Alternatively, hUBP8 antigens are assembled as multi-antigenic complexes (see, e.g., European Patent Application 0339695, published November 2,

1989) and employed to elicit high titer antibodies capable of binding the hUBP8. Further provided by the present invention are anti-idiotype antibodies (Ab2) and anti- anti-idiotype antibodies (Ab3). Ab2 are specific for the target to which anti-hUBP8 antibodies of the invention bind and Ab3 are similar to hUBP8 antibodies (Abl) in their binding specificities and biological activities (see, e.g., M. Wettendorff et al,

1990 "Modulation of anti-tumor immunity by anti-idiotypic antibodies." In Idiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux J, Am Soc. Microbiol., Washington DC: pp. 203-229). These anti-idiotype and anti-anti-idiotype antibodies are produced using techniques well known to those of skill in the art. Such anti- idiotype antibodies (Ab2) can bear the internal image of hUBP8 and are thus useful for the same purposes as hUBP8.

In general, polyclonal antisera, monoclonal antibodies and other antibodies that bind to hUBP8 as the antigen (Abl) are useful to identify epitopes of hUBP8 to separate hUBP8 and its analogs from contaminants in living tissue (e.g., in chromatographic columns and the like), and in general as research tools and as starting material essential for the development of other types of antibodies described above. Anti-idiotype antibodies (Ab2) are useful for binding the same target and thus may be used in place of hUBP8 to induce useful ligands to hUBP8. The Ab3 antibodies are useful for the same reason the Abl are useful. Other uses as research tools and as components for separation of hUBP8 from other contaminants, for example, are also contemplated for the above-described antibodies.

3. Chemical compound inhibitors

Other ligands may include small chemical compounds that are screened in a suitable assay, such as the high throughput screening assay described below and that are found to inhibit the deubiquitylation enzymatic activity or other activities of hUBP8. Such hUBP8 ligands or inhibitors may be identified and developed by the drug screening methods discussed in detail below.

Similar chemical compounds may be found that enhance the enzymatic activity of hUBP8, and thus may be used in situations where upregulation of the enzymatic activity is desired, such as in the case in which the gene to be regulated is a tumor suppressor. The enhancing effect of a compound may be determined in the same type of enzymatic assay as discussed above.

4. Inhibitors as diagnostic reagents and kits For use in diagnostic assays and kits, the above-described inhibitors or enhancers of the hUBP8 gene and hUBP8 polypeptide and/or substrates in an enzymatic assay are preferably associated with a detectable label that is capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Where more than one reagent sequence or hUBP8 inhibitor is employed in a diagnostic method, the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. colorimetrically. A variety of enzyme systems operate to reveal a colorimetric signal in an assay, e.g., glucose oxidase (which uses glucose as a substrate) releases peroxide as a product that in the presence of peroxidase and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color. Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase in conjunction with glucose-6-phosphate dehydrogenase that reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength. Other label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles (Bangs Laboratories, Indiana) in which a dye is embedded may be used in place of enzymes to form conjugates with the inhibitor sequences or ligands and provide a visual signal indicative of the presence of the resulting complex in applicable assays. Still other labels include fluorescent compounds, radioactive compounds or elements. Preferably, each reagent or ligand is associated with, or conjugated to detectable fluoro chromes, e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). All of these fluorescent dyes are commercially available, and their uses known to the art.

Detectable labels for attachment to reagent sequences and antibodies and substrates useful in diagnostic assays and enzymatic assays of this invention may be easily selected from among numerous compositions known and readily available to one skilled in the art of diagnostic assays. The reagents and ligands of this invention are not limited by the particular detectable label or label system employed.

Methods for coupling or associating the label with the reagent sequence or ligand are similarly conventional and known to those of skill in the art. Known methods of label attachment are described (see, for example, Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., R.P. Haugland, Molecular Probes, Inc.,

Eugene, OR, 1996; Pierce Catalog and Handbook, Life Science and Analytical Research Products, Pierce Chemical Company, Rockford, IL, 1994/1995). Thus, selection of the label and coupling methods do not limit this invention.

For convenience, the conventional reagents for immunoassays, e.g., ELISA or enzymatic assays, e.g., the high throughput enzymatic assays described below or other diagnostic assays according to this invention may be provided in the form of kits. Such kits are useful for determining the absence or reduction (e.g., inactivation) or presence or overproduction of hUBP8 gene or hUBP8 polypeptide in a cell, particularly a tumor cell. Such kits are useful for determining the levels of enzymatic activity of hUBP8 in a cell. Thus, such a kit will be useful in conducting the diagnostic assays discussed below, e.g., in determining if a cell is tumorigenic, in determining the status of treatment of a cancer, etc. Such a diagnostic kit contains a nucleotide reagent sequence (e.g., &hUBP8 antisense sequence), or MJBP8 inhibitor (e.g., an antibody or compound capable of binding hUBP8) or hUBP8 agonist (e.g., a chemical compound that can mimic some of the activity of hUBP8) of this invention.

Such kits may contain the hUBP8 substrate, e.g., a ubiquitylated histone, such as ubH2B. Such kits also contain labels, exemplified above, pre-attached to the other components of the assay, or provided separately for attachment to a selected component, e.g., a substrate. Alternatively, such kits may contain a simple mixture of such compositions or means for preparing a simple mixture. The kits also include instructions for performing the assay, microtiter plates to which the inhibitors or nucleic acid sequences of the invention have been pre-adsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound compositions and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens. Other components may include indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, and a sample preparator cup. Such kits provide a convenient, efficient way for a clinical laboratory to screen possible diagnostic and therapeutic compounds targeted to hUBP8 and its activity, or to diagnose the tumorigenic potential of a mammalian cell according to this invention.

Still another variant of a diagnostic kit for detecting the tumorigenic potential of a cell contains the components necessary for a hUBP8-mediated deubiquitylation assay such as the assay described below in Example 4. Such components may include a hUBP8 protein or homolog thereof such as yUBP8. Still other components of a kit include a substrate for hUBP8, e.g., ubH2B; ubiquitin, an anti-ubiquitin antibody, a label for association with the substrate, an immobilized agent capable of binding labeled UBP8, as well as reagents necessary for performing gel electrophoresis and immunoblotting. Similarly, the non-biologic materials necessary for performing such an assay (as described above) may be included in this kit. One of skill in the art may be expected to vary the components of these diagnostic kits in obvious ways based on the knowledge in the art coupled with this disclosure. Such varied components are considered to be encompassed in this embodiment of the invention. 5. Agonists or Antagonists ofhUBP8 as Therapeutic

Compositions of this Invention

Alternatively, an above-described inhibitor of hUBP8 of this invention may be employed therapeutically, and as such, is encompassed in a pharmaceutical composition. Such a composition includes a hUBP8 agonist or antagonist (nucleotide or polypeptide or protein, or a small chemical compound) and a pharmaceutically- acceptable carrier. As used herein, the term "pharmaceutically-acceptable carrier" means a chemical composition with which an appropriate hUBP8 agonist or antagonist may be combined and which, following the combination, can be used to administer the appropriate composition to a mammal. For example, suitable carriers include saline, buffered saline, and the like. In addition to the appropriate hUBP8 agonist/antagonist, such pharmaceutical compositions may also contain other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer an appropriate hUBP8 agonist/antagonist according to the methods of the invention.

Also, as noted herein, pharmaceutical compositions of this invention may include a combination of compounds comprising a hUBP8 agonist/antagonist and another chemotherapeutic agent. Among such agents include, without limitation, alkylating agents, antibiotics, antimetabolitic agents, plant-derived agents, and hormones. Among the suitable alkylating agents are nitrogen mustards, such as cyclophosphamide, aziridines, alkyl alkone sulfonates, nitrosoureas, nonclassic alkylating agents, such as dacarbazine, and platinum compounds, such as carboplatin and cisplatin. Among the suitable antibiotic agents are dactinomycin, bleomycin, mitomycin C, plicamycin, and the anthracyclines, such as doxorubicin (also known as adriamycin) and mitoxantrone. Among the suitable antimetabolic agents are antifols, such as methotrexate, purine analogues, pyrimidine analogues, such as 5-fluorouracil (5-FU) and cytarabine, enzymes, such as the asparaginases, and synthetic agents, such as hydroxyurea. Among the suitable plant-derived agents are vinca alkaloids, such as vincristine and vinblastine, taxanes, epipodophyllotoxins, such as etoposide, and camptothecan. Among suitable hormones are steroids. Other suitable chemotherapeutic agents, or therapies such as radiation, and including additional agents within the groups of agents identified above, may be readily determined by one of skill in the art depending upon the type of tumor being treated, the condition of the human or veterinary patient, and the like. Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically by conventional therapeutic routes, e.g., intravenously, intraperitoneally, orally, via the mucosa, by inhalation, intramuscularly, subcutaneously, transdermally, topically, etc. Formulations suitable for the selected route can include, among others, oral solid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations that may be designed using information known to one of skill in the pharmaceutical formulations art. Selection of the formulations and routes are within the skill of the art, and are not a limitation of this invention. See, also, Milner, cited above.

E. Methods For Using the Compositions of this Invention According to the present discovery, hUBP8 is required for mamtaining a low ubiquitylated level of histones, such as H2B, and subsequent regulation of relevant SAGA complex genes among other genes, including oncogenes, tumor suppressors, cytokines such as IFN-β, as discussed above. hUBP8 mediates the transcription of such genes in cells. Thus, the absence or reduction of functional hUBP8 in a cell, or the presence of insufficient hUBP8 or the over-production of hUBP8 enzymatic activity in a cell has ramifications with respect to whether relevant gene products will be expressed or inhibited in a cell. The ability of UBP8 to turn off or on such expression can determine whether a cell will become a tumor cell, whether an inflammatory response will be enhanced or inhibited, whether viremia will be enhanced or reduced, whether fungal infections will be enhanced or inhibited, depending upon the identity of the gene which is regulated.

Methods of this invention employ nucleotide sequences encoding the UBP8 polypeptides, UBP8 polypeptide sequences, fragments thereof, as well as the hUBP8 agonist/antagonists in drug screening, diagnostic and therapeutic protocols.

7. Diagnostic Methods of the Invention Because cells that carry oncogenes or that have insufficiently expressed tumor suppressor genes are more likely to develop into tumor cells, the invention includes methods of identifying a cell that is likely to become a tumor cell using the above-described compositions. In one embodiment, a method of determining tumorigenic potential of a mammalian cell includes examining the cell for the presence of, or mutations in, the hUBP8 nucleic acid sequence. The substantial absence of, or mutation in, ahUBP8 nucleic acid sequence in a cell containing a tumor suppressor gene indicates that the cell may be predisposed to tumorigenesis, particularly upon exposure to a carcinogenic agent or environment.

The detection of ahUBP8 gene in a cell may be assessed in any ordinary nucleic acid expression assay, including techniques such as, Northern blotting with a suitable nucleic acid probe, Southern blotting, polymerase chain reaction (PCR), reverse transcriptase-PCR, RNase protection assays and in situ hybridization and the like. Such assays may readily be employed in vitro by exposing a sample of tissue to be examined for tumorigenic potential to an anti-sense oligonucleotide, PCR primer or other hUBP8 agonist/antagonist of this invention. Such assay techniques are conventional and the protocols for these assays are found in standard texts, such as Sambrook et αl, cited above. Another embodiment of a nucleic acid assay for use in determining the tumorigenic potential of a cell includes the steps of examining the cell for mutations in the hUBP8 gene that result in over-expression or under-expression of the hUBP8 enzymatic activity. The presence of mutations in the gene along with the presence in the cell of a hUBP8-regulated oncogene or tumor suppressor gene can indicate predisposition of the cell to tumorigenesis. This method involves isolating nucleic acid from the cells of selected species of mammal (preferably human) or other animal. This can be accomplished using either RNA or genomic DNA and using fragments of the hUBP8 gene of this invention as the primers. The sequences obtained from the cells using RT-PCR for RNA or PCR for DNA are then amplified and the resulting gene sequenced to uncover any mutations. In order to examine the sequence for mutations, any conventional technique may be used, such as in situ hybridization. By this means the sequence from the cell under examination is compared to the sequence of a normal hUBP8 gene to determine if the hUBP8 gene of the cell bears a mutation. Techniques for comparison include conformation sensitive gel electrophoresis or single strand polymorphism analysis, among others. (See, Sambrook et al, or other conventional texts). If desired, the sequence may be used to express a polypeptide, and that polypeptide may be tested to determine if it retains a function of hUBP8, such as hUBP8-mediated deubiquitylation activity, or other functions as disclosed herein. Any mutations in these sequences that inactivate or overexpress the hUBP8 function may be employed in methods and compositions of this invention.

In another embodiment, the invention provides a method of determining tumorigenic potential of a cell comprising examining the cell for the presence of hUBP8 polypeptide expression. The absence or reduction of a detectable level of hUBP8 polypeptide indicates that the cell is predisposed to tumorigenesis upon exposure to mitotic stress. The method also comprises determining whether or not hUBP8 is expressed at a lower or higher than normal level in a cell, wherein a lower or higher level of expression of hUBP8 in the cell, compared with expression of hUBP8 in an otherwise identical normal cell, is an indication that the cell will develop into a tumor cell. Cells may be examined for expression of hUBP8 polypeptide using conventional protein and immunological assays, such as, without limitation, western immunoblotting with a suitable antibody, ELISA, immunofluorescence and immunochemistry (see, e.g., Sambrook et al, and other texts for such assay steps). Such assays may readily be employed in vitro by exposing a sample of tissue to be examined for tumorigenic potential to a hUBP8 agonist or antagonist, e.g., an antibody of this invention as described above.

Preferably, enzymatic assays are used to detect over-expression or under-expression of the enzymatic activity of hUBP8 and thereby determine the tumorigenic potential of a cell. Such assays involve examining the cell for HUBP8- mediated deubiquitylation activity. In one embodiment, an in vitro assay format involves using purified ubiquitylated histone H2B. The ubH2B is obtained by purification from a yeast strain that has a FLAG (Sigma)-tagged histone H2B (or tagged H2A and H2B in mammalian cells) and hemagglutinen (HA)-tagged ubiquitin. Purification is accomplished with an immunoprecipitation of cell lysates with anti-

FLAG antibody followed by elution to yield all tagged histone H2B or H2A, ubiquitylated or not. That immunoprecipitate (IP) fraction is then subject to immunoprecitation with anti-HA antibody, which results in the purification of only ubiquitylated histones. Another method involves obtaining purified, tagged histone H2B (by IP as above) and subjecting it to in vitro ubiquitylation with recombinant S. cerevisiae Rad6 enzyme using HA-tagged (or 1-125 labeled) ubiquitin followed by FLAG-histone/HA-ubiquitin (or anti-ubiquitin) IP. This method has been used before to determine if Rad6 has in vitro histone ubiquitylation activity. While the latter purification method requires more steps, it likely yields more protein, since only about 5 to 10% of histones are ubiquitylated in vivo. It is anticipated that a much greater yield is obtainable using in vitro ubiquitylation of H2B.

The steps of the enzymatic assay thereafter include binding HA- Ub:Flag-H2B to a surface or bead, such as by using the FLAG epitope on H2B. The FLAG epitope binds to anti-FLAG affinity resin. Alternatively, the anti-FLAG antibodies are conjugated to the surface of a 96- or 384- well assay plate. Thereafter, deubiquitylation assay buffer [lOOmM Tris-HCl (pH 8.0), ImM EDTA ImM DTT, 5% glycerol, lμg/ml pepstatin A] is added. The test compounds, e.g., potential Ubp8 inhibitors that are being screened, are added at varying concentrations, along with appropriate positive and/or negative controls to respective wells. Ubp8 or HUBP8 protein (yeast or human, respectively) is then added to each well and the wells are incubated at 37°C for various timepoints. The reaction is terminated by placing samples on dry ice, adding 1 volume of 2X sodium dodecylsulfate (SDS) sample buffer and boiling. The resulting samples are run on 15%) SDS-PAGE gel for 1 to 1.5 hours at 100V and transferred to membrane. Western analysis using anti-FLAG antibody is performed. The presence of an ~ 25kDa band indicates the persistence of Ub-H2B

(inactive Ubp8 activity), while an ~15kDa band indicates H2B (active Ubp8). The presence of an ~10kDa band detected by anti-ubiquitin or anti-HA Western analysis is indicative of HA-ubiquitin cleavage. The presence of two bands indicates partial activity/partial inhibition or incomplete reaction. If the cell has such activity, the cell contains functional hUBP8. The absence (or substantial reduction) of such activity coupled with a regulated tumor suppressor or the over-production of the enzymatic activity coupled with a regulated oncogene indicates that the cell is therefore predisposed to tumorigenesis. Such cells may be more sensitive to carcinogenic agents. Thus, the invention further includes a method of determining the sensitivity of a tumor cell in a mammal to chemotherapeutic agents. The methods described in detail above can be used to assess the cell for one or more of the characteristics including the substantial overproduction of hUBP8 activity or expression, the substantial absence or a mutation in a hUBP8 gene; the substantial absence of hUBP8 deubiquitylation activity. The identification of any of these characteristics along with the presence of a regulated tumor suppressor or oncogene indicate that the tumor cell is sensitive to a carcinogen, e.g., radiation. Thus, for example, the method can include assessing ex vivo the level of hUBP8 expression at the nucleic acid or protein level in the mammalian cell, which has been identified as a tumor cell. This experimental level is then compared to the level of hUBP8 expression in a non-tumor cell of the mammal. An abnormal level of activity of hUBP8 in the cell compared with the level of expression or activity of hUBPS in an otherwise identical mammalian non-tumor cell, is an indication that the cell is overexpressing a gene product, such as an oncogene, or underexpressing a gene product, e.g., a tumor suppressor. This method can include assessing the cell for KUBP8 gene mutations, as described above. Further, this method can include assessing the cell for hUBP8- mediated enzymatic activity, as described above.

Knowledge of the sensitivity of such a tumor cell in a mammal to a chemotherapeutic agent or therapy, such as radiation, may be used to determine the type of chemotherapeutic agent that might be administered to the mammal to kill the tumor cell. For example, the cells so identified may thereafter be exposed to a battery of such chemotherapeutic agents to enable the selection of the agent most effective in killing the tumor cells in an ex vivo or in vivo therapeutic context.

Similarly, as described above for nucleic acid assays, amplified RNA or DNA from the cells of a variety of mammalian (or other animal) species may be examined and/or expressed and assayed to detect mutations that inactivate or enhance the function of HUBP8, as appropriate.

Still other assays may employ the nucleic acid sequences encoding the homologs of hUBP8 for the determination of the level of expression of selected genes implicated in the SAGA complex. Such assays may be useful as diagnostic assays to evaluation over-expression or under-expression of selected genes. 2. Therapeutic Methods of this Invention

Thus, the present invention also provides a therapeutic method of retarding the growth of, or killing, tumor cells, by inhibiting expression and or activity of hUBP8 in cells that are tumor cells or contain an oncogene regulated by hUBP8 activity. The present invention also provides a therapeutic method of inhibiting the growth of, or killing, such tumor cells, by enhancing the expression and/or activity of hUBP8 in cells that are tumor cells and contain a tumor suppressor gene regulated by hUBP8 activity. Since the development of tumor cells occurs via a vast number of mechanisms, the tumor cells to be killed need not necessarily have arisen due to an abnormal expression or activity of hUBP8. Indeed, the method of killing tumor cells is likely to be more effective in cells in which hUBP8 is expressed, and which have developed into tumor cells via a hUBP8-independent mechanism.

Thus, in another embodiment a therapeutic method of the invention comprises administering to a mammalian tumor cell, in vitro, ex vivo or in vivo, an agonist or antagonist of hUBP8 expression or biological activity, as appropriate depending on the identity of the regulated gene. Such agonists or antagonists include, without limitation, the reagent antisense sequences and/or the protein ligands, and/or small chemical compounds described above in a dosage that is suitable to inhibit or enhance expression or function of hUBP8 in the cell. This inhibition of hUBP8 activity results in reduced expression of an oncogene. The enhancement of hUBP8 activity results in enhanced expression of a tumor suppressor. Such methods are also useful for killing a tumor cell. Thus, an optional step in this therapeutic method is aciministering to the tumor cell, or to the mammal bearing the tumor cell a chemotherapeutic agent or radiation in a suitable dosage selected for therapy. The administration of this second reagent may occur simultaneously with the hUBP8 agonist/antagonist composition, or the administration of the chemotherapeutic agent/radiation therapy may occur at some time after the hUBP8 agonist/antagonist has produced its effect on the tumor cells. This method is useful in some embodiments in killing the cancer cell.

This method may be performed by administering the pharmaceutical compositions described above via any suitable therapeutic route, and selection of such route is not a limitation of this invention. Similarly the appropriate dosage of such pharmaceutical compositions may be determined by a physician, based on typical characteristics such as the physical condition of the patient, the disease being treated, the use of other therapeutic compositions, etc. In one embodiment, the pharmaceutical compositions useful for practicing the therapeutic methods of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. The dosages of the chemotherapeutic agents or radiation or other anti-cancer therapies, are known to those of skill in the art. This invention is therefore not limited by the dosage selection, which is within the skill of the art.

In other embodiments of therapeutic methods, a suitable UBP8 homolog, e.g., human, yeast or other, may be introduced to a cell (in vitro, ex vivo or in vivo) that lacks appropriate de-ubiquitylating activity. This method permits regulation or expression (or inversely, inhibit the expression) of genes related to Aizheimers, viral or fungal infection, inflammation, cancer, etc. as described above. F. Drug Screening and Screening for Agonist/Antagonists ofh UBP8 Enzymatic Activity The hUBP8 nucleic acid sequences and hUBP8 polypeptides of this invention and/or homologs thereof, such as the homologous yeast UBP8 sequence and polypeptide, may also be used in the screening and development of chemical compounds, proteins or other compounds that have utility as therapeutic drugs. As one example, the compositions of this invention may be useful for screening for compound useful in the treatment or diagnosis of cancer, treatment or diagnosis of imlammatory diseases, treatment or diagnosis of viral diseases, such as HIV, the regulation of interferon-β, the treatment or diagnosis of Alzheimer's disease, the treatment or diagnosis of fungal infections, the treatment or diagnosis of high cholesterol, and the treatment other diseases related to the abnormal activity of hUBP8. Suitable assay methods for screening such potential drug compounds may be readily determined by one of skill in the art. As described below, while the description relates primarily to hUBP8, the yeast UBP8 sequences or other homologs that share activity with the hUBP8, may be used in place of the human sequences in these assay methods. As one example, assays employing hUBP8 can be employed to screen for compounds inter alia that inhibit HDAC, that identify or inhibit HAT translocations in leukemia, that identify or inhibit HMT overexpression in transition from benign to malignant prostate, and that identify or inhibit targets in hematologic malignancies. See, e.g., Dutnall, R.N. and Pillus, L. 2001 Cell 705:161-4; Johnstone RW. 2002 Nat. Rev. DrugDiscov. 7:287-99; Melnick A, Licht JD. 2002 Curr. Opin. Hematol

9:322-32; and Vafambally, S. et al, 2002 Science. 419:624-29.

However, in an embodiment a method for identifying an agonist/antagonist of hUBP8 expression or activity involves adding a test compound to a cell that is known to have hUBP8 enzymatic activity at a specified level. The cell in which hUBP8 is expressed may be any cell found to express the hUBP8 gene. Alternatively the cell may be one in which hUBP8 is not normally expressed, but into which hUBP8 has been introduced, by way of, for example, a plasmid or other vector, thereby enabling the expression of hUBP8 within the cell. After sufficient exposure to the test compound, the level of expression of hUBP8 mRNA or protein or hUBP8 activity is assessed according to the assays described herein. This experimental level is then compared with the level of expression/activity of hUBP8 nucleic acid or hUBP8 protein in an otherwise identical cell to which the test compound has not been added. An abnormal level of expression of hUBP8 nucleic acid, protein or enzymatic activity in a cell to which the test compound has been added, compared with the level in a cell to which the test compound has not been added, is an indication that the test compound is capable of affecting the abnormal hUBP8 expression.

Agonists/antagonists of hUBP8 activity may also be screened by resort to assays and techniques useful in identifying drugs capable of binding to or interacting with the hUBP8 polypeptide and thereby affecting its biological activity in a positive or negative fashion in a cell that expresses hUBP8. For example, another method of identifying a hUBP 8 agonist/antagonist comprises the steps of screening a test compound in a hUBP8-mediated ubiquitylation/deubiquitylation assay, such as the in vitro assay described above and variants thereof. The substantial alteration of the ubiquitylation/deubiquitylation activity in the assay in the presence of the test compound indicates that the test compound inhibits or enhances hUBP8 activity. In one embodiment, the hUBP8-mediated enzyme in vitro assay may be performed to screen small chemical compounds as agonists/antagonists. To develop or screen small chemical compounds that agonize/antagonize hUBP8-mediated enzymatic activity, it is preferred to employ purified, recombinantly-produced labeled hUBP8 protein (e.g., glutathione S-transferase (GST)-hUBP8 or FLAG-tagged-hUBP8). These proteins may be conventionally recombinantly produced in, e.g., bacterial cells, insect cells or any of the cells described above for recombinant production above. This assay may be performed by contacting a mixture that normally demonstrates hUBP8-mediated enzymatic activity with a test compound; and assaying said mixture and test compound for the activity. This mixture can contain, among other things, hUBP8 protein, conventional beads or assay plates for immobilization, the substrate H2B, preferably tagged with FLAG (Sigma) and/or associated with HA-ubiquitin, anti-FLAG antibodies, the above-described deubiquitylation assay buffer, appropriate positive or negative controls, FLAG peptide (Sigma) as well as suitable buffers and buffering agents. The assay can include the further steps of separating the labeled hUBP8 protein or ubiquitylated substrate or non-ubiquitylated substrate from the mixture, and performing gel electrophoresis thereon. Immunoblotting the gel with an anti-ubiquitin or anti-HA antibody permits detection of ubiquitinated H2B in the gel. Identification of the presence of ubiquitin on the H2B protein by the antibody demonstrates hUBP8- mediated deubiquitylation activity. If the antibody cannot bind any ubiquitylated H2B in the gel, the cell has no functional hUBP8. The performance of such an assay when the mixture is in the presence or, or absence of a test compound and the comparison of the results obtained identifies the test compound as ahUBP8 agonist/antagonist, as appropriate. Similarly assays that measure the response of such cells to carcinogenic compounds or carcinogenic modalities, e.g., radiation, may also be used for screening of chemotherapeutic drugs or therapies according to this invention.

Other conventional drug screening techniques may be employed using the proteins, antibodies or polynucleotide sequences of this invention. As one example, a method for identifying compounds that specifically bind to a hUBP8 polypeptide of this invention can include simply the steps of contacting a selected cell expressing hUBP8 with a test compound to permit binding of the test compound to hUBP8 and determining the amount of test compound, if any, which is bound to the hUBP8. Such a method may involve the incubation of the test compound and the hUBP8 polypeptide immobilized on a solid support. Typically, the surface containing the immobilized ligand is permitted to come into contact with a solution containing the protein and binding is measured using an appropriate detection system. Suitable detection systems include those described above for diagnostic use.

Thus, through use of such methods, the present invention is anticipated to provide compounds capable of interacting with hUBP8 or the hUBP8 gene or portions thereof, and either enhancing or decreasing hUBPδ's biological activity, as desired. Compounds effecting homologs of hUBP8, such as the yeast or other mammalian Ubp8 proteins may also be provided. Still other methods of drug screening for novel compounds that affect hUBP8 expression and/or activity at the nucleic acid or protein level involve computational evaluation and design. According to these methods, the three dimensional structure of the hUBP8 gene and/or the polypeptide is determined and chemical entities or fragments are screened and selected for their ability to associate with the three dimensional structures. Suitable software for such analysis include docking software such as Quanta and Sybyl, molecular dynamics and mechanics programs, such as CHARMM and AMBER, the GRID program available from Oxford University, Oxford, UK (P. J. Goodford, 1985 "A Computational

Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 25:849-857); the MCSS program available from Molecular Simulations, Burlington, MA (A. Miranker and M. Karplus, 1991 "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method", Proteins: Structure, Function and Genetics, 77:29-34); the AUTODOCK program available from Scripps Research Institute, La Jolla, CA (D. S. Goodsell and A. J. Olsen, 1990 "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure, Function, and Genetics, 5: 195-202); and the DOCK program available from University of California, San Francisco, CA (I. D. Kuntz et al, 1982 "A Geometric Approach to Macromolecule-Ligand Interactions", J. Mol. Biol,

767:269-288). Additional commercially available computer databases for small molecular compounds include Cambridge Structural Database, Fine Chemical Database, and CONCORD database (for a review see Rusinko, A, 1993 Chem. Des. Auto. News, 8: 44-47). Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or HUBP8 agonist/antagonist. Assembly may proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the 3D structure of HUBP8. This would be followed by manual model building using software such as Quanta or Sybyl software, CAVEAT program (P. A. Bartlett et al, 1989 "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc. 78, pp. 182-196), which is available from the University of California, Berkeley, CA; 3D Database systems such as MACCS-3D database (MDL Information Systems, San Leandro, CA) (see, e.g., Y. C. Martin, 1992 "3D Database

Searching in Drug Design", J. Med. Chem., 35:2145-2154); and the HOOK program, available from Molecular Simulations, Burlington, MA.

Other molecular modeling techniques may also be employed in accordance with this invention. See, e.g., N. C. Cohen et al, 1990 "Molecular Modeling Software and Methods for Medicinal Chemistry", J. Med. Chem., 33:883-894. See also, M. A

Navia and M. A. Murcko, 1992 "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2:202-210. For example, where the structures of test compounds are known, a model of the test compound may be superimposed over the model of the structure of the invention. Numerous methods and techniques are known in the art for performing this step, any of which may be used. See, e.g., P.S. Farmer, Drug Design, Aliens, E.J., ed., Vol. 70, pp 119-143 (Academic Press, New York, 1980); U.S. Patent No. 5,331,573; U.S. Patent No. 5,500,807; C. Verlinde, 1994 Structure, 2:577-587; and I. D. Kuntz, 1992 Science, 257: 1078- 1082. The model building techniques and computer evaluation systems described herein are not a limitation on the present invention.

Thus, using these computer evaluation systems, a large number of compounds may be quickly and easily examined and expensive and lengthy biochemical testing avoided. Moreover, the need for actual synthesis of many compounds is effectively eliminated. Once identified by the modeling techniques, the HUBP8 agonist/antagonist may be tested for bioactivity using the assays described herein.

The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. EXAMPLE 1 - THE ROLE OF DEUBIOUITINATED UBP8 IN TRANSCRIPTIONAL REGULATION IN THE EXPRESSION OF SAGA- DEPENDENT GENES A. To determine if UBP8 protein is a stable component of the three Gcn5 complexes, SAGA and the other Gcn5/Ada2/Ada3 containing complexes, SALSA and ADA, multi-step purification was employed. Ubp8 was tagged with two copies of the FLAG epitope (Ubp8-2FLAG) and transformed into a strain in which Ada2, a core component of each of the ADA, SAGA and SALSA complexes, was tandem affinity purified (TAP)-tagged. Whole cell extracts containing Ada2-TAP and UBP8-FLAG were chromatographically fractioned first via the TAP purification method (Puig et al, 2001 Methods, 24:218-229) to allow for straightforward purification and detection of Ubp8 in Ada2-containing complexes. TAP-purification was followed by MonoQ ion exchange chromatography to separate the three Gcn5/Ada2 complexes (Grant et al, 1997 Genes Dev. , 11:1640), resulting in near homogenous purification of the complexes. Even numbered fractions (14-32) from the MonoZ column were subjected to Western blotting to detect UBP8-FLAG, which was compared to ADA/SALSA/SSAGA-associated Ada3 and Gcn5, SALSA/SAGA-associated Spt3, and SAGA-specific Spt8. Samples from inputs and flowthroughs for calmodulin-bead binding and MonoQ column purification steps were included.

The polyacrylamide electrophoretic gel (not shown) illustrated that UBP8- FLAG cofractionates with the SAGA (fractions 34-36) and related SALSA(fraction 28) histone acetyltransferase (HAT) complexes but not with the ADA complex (fractions 18-22). To confirm that Ubp8 is stably and specifically associated with SAGA, FLAG immunoprecipitation of UBP8-FLAG was performed using the SAGA or ADA fractions. UBP8-FLAG was detected in the SAGA, but not ADA, fraction, and co-precipitated with Gcn5 and Ada3.

Thus, H2B ubiquitylation is involved in transcription of certain SAGA- dependent genes and Ubp8 is a component of the transcriptionally relevant SAGA complex. B. To confirm that Ubp8 is a stable component of SAGA Ubp8 was immunoprecipitated from fractions that contained the ADA and SAGA complexes (fractions 22 and 35, respectively) using anti-FLAG affinity resin (Sigma, St. Louis, MO) followed by Western analysis. The resulting gel (not shown) indicated that while protein input levels were similar, only Ada3, TAF60, Spt20 and Gcn5 co- immunoprecipitated in the SAGA-containing fraction but not in the ADA fraction. Thus, Ubp8 is a stable component of the SAGA complex, but not ADA.

C. The GALl and SUC2 genes were tested because they are well- characterized, highly inducible, and dependent upon chromatin regulation, making them candidates for dependence on additional histone modifications, such as ubiquitylation. Quantitative SI nuclease RNA protection analysis was performed on RNA isolated from cells grown in non-inducing or inducing conditions for each gene. The strains were either wild type (WT HTB1+) for histone H2B, or bore a single substitution of the ubiquitylation target residue in H2B (htbll-K123R, hereafter called htbl-KR) (Robzyk et al, 2000 Science 257:510). Fold induction was calculated as the level of expression under inducing conditions compared to repressing conditions and were presented as the percentage of WT induction (set at 100%). Repressing and inducing conditions respectively, were: GALl, 2% glucose and 2% galactose for 2.5 hours at OD₆5_0nm0.8; SUC2, 2% glucose and 0.05% glucose for 2.5 hours at OD₆₅o_nm <0.5. GALl RNA levels were reduced four-fold, while SUC2 levels were less affected by the htbl-KR substitution, suggesting that H2B ubiquitylation is involved in gene activation in addition to its role in telomeric silencing (Wood et al, 2003, Mol. Cell, 77:267; Sun and Allis, 2002 Nature, 418:104).

D. To determine whether ubiquitylation at K123 of histone H2B is linked to gene activation, chromatin double immunoprecipitation (ChDIP) and quantitative

PCR in real-time were performed. Formaldehyde crosslinked chromatin was obtained from a wild type S. cerevisiae strain HTB1+ or htbl-KR strain (which carries a substitution in the known H2B ubilquitylation site) bearing FLAG-tagged H2B and HA-tagged ubiquitin in glucose medium or during a timecourse in galactose-containing medium. The protocol for the ChDIP was as follows: Sonicated chromatin was immunoprecipitated initially with anti-FLAG antibody (M2, Sigma), then eluted with 3x FLAG peptide (Sigma). The eluates were then immunoprecipitated with anti-HA antibody (12CA5, Roche) before elution by boiling in 1% SDS, 50 mM Tris, pH8 buffer. Quantitative PCR analysis was done in real-time to examine the co-precipitated GALl promoter.

The level of immunoprecipitated chromatin from the GALl promoter and Int. V region is shown in FIG. 2 for WT strain and in FIG. 3 for htbl-KR strain as relative immunoprecipitation as a ratio of immunoprecipitated material to input chromatin. Immunoprecipitation of the GALl promoter was low in glucose, increased at 30 and 60 minutes after galactose induction, and then decreased at 90 and 120 minutes. PCR of samples from the htbl-KR strain exhibited a greatly reduced signal, identifying H2BK123 as a major ubiquitylated species in the ChDIP of WT samples. In addition PCR analysis of the first FLAG-H2B immunoprecipitate showed similar GAL 1 levels in all samples (not shown), indicating that promoter-associated H2B was constant during the time course. The PCR signal of a non-transcribed region of chromosome V (Int.V) was very low in all immunoprecipitated samples.

E. Since H2B ubiquitylation at the GALl promoter increased and then decreased during growth in galactose, it was compared to the pattern of transcription during the time course. RNA collected from cells grown under the ChDIP conditions was analyzed by SI nuclease assay. RNA was detected at 60 minutes and continued to increase to 120 minutes and the level was lower at each time point in the htbl-KR strain. Data from Figs. 2 and 3, as well as data from a GALl RNA SI nuclease assay (not shown) measuring GALl transcription during galactose induction in 2% galactose for 2.5 hours at OD₆oo_nm 0.8 in the same strains, were graphed together in FIG. 4. Thus, H2b ubiquitylation decreased during the same period in which GALl RNA showed substantial accumulation. These data indicate that H2B ubiquitylation increases early in induction and may be targeted for deubiquitylation prior to the buildup of high RNA levels.

F. Deletion of the gene encoding the ubiquitin hydrolase was not essential for SAGA integrity since deletion of the gene that encodes the ubiquitin hydrolase Ubp8 did not alter the ability to co-immunoprecipitate SAGA complex components ADA, TATA box-associated factor 60 (TAF60), Spt20, Gcn5 and Ubp8 with Ada2- TAP (figure not shown).

EXAMPLE 2: THE ROLE OF DEUBIOUITINATED UBP8 IN

TRANSCRIPTIONAL REGULATION

In higher eukaryotes, the C-terminal domain of histone H2A and, to a lesser extent, H2B serve as targets for ubiquitylation. Histone ubiquitylation is associated with transcriptionally active regions of DNA. In yeast, it has been shown that the C- terminus of histone H2B is the predominant target of monoubiquitylation (uH2B) by the Rad6 ubiquitin conjugase (Robzyk, K et al, 2000 Science, 257:501-504). Histone ubiquitylation in mammalian cells has been linked with gene activation. Recent work has also shown that the ubiquitylation of histone H2B also has a role in H3 methylation and silencing of certain genes, so the relevant role of this modification on expression may, as is seen with other histone modifications, be related to chromatin context and cross talk.

While not wishing to be bound by theory, the inventors hypothesized that UBP8 serves as a regulator of gene expression by targeting H2B for removal of ubiquitin, i.e., deubiquitylation. The large ubiquitin moiety can either serve as a "tag" for the recruitment of other factors or may open up the chromatin to allow the binding of these factors but must be removed in order for the components of the transcriptional machinery to function at the promoter.

To investigate the role of this modification in transcriptional regulation, the GALl gene was used as a model promoter, as the expression of this gene is tightly- regulated, carbon source dependent (induced in galactose-containing medium), and dependent upon SAGA and SWI/SNF activity. A bulk histone hemagglutinin (HA) Western gel (not shown) was generated to compare expression of H2B in parental wildtype control S. cerevisiae cells (WT), to cells in which the ubiquitylation site was mutated or evidenced defective transcription of gene products in the absence of UBP8: S. cerevisiae cells in which Ubp8 is deleted (ubp8A strain), the mutant S. cerevisiae strain htbl K123R that lacked the ability to ubiquitylate histone H2B due to a mutation at the H2B ubiquitylation site, and the strain swi (the spt20A control). Cell lysates fromN-terminally FLAG-tagged H2B in these strains was subjected to Western blot analysis performed with M2 anti-FLAG antibody (Sigma) to determine the levels of FLAG-H2B (blots not shown).

Because the ubiquitin moiety is relatively large (76 amino acids), it causes a shift in the electrophoretic migration of H2B. Ubiquitylated H2B was detected in the WT extracts, and this ubH2B species was not present in extracts from the htbl-KR or rad6Δ strains. The level of ubH2B was significantly increased in extracts prepared from the ubp8Δ strain lysates compared to a parental control. This species was approximately 9 kDa larger than native H2B. These significantly increased levels of a modified histone H2B species was also eliminated in the htbl-KR/ubp8Δ strain. Since the ubiquitin molecule is approximately 8.5 kDa, the size increase suggested that the modification could be the result of addition on the 8.5 kDa ubiquitin moiety. The loss of Spt20, which is required for SAGA integrity, also resulted in an increase in ubH2B levels in spt20A cells (data not shown). This indicates that UBP8 targets H2B- Ub for hydrolysis and controls bulk intracellular ubH2B levels specifically as a component within the SAGA complex.

The strain K123R had reduced levels of GALl RNA (less than 50% of wild type controls) under inducing conditions. Loss of Ubp8 resulted in a complete loss in

GALl transcription, which was also seen in the spt20A control. Defective growth of S. cerevisiae on galactose media was demonstrated in the absence of Ubp8. Following TAP-Ada2 purification and Western gel electrophoresis of Ubp8+ or bp8A strains of S. cerevisiae, it was shown that loss of Ubp8 does not alter SAGA stability, since the SAGA components Ada3, Gcn5 and TAF60 remain associated with Ada2 in both strains (gel not shown).

Since histone H2B ubiquitylation in S. cerevisiae was found to have a positive role in gene expression, it was expected that the deletion of Ubp8 would perpetuate GALl expression because of the inability to remove the ubiquitin moiety and re- establish chromatin structure. Surprisingly, the disruption of ubp8 was found to result in a dramatic decrease (ie., complete loss) of induced GALl transcription. In addition, growth of the ubp8A strain on galactose-containing medium showed growth that was substantially reduced from that observed in the htbl K123R strain, which itself showed poor growth compared to wild type. It is known that the expression of GALl is dependent upon the presence of the

SAGA complex and that a loss of SAGA results in a loss of GALl transcription. Deletion of the SAGA subunit Spt20, which is required for SAGA integrity (Bhaumik and Green, 2001 Genes Dev. 75(15): 1935-45) results in a loss f GALl. This did not seem to be the case for Ubp8 since the loss of that protein from SAGA did not result in any detectable decrease in the association of the SAGA subunits Ada3, Gcn5,

TAF60 and Spt3 from Ada2-TAP-containing co-activators. The data presented here indicates that Ubp8 is also essential for GALl transcription.

To confirm that this altered species was ubH2B, HA-immunoprecipitation of the lysates of the cells listed above with anti-FLAG antibody was followed by a Western blot against both FLAG and ubiquitin (not shown). These results revealed that the higher molecular weight species that was increased in ubp8Δ extracts was indeed monoubiquitinated H2B (ubH2B). This modification was not observed in strains in which the ubiquitylation site was mutated (htbl K123R) or in a strain in which RAD6 was deleted (radό- and htbl K123R rad6-). This data reveals that the SAGA-associated Ubp8 targets ubiquitinated histone H2B uH2B for deubiquitylation and hydrolysis.

Another approach examined the activity of UBP8 on ubH2B in vitro. SAGA complex was purified from Ada2-TAP -tagged UBP8+ or ubp8Δ strains using TAP- affinity followed by MonoQ fractionation. Equivalent amounts were incubated for 30 minutes with ubH2B, which bore either single or double tages in buffer containing 100 mM tris, pH8, ImM EDTA, ImM DTT, 5% glycerol, ImM PMSF, lμg/ml each of aprotinin and pepstatinA. Western blotting was perfomed with anti-FLAG to detect H2B or with anti-HA to detect ubH2B. SAGA-Ubp8+ deubiquitylated ubH2B (both 3HA-ub-FLAG-H2B and ub-FLAG-H2B), but SAGA lacking UBP8 did not have this activity (data not shown). As a third test of UBP8's deubiquitylating activity, the ChDIP assay was repeated to compared the level of ubH2B at the GALl promoter in the WT and UBP8Δ strains, using FLAG-H2B-HA-ub double immunoprecipitation. The ubH2B level was examined at 120 minutes of galactose induction, when ubiquitylation has decreased and the RNA level has risen. An increased level of ubH2B was detected in UBP8Δ relative to the WT strain (FIG. 5), indicating the ubH2B is a target of UBP8 at the GALl promoter and, in the absence of UBP8, H2B ubiquitylation remains high.

Thus, H2B ubiquitylation is required for full GAL 1 activation and MJBP8 modulates the level of ubH2B.

EXAMPLE 4 - ANALYSIS OF GALl GENE EXPRESSION IN THE ubp8Δ STRAIN.

The requirement for UBP8 in GAL 1 transcription was investigated. SI nuclease assay was performed as described above. GAL 1 expression was analyzed in WT, UBP8Δ, htbl-KR, htbl-KR/ubp8Δ double mutant, or spt20Δ strains. In the ubp8Δ strain, there was a dramatic reduction in mRNA levels, an effect that was much stronger than that observed in the htbl-KR strain (not shown); in the bp8Δ mutant, transcription was not detected even after 180 minutes of induction (not shown). This suggests that failure to ubiquitylate H2B is not as detrimental to transcription as the failure to remove the ubiquitylating moiety. Furthermore, the absence of transcription in ubp8Δ was partially suppressed in an htbl-KR/ubp8Δ double mutant (not shown). This partial suppression suggests that ubH2B is one target of UBP8 and that other targets of UBP8 exist in the GALl activation pathway. Expression of GALl is dependent on Spt20 in SAGA and transcription was as low in spt20Δ as in the ubp8Δ strain. Thus, UBP8, similar to Spt20, is critical for GALl transcription and both are involved in controlling ubH2B levels (the Spt20 effect of H2B ubiquitylation at GALl is presumably through its role in maintaining SAGA integrity).

In addition growth and doubling times in galactose medium correlated with the transcription results: strains bearing substitution of the H2B ubiquitylation site or disruption of UBP8 resulted in the reduced growth compared to WT. Generation times for the indicated strains were calculated from cultures grown in YP-2% galactose medium at 23°C: for WT, 6.7 hours; for ubpSΔ, 10 hours; for htbl-KR, 8.3 hours; and for htbl-KR/ ubp8Δ, 8.1 hours. These growth phenotypes were suppressed in the htbl-KR/ubp8Δ double mutant. These effects are likely due to catalytic activity of UBP8, since a substitution in one of the highly conserved residues within the protease domain had a comparable deleterious effect on growth as did deletion of UBP8, and was also suppressed by the htbl-KR mutation. A second SAGA- dependent gene, SUC2 also required UBP8 for full transcription, although the dependency was not as great as GALl (see FIG. 6). The apparent role of UBP8-mediated deubiquitylation in transcriptional activation of GALl suggests that, as a component of SAGA, UBP8 would also be present at the GALl promoter. Previous results have shown that Gcn5 increases at GALl during galactose induction. ChIP analysis was performed on NA-tagged Gcn5 or UBP8. Gcn5-3HA and Ubp8-3HA binding in WT strains was analyzed by ChIP at the GALl promoter in glucose (0 timepoint) and in galactose (60 and 120 minute timepoints). ChIP samples were normalized to input levels and glucose values were set to 1. The galactose samples were then calculated relative to the glucose values for fold relative IP. Association was compared to Int. V (Gen5-3HA sample) or Ubp8- 3HA. Both proteins increased at the GAL 1 promoter (but not at Int.V) in galactose compared to glucose and exhibited similar timing of increased association (FIG. 7).

The greater Ubp8 presence at 120 minutes compared to 60 minutes is consistent with a role in H2B deubiquitylation at the later time.

EXAMPLE 5 - EFFECT OF UBP8 DELETION ON SAGA FUNCTION. GAL4 STABILITY AND ON METHYLATION OF HISTONE H3 AT LYS-4

Several explanations for the severe GALl transcriptional reduction caused by a potential indirect effect of deleting ubp8 were examined. First deletion of UBP8, much like loss of Spt 20 could disrupt SAGA. To examine this possibility, Ada2-TAP was immunoprecipitated from WT and UBP8Δ extracts, followed by Western blotting to detect components of SAGA. The levels of Ada3, Gcn5, Spt20, and Taf560 were comparable in normal SAGA and in SAGA lacking UBP8 (not shown). Isolation of SAGA by TAP affinity (Ada2-TAP) and MonoQ purification following by analysis by Western blotting using 2 -fold serially diluted samples showed stability of both complexes (not shown). Previous data indicated that SPT7 in SAGA is ubiquitylated and that it, or other SAGA components, could be targeted by UBP8. Such ubiquitylation within SAGA could affect HAT activity. Threefold serial dilutions of equivalent amounts of WT or mutant SAGA were assayed for HAT activity on core histones (Sigma) by conventional methods. Background used was incorporation of ³H-acetate without added SAGA complex. Comparable amounts of wild type SAGA or SAGA lacking

UBP8 exhibited similar HAT activity using either core histones (FIG. 8) or nucleosomal histones (data not shown) as substrates. Thus, reduced GALl transcription in the ubp8A strain is not caused by either SAGA destabilization or reduced HAT activity. A third explanation for defective GALl transcription is that the absence of

UBP8 could alter GALl activator levels. Activators have been seen to be monoubiquitylated during gene activation, as well as polyubiquitylated during proteolysis. To test this, the Gal4 activator was 3HA -tagged in the WT or ubp8Δ strains. Equivalent protein samples from lysates collected from cells in glucose (0 minute) or induced in galactose (30, 60, 90 minutes) were immunoprecipiatated with anti-HA antibody and analyzed by Western blotting with anti-Gal4 DNA binding domain antibody (Upstate Biotechnology). The levels of Gal4 increased following galactose induction and stayed constant in both strains over time in galactose (not shown) indicating the UBP8 does not regulate Gal4 levels. It was recently shown in yeast that promoter-associated trimethylation of K4

(3meK) on histone H3 increases during gene activation. Moreover, analysis of bulk nucleosomal histones revealed that ubK123 on H2B is required for meK4 on H3. These observations suggested that 3meK4 might increase at the GALl promoter in a manner dependent upon ubiquitylation of K123. This was examined by 3meK4 ChIP analysis in WT and ubp8Δ strains. An antibody specific for 3mek4 of histone H3 (Abeam) was used in ChIP at the GALl promoter. Samples were taken from glucose (0 min) and at the indicated times after the switch to galactose. Samples were analyzed from WT, ubpδzl and htbl-KR strains as shown in FIG. 9. The level of trimethylation increased during galactose induction and this increase was abolished in the htbl-KR mutant. This regulation of 3meK4 by ubH2B suggested that UBP8 might function to regulate K4 methylation levels. Indeed, the level of 3meK4 was increased at each time point in the absence of UBP8 relative to its level in wild type cells. The level of 3meK4 was somewhat higher in glucose in ubp8Δ (less than two-fold); however, the 3meK4 effect was magnified by galactose (nearly four-fold at 90 minutes) when higher levels of UBP8 were detected at the GALl promoter.

These observations suggest that both ubiquitylation and deubiquitylation of H2B are involved in GALl transcriptional activation and function to regulate H3 K4 methylation levels at the GAL 1 promoter. H2B ubiquitylation levels increase early during gene induction, and are reduced when RNA accumulation reaches a high level. Furthermore, ubiquitylation of H2B is required for H3 K4 trimethylation at the promoter, and deubiquitylation functions to maintain a lower level of K4 trimethylation. Thus, either high levels of ubiquitylation or methylation may be detrimental to transcription.

UBP8, a stable component of the SAGA (and SALSA/SLIK) complex, targets histone H2B for deubiquitylation. The finding the UBP8-mediated H2B deubiquitylation is involved in transcriptional activation is surprising, since removal of histone modifications has previously been observed to oppose the effect of their addition, such as acetylation/deacetylation. However, recent observations indicate that deacetylases themselves are involved in transcriptional activation through histone targets, possibly through regeneration of a permissive chromatin state during multiple rounds of initiation. Ubiquitylation/deubiquitylation may serve a similar role during transcription of highly induced genes.

Loss of Ubp8 has a more severe transcriptional phenotype than loss of the ubiquitin site in H2B. This might be the case if ubiquitylation and another alteration, such as another histone modification, were functionally redundant, and also if deubiquitylation were absolutely required for a subsequent step in transcription. The ubiquitin moiety may serve a distinct mechanistic rose compared to acetyl, phosphate and methyl groups. The ubiquitin moiety is large relative to the other covalent modifications and to H2B itself. One plausible model as illustrated in FIG. 10, is that ubiquitylation opens chromatin at the promoter due to its bulkiness, to allow access by other chromatin enzymes such as methylases, but then must be removed for subsequent steps in initiation to occur. The high level of ubiquitin and/or methylation in the absence of UBP8 may thus be detrimental to progression to transcriptional initiation. These data suggest that ubiquitin is added and then obligatorily removed during the transcription cycle.

EXAMPLE 5 - THE HUMAN ORTHOLOG OF YEAST UBP8 IS PRESENT IN A HISTONE ACETYLTRANSFERASE COMPLEX IMPLICATED IN TRANSCRIPTIONAL REGULATION. An epitope-tagged version of hUBP8/USP22 SEQ ID NO: 1 was expressed in human cells. After lysis and immunoprecipitation, hUBP8/USP22 was assessed for association with known acetytransferase complex subunits. As evident, western blot analysis revealed that hUBP8/USP22 binds to both TRRAP and hGCN5 in vivo in human cells. TRRAP and hGCN5 are the direct orthologs of subunits present in the yeast SAGA complex, where yeast UBP8 resides. Further analysis of hUBP8/USP22 immunoprecipitates demonstrated that histone acetyltransferase activity was also associated with this complex, as true for the yeast ortholog.

EXAMPLE 6 - THE ROLE OF HUMAN UBP8 IN CANCER. Several parallel approaches are taken to assess the role of Ubp8 (Ubp22) in cancer.

A. Knock-Out Mice

In the first of such approaches, using mouse genetics, a mouse strain is produced which "knocks-out" the ubp8 gene. Tumorigenesis is assessed in the resulting animals. This involves folio wing the animals for the duration of their lifespan and assessing causes of mortality. Wild-type mice of the same strain will be followed as well to control for spontaneous tumor formation. If no tumors arise in ubp8 knockout mice, the ubp8 knockout is crossed onto a tumor-prone background to mimic the multi-hit genetics found in human cancer. Tumor-prone mice to be utilized for these studies have deletions in the genes encoding the major tumor suppressor proteins, p53, pl9ARF, p21CIP etc. If loss of the ubp8 gene is lethal, conditional knockouts are generated such that the gene is not eliminated until adulthood. This is accomplished by flanking the ubp8 gene with lox recombination sites. The mice are then crossed with a commercially available strain in which expression of the recombinase, CRE, can be induced by administering tetracycline to the animals.

B. Deletion ofubp8 in Cells

In a second, faster, cellular approach, ubp8 levels are reduced in cultured human cells by RNA treatment. The effectiveness of RNA is assessed by immunoblotting for the Ubp8 protein. Once conditions yielding the efficient loss of ubp8 are identified, the loss of Ubp8 function is assessed for its role in cell cycle progression. For example, the ubp8 deficient cells are stained using propidium iodide and their cell cycle profile determined. A block in a particular phase of the cell cycle, eg. Gl or S, which results from loss of Ubp8 function, indicates that Ubp8 is critical for cellular proliferation.

C. Analysis of Expression in Human Tumors

In addition, Ubp8 expression levels are examined in a panel of human tumors and these levels are compared to those of normal tissues. This is studied at the protein level, the enzymatic activity level and at the mRNA level. Obviously, a gain or loss of Ubp8 expression that is specific to tumor cells indicates a causal role in tumorigenesis.

In addition, loss of heterozygosity (LOH) mapping is conducted in human tumor cells to determine whether any non-random segregation of the ubp8 locus occurs. LOH at a given locus in cancer cells is a fairly accurate predictor of a role in the tumorigenesis process. Human Ubp8 RNA may also be employed in tumor cells to determine if inhibition of hUbp8 results in loss of tumorogenic properties.

EXAMPLE 7 - THE ROLE OF MJBP8 IN FUNGAL INFECTIONS

Fungal infections, such as those caused by the pathogenic yeast Candida albicans, are generally treated with drugs that target the cell membrane, either by inhibiting sterol biosynthesis enzymes (sterol biosynthesis inhibitors, SBI) or through membrane disruption by binding to ergosterol (polyenes), the principal sterol of fungal cell membranes. Due to toxicity issues associated with polyenes, most non-topical cases are treated with SBI that reduce cell growth allowing the host immune system time to clear the infection. In the immuno compromised patient where fungal infections are life-threatening, treatment with SBI often results in resistance and recurrent infection (Lortholary and Dupont, 1997 Clin. Microbiol Rev., 70:477-504). Characterization of SBI-resistant clinical isolates indicate that there are several mechanisms of resistance including increased expression of genes encoding multidrug efflux proteins and the SBI target enzyme (Franz et al, 1998 Antimicrob. Agents Chemother., 42:3065-12; Lopez-Ribot et al, 1998 Antimicrob. Agents Chemother.

42:2932-31). Study of many processes in Candida has been aided by the use of the non-pathogenic yeast Saccharomyces. cerevisiae as a model system (Pla etal, 1996 Yeast, 72:1677-1702). It has been shown in C. albicans and S. cerevisiae that exposure to SBI results in increased expression of genes in the ergosterol biosynthesis pathway (ERG genes) (Henry et al, 2000 Eukaryot. Cell, 7:1041-4; Bammert and Fostel, 2000, cited above).

The possible role of chromatin modifications on ERG gene regulation and SBI- susceptibility has been demonstrated previously. It has been shown that there is an inverse relationship between ROX1 and ERG expression following exposure to SBI and that the loss of Roxl results in hypersensitivity to SBI (Henry et al, 2002, cited above). Tupl/Ssn6 are general repressor proteins that are recruited to DNA by Roxl and are then able to bring histone deacetylases (HDAC) to regions of the genome to aid in transcriptional repression. Additionally, the inventors have found evidence for chromatin modifications in antifungal susceptibility as the loss of the histone H2B ubiquitylation sites results in a two-fold decrease in the SBI concentration required to inhibit growth to 50% (IC₅o) and 90% (MIC) of drug-free growth. Further, the loss of Ubp8 also results in up to a four-fold decrease, while the combination (htbl- KR/ubp8Δ) results in a synergistic effect where the IC₅₀ and MICs have decreased up to eight-fold.

Thus, targeting ubiquitylation in fungi has a synergistic effect on antifungal therapy and aids in treatment of fungal infections. In addition, since many of the enzymes in the ergosterol pathway are also utilized by humans for the production of cholesterol and anticholesterol drugs (lovastatin) and can also inhibit ergosterol biosynthesis in yeast, compounds that inhibit chromatin modifying enzymes have a role in cholesterol reduction through reduced gene expression of sterol biosynthesis enzyme (Lorenz and Parks, 1990 Antimicrob. Agents Chemother., 34: 1660-5).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.

Claims

CLAIMS:

1. A composition that alters the expression of human ubiquitin protease 8 (UBP8) or a homolog thereof and reduces, eliminates or enhances the deubiquitylating activity thereof in a cell.

2. The composition according to claim 1, which is an antisense oligonucleotide that hybridizes to a nucleic acid sequence encoding SEQ ID NO: 1 or a fragment thereof, and inhibits expression of human UBP8 in a cell.

3. The composition according to claim 1, which is a double stranded ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding SEQ ID NO: 1, which ribonucleotide sequence inhibits expression of human UBP8 in a cell.

4. The composition according to claim 1, further comprising a detectable label.

5. The composition according to claim 1, which is a protein ligand of hUBP8.

6. The composition according to claim 5, wherein said ligand is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or a recombinant antibody of classes IgG, IgM, IgA, IgD and IgE, a Fab, Fab' or F(ab')2, or Fc antibody fragment thereof which binds hUBP8, a single chain Fv antibody fragment, a recombinant construct comprising a complementarity deterrnining region of an antibody, a synthetic antibody or a chimeric antibody or a humanized antibody construct which shares sufficient CDRs to retain functionally equivalent binding characteristics of an antibody that binds said hUBP8.

7. A diagnostic kit comprising a composition that inhibits the expression of human ubiquitin protease 8 (UBP8) SEQ ID NO: 1 or ahomolog thereof and reduces or eliminates the deubiquitylating activity thereof in a cell, a detectable label, and suitable components for detection of said label.

8. The kit according to claim 7, wherein said composition is an antisense oligonucleotide that hybridizes to a nucleic acid sequence encoding SEQ ID NO: 1 or a fragment thereof, and inhibits expression of human UBP8 in a cell.

9. The kit according to claim 7, wherein said composition is a double stranded ribonucleotide sequence made up of a sense and antisense region corresponding to a sequence within the nucleotide sequence encoding SEQ ID NO: 1, which ribonucleotide sequence inhibits expression of human UBP8 in a cell.

10. The kit according to claim 7, wherein said composition is a protein ligand of hUBP8.

11. The kit according to claim 10, wherein said ligand is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or a recombinant antibody of classes IgG, IgM, IgA, IgD and IgE, a Fab, Fab' or F(ab')2, or Fc antibody fragment thereof which binds hUBP8, a single chain Fv antibody fragment, a recombinant construct comprising a complementarity determining region of an antibody, a synthetic antibody or a chimeric antibody or a humanized antibody construct which shares sufficient CDRs to retain functionally equivalent binding characteristics of an antibody that binds said hUBP8.

12. The kit according to claim 7, designed for detecting the tumorigenic potential of a cell.

13 The kit according to claim 7, further comprising components for a hUBP8-mediated deubiquitylation assay.

14. A recombinant molecule comprising a nucleic acid sequence encoding SEQ ID NO: 1 under the regulatory control of sequences that direct the expression of hUBP8 in ahost cell.

15. A method of identifying an agonist or antagonist of hUBP8 activity or expression, said method comprising the steps of:

(a) contacting a cell capable of expressing hUBP8 with a suitable amount of a test compound, and assessing the level of expression or activity of hUBP8 in said cell;

(b) assessing the level of expression or activity of hUBP8 in an otherwise identical cell that has not been contacted with said test compound; and

(c) comparing the levels of hUBP8 expression or activity, wherein an altered level of expression or activity of said hUBP8 in said cell (a) compared with the level of expression or activity of MJBP8 in said cell (b) indicates that said test compound is an agonist or antagonist of hUBP8 activity.

16. A method of identifying an agonist or antagonist of hUBP8 activity, said method comprising the steps of: screening a test compound in ahUBP8-mediated deubiquitylation assay, and measuring the deubiquitylation activity in said assay; wherein the substantial absence of, or reduction in, said enzymatic activity in said assay in the presence of said test compound indicates that said test compound is an antagonist of hUBP8 activity; and wherein a substantial increase in said enzymatic activity in said assay in the presence of said test compound indicates that said test compound is an agonist of hUBP8 activity

17. The method according to claim 16, further comprising the step of contacting a mixture that normally demonstrates hUBP8-mediated enzymatic activity with a test compound; and assaying said mixture and test compound for said activity, wherein the absence or reduction of said activity in the presence of said test compound indicates that said test compound inhibits hUBP8 activity.

18. The method according to claim 16, further comprising the step of contacting a mixture that normally demonstrates hUBP8-mediated enzymatic activity with a test compound; and assaying said mixture and test compound for said activity, wherein an increase in said activity in the presence of said test compound indicates that said test compound enhances hUBP8 activity.

19. The method according to claim 17 or 18, wherein said mixture comprises hUBP8 protein, a histone H2B substrate, a label, and a buffer.

20. The method according to claim 17 or 18, wherein said assaying step comprises separating said labeled hUBP8 protein from said system, and performing gel electrophoresis thereon, and immunoblotting said gel with an anti-ubiquitin antibody, wherein the detection of ubiquitin in the gel by said antibody demonstrates hUBP8- mediated enzymatic activity.

21. The method according to claim 17 or 18, wherein said assay is an in vitro assay..

22. A method of retarding the growth of a cancer cell, said method comprising administering to said cell a hUBP8 inhibitor that reduces oncogene transcription mediated by hUBP8 enzymatic activity.

23. The method according to claim 22, further comprising administering to said cancer cell a chemotherapeutic agent or therapy.

24. A method of retarding the growth of a cancer cell, said method comprising administering to said cell ahUBP8 agonist that increase transcription of a tumor suppressor gene mediated by hUBP8 enzymatic activity.

25. The method according to claim 24, further comprising administering to said cancer cell a chemotherapeutic agent or therapy.

26. A method of assessing the sensitivity of a tumor cell to an agent that disrupts deubiquitylation function, said method comprising examining said cell for a characteristic selected from the group consisting of:

(a) the presence of a h UBP8 gene;

(b) the presence of hUBP8 protein; and

(c) the presence of hUBP8-mediated deubiquitylation activity; wherein the identification of any of said characteristics provides an indication that said tumor cell is sensitive to said agent.

27. A method of determining tumorigenic potential of a cell comprising examining said cell for the over-expression of hUBP8 enzymatic activity in said cell, wherein the over-expression of said activity indicates that said cell is predisposed to tumorigenesis.

28. The method according to claim 27, wherein said examining step is selected from the group consisting of an enzymatic assay, Western immunoblotting, enzyme-linked immunoassay, immunofluorescence and immunohisto chemistry.

29. A method for determining tumorigenic potential of a cell comprising examining said cell for hUBP8-mediated deubiquitylation activity, wherein the absence or underexpression of said activity in a cell carrying a tumor suppressor gene indicates that the cell is predisposed to tumorigenesis.

30. The method according to any of claims 15-29, wherein said hUBP8 is selected from the group consisting of

(a) SEQ ID NO: 1 or a complementary sequence thereof, (b) a sequence having a homology of at least 50% to the sequence (a) according to a selected algorithm and comprising a protein or peptide having ubiquitylation/deubiquitylation activity, and

(c) a fragment of (a) or (b) comprising a peptide having deubiquitylation activity.

31. A method for treating or diagnosing a disease characterized by expression of a gene that relies upon deubiquitylation for transcription, comprising the step of: inhibiting the expression or activity of hUBP8 SEQ ID NO: 1 in cells of a subjuct have said disease.

32. The method according to claim 31, wherein said disease is selected from the group consisting of: inflammation, viral infection, fungal infection, and cancer.