WO2003035844A9 - Novel histone methyltransferase and methods of its use - Google Patents

Abstract

Disclosed herein is a novel histone methyltransferase and nucleic acid molecules encoding it, as well as variants of these molecules. Also provided are methods of using these molecules to influence histone methylation and/or DNA methylation, as well as methods of screening for compounds that influence histone and/or DNA methylation. This disclosure also provides various kits.

Description

NOVEL HISTONE METHYLTRANSFERASE AND METHODS OF ITS USE

STATEMENT OF GOVERNMENT SUPPORT

The United States Government may have certain rights in this application pursuant to grants (including Grant No. GM356900) awarded by the National Institutes of Health.

FIELD This disclosure relates to histone methyltransferases, nucleic acids encoding such, and methods for the use of these molecules. It also relates to methods for influencing DNA methylation and gene activation, as well as systems and methods for identifying molecules that influence DNA methylation.

BACKGROUND

DNA methyltransferases (also referred to as DNA methylases) transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on a DNA molecule. Several biological functions have been attributed to the methylated bases in DNA. The most established biological function is the protection of the DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has been observed only in bacteria. Mammalian cells possess at least three methyltransferases; one of these (DNMT1) preferentially methylates cytosine residues on the DNA that are 5' (upstream) neighbors of guanine (forming the dinucleotide CpG). This • methylation has been shown by several lines of evidence to play a role in influencing gene activity (e.g., activation and/or silencing), cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin and Riggs, eds. in DNA Methylation Biochemistry and Biological Significance, Springer-Verlag, New York, 1984).

When gene sequences contain multiple methylated cytosines, they are less likely to be expressed (Willson, Trends Genet. 7: 107-109, 1991); if a site in the promoter of the gene is methylated, gene silencing often occurs. Hence, in general if a maternally inherited copy of a gene is more highly methylated than the paternally inherited copy, the paternally inherited copy will be expressed more effectively. Similarly, when a gene is expressed in a tissue-specific manner, that gene often will be unmethylated in the tissues where it is active but highly methylated in the tissues where it is inactive.

Incorrect methylation is believed to be the cause of some diseases such as Beckwith- Wiedemann syndrome and Prader-Willi syndrome (Henry et al., Nature 351.665, 1991 ; Nicholls et al, Nature 342:281 , 1989), as well as a contributing factor in many cancers (Laird and Jaenisch, Hum. Mol. Genet. 3 Spec. No.:1487-1495, 1994). Expression of a tumor suppressor gene can be abolished by de novo DNA methylation of a normally unmethylated 5' CpG island (lssa et al, Nature Genet., 7:536, 1994; Herman et al., Proc. Natl. Acad. Sci., U.S.A., 91 :9700, 1994; Merlo et al., Nature Med., 1 :686, 1995; Herman et al, Cancer Res., 56:722, 1996; Graff et al, Cancer Res., 55:5195, 1995; Herman et al, Cancer Res., 55:4525, 1995). Such hypermethylation has now been associated with the loss of expression of VHL, a renal cancer tumor suppressor gene on 3p (Herman et al, Proc. Natl. Acad. Sci. USA, 91 :9700-9704, 1994), the estrogen receptor gene on 6q (Ottaviano et al, Cancer Res., 54:2552, 1994) and the H19 gene on 1 lp (Steenman et al, Nature Genetics, 7:433, 1994). Similarly, a CpG island has been identified at 17pl 3.3, which is aberrantly hypermethylated in multiple common types of human cancers (Makos et al, Proc. Natl. Acad. Sci. USA, 89:1929, 1992; Makos et al, Cancer Res., 53:2715, 1993; Makos et al, Cancer Res. 53:2719, 1993). This hypermethylation coincides with the timing and frequency of 17p losses and p53 mutations in brain, colon, and renal cancers. Many effects of methylation are discussed in detail for instance in published International patent application PCT/US00/02530.

Because of the importance of DNA methylation in disease, understanding the mechanisms controlling such methylation and being able to influence it would be vital scientific and medical advances.

SUMMARY OF THE DISCLOSURE

A novel histone methyltransferase (HMTase) has been identified in Neurospora, and is termed herein DIM-5. Nucleic acids encoding this enzyme, and the protein itself, are provided herein. Through characterization of DIM-5, it has been surprisingly discovered that the methylation of histones influences and controls the methylation of DNA presumably in regions proximal to the methylated histones. Thus, the systems provided herein illustrate for the first time a pathway involved in influencing and controlling DNA methylation and thereby controlling gene expression in eukaryotic cells. This disclosure provides methods and compositions useful in regulating and influencing histone methylation, for instance methylation of the lysine 9 residue of histone H3, and particularly trimethylation in some embodiments, and thereby altering DNA methylation in eukaryotic cells. Also provided are methods for identifying molecules that interact with HMTases, for instance which inhibit or enhance the activity or histone-binding affinity or specificity of a HMTase, and therefore which are useful in influencing histone and/or DNA methylation in a target cell.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Growth deficiencies of dim-5 strains. Rates of apical growth often wildtype (dim*) and ten mutant (dim-5) progeny of N2140 (dim-5, leu-2, pan-2, A) X N185 (trp-4, a) were measured at 32 C using "race tubes" containing 1.5% sucrose Vogel's medium with pantothenate. The average growth rates and standard deviations ofthe wildtype and mutant strains were 4.9 +/-0.1 and 2.4 +/- 0.7 mm/hour, respectively. The growth rates ofthe ten wildtype strains were so similar that their plots are virtually superimposed.

Figure 2. DNA methylation defect of dim-5 strains. Genomic DNA of wildtype (wt), a DNA methyltransferase mutant (dim-2) and the dim-5 mutant were digested with Dpnll (D) or Sau3A\ (S) and analyzed by gel electrophoresis and Southern hybridization using probes for the indicated five methylated chromosomal regions. The DNAs were stained with ethidium bromide (total) to reveal the total digestion profiles generated with these isoschizomers. Blots were reprobed for unmethylated regions to confirm that digests were complete. The 9A20 region in the dim-5 strain shows an RFLP relative to the wild-type and dim-2 strains.

Figure 3. Identification of dim-5 by genetic mapping and complementation. FIG 3A, Map of genes revealed by BLASTx in leu-2/trp-4 interval of contig 1.1 18 (Assembly version 1. Neurospora Sequencing Project, Whitehead Institute/MIT Center for Genome Research, 2001). Regions of marked similarity (alignment scores >80) to genes in NCBI database are indicated (rectangles). A segment that was amplified to test two dim-5 candidates (hibD gene; blue; homologue of S. pombe clr4; red with white intron) is shown expanded. FIG 3B illustrates complementation ofthe dim-5 mutation, dim-5 strain N2145 was co-transformed with pBT6 and 2.0kb Pstl-Xbaϊ or 1.4 kb Mlul-Xbal fragment. Genomic DNA from representative bml^R transformants was analyzed by Southern hybridization for DNA methylation in the ψ63 region using £coRI (E) and SαwHI (B). Methylation ofthe Ba Kl site (Margolin et al, Genetics 149:1787-1797, 1998) gives a 6.4 kb fragment, as illustrated. Results for representative transformants are shown with positive (wt) and negative (dim-5) controls.

Figure 4. Quelling of dim-5 relieves silencing of hph in N644. FIG 4A, Cartoon of methylated (hyg^s) or unmethylated (hyg^R) hph gene, flanked by methylation-inducing DNA segments that had been subjected to RIP (Irelan & Selker et al, Genetics 146:509-523, 1997). FIG 4B,

Conidia of 24 random transformants of strain N644 generated with pBT6 alone or with pBT6 plus EcoRV digested PCR fragment containing dim-5 were spot-tested for growth in the absence or presence of hygromycin. FIG 4C, Effect of dim-5 gene fragments on methylation at ψ63. The transformants illustrated in FIG 4B were analyzed for methylation as in FIG 3. Figure 5. FIG 5A. Amino acid alignment of conserved regions of N. crassa DIM-5

(accession AF419248), S. pombe Clr4 (accession 060016), Mus musculus Suv39h2 (accession NM 022724.1) and Drosophila melanogaster Su(var)3-9 (accession S47004) generated with CLUSTALW (available through various public sources, including the University of Illinois Biology Workbench and the National Laboratory for Computational Science and Engineering at the University of California, San Diego). Amino acid coordinates and species abbreviations are shown at the left. The position of a nonsense mutation in allele dim-5WT\ is indicated (*). Amino acid residues conserved among all four proteins are highlighted in red and indicated in "consensus"; residues conserved in three ofthe four proteins are highlighted in yellow; dashes indicate gaps in the alignment. The black and orange lines mark the extent ofthe cysteine-rich and SET domains, respectively. FIG 5B. Protein domain organization of DIM-5 and related proteins aligned at their C-termini with predicted number of amino acids and locations of Chromo, SET, and cysteine-rich (C-rich), domains indicated. The N-terminal endpoints of recombinant proteins made in this study or previously (Rea et al, Nature 406:593-599, 2000) are indicated by vertical dashed lines.

Figure 6. Histone methyltransferase activity of recombinant dim-5 protein. Purified histones (-20 μg; Boehringer Mannheim) were incubated for 6 hours at 20° C with or without purified GST-DIM-5 fusion protein (GST-DIM-5; ~lμg) and 2.75 μCi S-adenosyl-[methyl-³H]-L- methionine, as methyl donor. Reaction products were fractionated by PAGE (16.5%), stained with Coomassie Blue (left) and then fluorographed (right) to detect methylation. The positions of selected size standards, intact recombinant protein (*) and core histones are indicated.

Figure 7. Reactivation of hph and loss of DNA methylation induced by transformation of a dim⁺ strain with mutant alleles of histone H3 gene (hH3). FIG 7A, Sequence of N-terminal segment of Neurospora histone H3 with residues presumed to be subject to methylation (m), acetylation (a) or phosphorylation (p) in red and residue implicated in silencing highlighted in yellow. FIG 7B,

Transformation experiment. Strain N644 (see FIG 4) was co-transformed with pBT6 and wildtype or mutant versions of hH3, bml^R transformants were selected en masse on bml and tested for drug resistance. FIG 7C, Southern analysis and sequencing of DNA from hyg^R transformants. DNA of representative transformants (T) and a wildtype (wt) control grown non-selectively was analyzed with £coRI and BamWλ for methylation (m) at ψ63 as in FIG 3 and for ectopic alleles of hH3. Direct sequencing of hH3 PCR products confirmed the presence of both the wildtype and mutant alleles in representative strains (sequencing chromatograms).

Figure 8: Structure-Based Sequence Alignment of SET Proteins. The alignment includes (1) all known members of human SUV39 family: SUV39H1 (accession NP_003164), SUV39H2 (accession NP_078946), G9a (accession S30385), Eu-HMTl (accession AAM09024), SETDB1

(accession NP_036564), and CLLL8 (accession NP_1 14121); (2) proteins (in bold) that have been shown to have HKMT activity from various species: N. crassa (Nc) DIM-5 (accession AAL35215), S. pombe (Sp) Clr4 (accession 060016), A. thaliana (At) SUVH4 or KRYPTONITE (accession AAK28969), S. cerevisiae (Sc) SET1 (accession P38827) and SET2 (accession P46995), human SET7 (accession XP_040150) and PR-SET7 (accession AAL40879); (3) three bacterial SET proteins: Xylella fastidiosa (Xf) SET (accession AAF84287), Bradyrhizobium japonicum (Bj) SET (accession Q9ANB6), and Chlamydophila pneumoniae (Cp) SET (accession AAD19016); and (4) human EZH2 protein, which appears inactive in vitro (Rea et al., Nature 406:593-99, 2000).

The residue number and secondary structural elements of DIM-5 (helices A-J and strands 1- 17) are shown above the aligned sequences. Dashed lines indicate disordered regions. Specific regions include the N terminus (residues 25-62), the pre-SET (residues 63-146), the SET (residues 147-236 and 248-277), the signature motifs (SET residues 237-247 and 278-285), and the post-SET (residues 299-308). The amino acids highlighted are invariant (white against black) and conserved (white against gray) among almost all members ofthe SUV39 family. The number in parentheses indicates the number of amino acids inserted relative to the alignment. The lowercase letters above the sequences indicate the structural/functional role ofthe corresponding DIM-5 residues: "h" indicates intramolecular hydrophobic interaction, "n" indicates intramolecular nonhydrophobic (polar or charge) interaction, "z" indicates zinc coordination, asterisk indicates structural residue Gly or Pro, and "s" indicates surface-exposed residues potentially important for cofactor or substrate binding or catalysis. The red circles mark the residues that were mutated in Example 1 1.

Figure 9: DIM-5 Structure. FIG 9A Front view of ribbons diagram (Carson, 1997) (top, stereo; bottom, mono). The protein is shaded according to the regions indicated in FIG 8, and the three zinc ions are shown as balls (as in FIG 9C). The difference electron density map (black), contoured at 5.5σ above the mean, indicates the presumed cofactor binding site (supported by tests on mutant forms).

FIG 9B Side view. A dashed line indicates the disordered amino acids between strand βl7 (magenta) and the post-SET segment. FIG 9C Stereo diagram ofthe triangular zinc cluster. Three zinc ions are shown as three numbered balls, the bridging (B) and nonbridging (NB) cysteine residues are indicated. The pre-SET sequence of DIM-5 is shown above. Both Cys-rich segments coordinate the one (red) and two (blue) zinc ions jointly, while the three (green) zinc ion is coordinated solely by the fϊve-Cys segment. Figure 10: Enzymatic Properties of Recombinant DIM-5. This figure shows HKMT activity as functions of (FIG 10A) temperature, (FIG 10B) salt concentration, (FIG 1 OC) pH, and

(FIG 10D) AdoMet crosslinking as a function of pH. The buffers used were 50 mM Na citrate for pH 5.0-6.0, MES for pH 6.0-6.5, HEPES for pH 7.0-7.5, Tris for pH 8.0-8.5, Bicine for pH 9.0, and glycine for pH 9.35-10.7. To rule out the potential inhibitory effect of Na citrate, both Na citrate and Mes are used for pH 6.0. FIG 10E shows relative activities of DIM-5 mutants with conservative point mutations. All mutant proteins were expressed to level similar to that ofthe wild-type, though some were less soluble, and all were monomeric, suggesting that none ofthe mutations caused gross aggregation of the protein. Various amounts of mutant enzymes were used, the activities were compared to that of serial dilutions of wild-type enzymes purified in the same way, and the specific activity of mutant proteins relative to wild-type was estimated. The activities shown are averages of at least two measurements.

FIG 10F shows fluorographic results of an AdoMet crosslinking experiment at pH 8.0, along with results of Coomassie staining to control for the amount of mutant protein tested in FIG 10E.

Figure 11: The Cofactor Binding and Active Site in DIM-5. Close-up view ofthe proposed cofactor binding site and the adjacent active site (top, stereo; bottom, mono). The difference electron density map (grey hatch structure) is contoured at 5.5σ; the water molecules are numbered 1-4. Dashed lines indicate the hydrogen bonds. The water at site 2 is hydrogen bonded to the main chain carbonyl oxygen atom of R238 and to the water molecules at sites 1 and 3, which in turn interacts with the side chain carbonyl oxygen of N241 and the side chain hydroxyl oxygen of Y204, respectively.

Figure 12: Putative Peptide Binding Cleft of DIM-5

FIG 12A is a front view of GRASP surface (Nicholls et al, Proteins 11 :281-296, 1991). The difference electron density map (black) is contoured at 5.5σ. Strand βlO includes L205, F206 and A207; N241, H242, and Y283 are shown below, and C244 includes Q5, T6, A7, R8, K9, and S10, each of which is shaded.

FIG 12B is a superimposition image of Drosophila HP1 β strand (Jacobs and Khorasanizadeh, Science 295:2080-2083, 2002; PDBcode 1KNA) and DIM-5 strand B10. Dashed lines indicate the hydrogen bonds between HP1 and H3 peptide. The DIM-5 residues on the other side ofthe HP1 peptide are Y283, V284, and N285. The dimethylated (methyl groups in black) target nitrogen atom occupies water site 2 (see FIG 1 1). The sequence of histone H3 peptide is shown at the bottom; both K4 and K14 are five residues away from K9.

FIG 12C shows the docked H3 peptide lying in the putative peptide binding cleft. The cleft extends in both directions following turns as indicated.

FIG 12D is a superimposition of active site NPPY residues of Taql DNA-adenine amino MTase (Goedecke et al, Nat. Struct. Biol. 8: 121-125, 2001; PDB code 1G38) and the proposed DIM-5 active site residues N241, H242, and Y283. The Tyr in both cases is hydrogen bonded to a main chain amide nitrogen atom (dashed bonds). Figure 13: Metal Chelators Inhibit DIM-5 Activity. FIG 13 A shows analysis of zinc content of DIM-5 with and without EDTA treatment. DIM-5 protein was incubated with 20 mM EDTA for two days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Expl) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. FIG 13B is a bar graph showing relative activity. Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hours at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present.

FIG 13C shows fluorographic results of AdoMet crosslinking in the presence ofthe indicated levels of EDTA.

Figure 14: DIM-5 trimethylates lysine 9 of histone H3 efficiently in vitro. FIG 14A illustrates DIM-5 activity with histone H3 peptide unmodified, dimethylated, or trimethylated at Lys9. 0.5μg unmodified, di- or trimethyl-Lys9 histone H3 peptide (ARTKQTARKSTGGKA; positions 1-15) was incubated for one hour at 16 °C with 0.5 μg purified recombinant DIM-5 protein (Zhang et al., Cell 111:117-127, 2002) and 1.1 μCi S-adenosyl-[methyl- 3H]-L-methionine (³H SAM). Reaction products were fractionated by SDS-PAGE (16.5%), fixed with 10% gluthalaldehyde for 15 minutes and fluorographed to detect methylation as described (Tamaru & Selker, Nature 414:277-283, 2001). Each peptide was assayed independently twice (1 & 2).

FIG 14B and 14C illustrate the determination of amino acid position of H3 peptides methylated by DIM-5. DIM-5 reactions were carried out as in panel A with either unmodified (FIG 14B) or dimethy I-Lys9 (FIG 14C) H3 peptides (ARTKQTARKSTGGKAPRKQL; positions 1 -20). Reaction products were subject to amino terminal sequencing and incorporation of labeled methyl groups into individual amino acid residues was detected by scintillation counting of each amino acid fraction. The amino acid sequence is shown below and lysine (K) residues are numbered. Fractions containing free ³H SAM are indicated in gray. FIG 14D through FIG 14G show mass spectrometry analyses of DIM-5 products from unmodified or dimethyl-Lys9 H3 substrates. Reactions were initiated by addition of 100 μM unmodified (FIG 14D) or dimethyl-Lys9 (FIG 14E) H3 substrate (TKQTARKSTGGKA; positions 3- 15) to a 20 μl mixture of 50 M Glycine (pH 9.8), 10 mM DTT, 750 μM S-adenosyl-L-methionine and 2 μg DIM-5. After incubation at room temperature for the indicated times, reactions were stopped by addition of TFA to 0.5%. Peptide masses were measured by MALDI-TOF on an Applied Biosystems Voyager System 4258 using α-Cyano-4-hydroxycinnamic acid as matrix. Relative intensity (%) of each mass was measured independently twice and plotted versus time. Mass identities ofthe H3 peptide with different Lys9 methylation status are indicated; H3, Mono, Di and Tri designate unmethylated, mono-, di- and trimethyl-Lys9, respectively. Examples of mass profiles at 30 minutes with unmodified H3 peptide (FIG 14F) and 2.5 minutes with the dimethyl-Lys9 H3 peptide (FIG 14G) are shown.

Figure 15: Characterization of antibodies and genomic regions to analyze by ChlP. FIG 15A illustrates the specificity of antibodies for methylated Lys9 of histone H3. Starting with l μg of unmodified, dimethyl-Lys9 or trimethyl-Lys9 H3(l-15) peptides, samples from a 2x dilution series were spotted onto a nitrocellulose membrane, stained with Ponceau S (bottom) and analyzed by immunoblotting (top) using anti-H3 dimethyl-Lys9 (Nakayama et al., Science 292:110- 1 13, 2001) or anti-H3 trimethyl-Lys9 (Cowell et al., Chromosoma 1 1 1 :22-36, 2002) antibodies. Antibody-peptide complexes were detected by horseradish peroxidase-conjugated goat anti-rabbit IgG and chemiluminescence (Pierce). FIG 15B shows Southern analyses of unmethylated (pen and hH4) and methylated (η and punt) chromosomal regions of Neurospora crassa. Genomic DNA ofthe wild type strain N150 (740R23-IVA), dim-2 strain N1877 (dim-2::hph his-3 mat a) (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001) and dim-5 strain N2269 (dim-5 mat A) (Tamaru & Selker, Nature 414:277-283, 2001) was digested with DpnW (D) or Sauikλ (S) and analyzed by gel electrophoresis and Southern hybridization as described (Foss e/ al, Science 262:1737-1741, 1993) using probes for the indicated regions. The pew, hH4, η and punt probes were generated by PCR from the wild-type strain N 150. FIG 15C is a diagram of endogenous euchromatic ura4 allele carrying deletion (ura4DS/E) and an ectopic heterochromatic ura4 allele integrated in cenl (cenl ^': :ura4) in Schizosaccharomyces pombe strain SPG 1355. PCR with primers ura4DS/E#\ and ura4DS/E#2 (Nakayama et al, Cell 101:307-317, 2000) (indicated by arrows) generates products of distinctive lengths from ura4DS/E and cenl::ura4. The central component (cntl) and part ofthe inverted repeats (imrlR and otrlR) of cenl are also represented.

Figure 16: Trimethyl-Lys9 H3, but not dimethyl-Lys9, is associated with methylated DNA regions of Neurospora.

ChIP with N. crassa and S. pombe extracts was carried out as described herein. Mixtures of extracts of N. crassa wild-type strain 740R23-IVA and S. pombe strain SPG 1355 were incubated with anti-H3 dimethyl-Lys4 (Upstate Biotechnology), anti-H3 dimethyl-Lys9 (Upstate Biotechnology) or anti-H3 trimethyl-Lys9 (Cowell et al., Chromosoma 111 :22-36, 2002) antibodies, or incubated without antibody (no antibody control). Total DNA, immunoprecipitated DNA and mock control DNA were subject to duplex PCR to amplify pairs of unmethylated and naturally methylated (m) DNA regions of N. crassa (FIG 16A and 16B); primers described herein SEQ ID

NOs: 22-29, or a pair of heterochromatic (cenl::ura4) and euchromatic (ura4DS/E) ura4 regions of S. pombe (FIG 16C); primers described in SEQ ID NOs: 22-29. Products were fractionated by gel electrophoresis and autoradiographed. No PCR product from N. crassa DNA was detected with anti- dimethyl H3 (Lys9) antibody. Bar graphs below the PCR products represent enrichment of η relative topcn (¥\G 16A), punt relative to hH4 (FIG 16B) and cenl::ura4 relative to ura4DS/E (FIG 16C) with the indicated antibody. The relative enrichment was normalized relative to the ratios obtained from total DNA. Results from two PCR reactions from each of two independent ChIP experiments were averaged.

Figure 17: DIM-5 is responsible for histone H3 Lys9 trimethylation associated with methylated DNA.

ChIP experiments with chromatin from the N. crassa wild-type strain 740R23-IVA, dim-5 strain #2269 (dim-5 mat A) (11) and dim-2 strain N1877 (dim-2::hph his-3 mat a) (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001) were carried out using the indicated antibodies as described in Fig. 3 but in the absence ofthe S. pombe chromatin. Duplex PCR was conducted to amplify (FIG 17A) pen and punt (top), hH4 and punt (bottom), (FIG 17B) pen and η (top) or hH4 and η (bottom) from the DNA samples. Enrichment of punt (FIG 17 A) and η (FIG 17B) relative to pen or hH4 are represented as in FIG 16.

SEQUENCE LISTING The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows a dim-5 encoding sequence. This sequence is believed to include an intron from position 49 through 172, which when spliced out produces DIM-5' (SEQ ID NO: 2). An alternative splicing variant is also possible, wherein the intron is from position 49 through 199; when spliced out, this produces DIM-5" (SEQ ID NO: 4).

SEQ ID NO: 2 shows the nucleic acid sequence of DIM-5', one of two splice variants, and the encoded amino acid sequence. SEQ ID NO: 3 shows the deduced amino acid sequence of one variant of DIM-5, encoded by the DIM-5' cDN A.

SEQ ID NO: 4 shows the nucleic acid sequence of DIM-5", one of two splice variants, and the encoded amino acid sequence.

SEQ ID NO: 5 shows the deduced amino acid sequence of one variant of DIM-5, encoded by the DIM-5" cDN A.

SEQ ID NOs: 6-21 show DNA primer pairs used for PCR in vitro amplification reactions as described herein. In particular, these primers were used to amplify region 1 D21 (SEQ ID NOs: 6 and 7); region 9a20 (SEQ ID NOs: 8 and 9); region hibDldim-5 (SEQ ID NOs: 10 and 11); region dim-5 ORF (SEQ ID NOs: 12 and 13); region 5'-GST-DIM-5 (SEQ ID NOs: 14 and 15); region H3L9 (SEQ ID NOs: 16 and 17); region H3R9 (SEQ ID NOs: 18 and 19); and region H3-ORF (SEQ ID NOs: 20 and 21).

SEQ ID NOs: 22-29 show DNA primers used for PCR in vitro amplification reactions of η (SEQ ID NOs: 22 and 23), punt (SEQ ID NOs: 24 and 25), pen (SEQ ID NOs: 26 and 27), and hH4 (SEQ ID NOs: 28 and 29) as described in Example 12.

DETAILED DESCRIPTION

Abbreviations

DMTase (DMeTase): DNA methyltransferase

HMTase (HMeTase): histone methyltransferase

//. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Discussions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review ofthe various embodiments ofthe invention, the following explanations of specific terms are provided:

Alcohol: This term refers to a chemical compound with the structure R-OH, wherein R is alkyl, especially lower alkyl (for example in methyl, ethyl, or propyl alcohol). An alcohol may be either linear or branched, such as isopropyl alcohol.

Alkyl: The term "alkyl" refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned contains one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1 -yl, or other functionality. The term "lower alkyl" refers to a cyclic, branched or straight chain monovalent alkyl radical of one to five carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, and n-amyl. Lower alkyl groups can also be unsubstituted or substituted, where a specific example of a substituted alkyl is 1,1 -dimethyl propyl. Alkoxy: The term "alkoxy" refers to a substituted or unsubstituted alkoxy, where an alkoxy has the structure -O-R, where R is substituted or unsubstituted alkyl. In an unsubstituted alkoxy, the R is an unsubstituted alkyl. The term "substituted alkoxy" refers to a group having the structure -O- R, where R is alkyl which is substituted with a non-interfering substituent.

Amino: The term "amino" refers to a chemical functionality -NR]R₂ where R| and R₂ are independently hydrogen, alkyl, or aryl groups.

Analog, derivative or mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a biomolecule that mimics the activity of another biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound. Animal: Living multi-cellular organisms, for instance a vertebrate (a category that includes, for example, mammals, and birds). The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects. Anti-proliferative activity: An activity of a molecule, e.g., a compound, which reduces proliferation of at least one cell type, but which may reduce the proliferation (either in absolute terms or in rate terms) of multiple different cell types (e.g., different cell lines, different species, etc.). In specific embodiments, an anti-proliferative activity will be apparent against cells (either in vitro or in vivo) that exhibit a hyper-proliferative condition, such as is characteristic of certain disorders or diseases.

In certain embodiments, an anti-proliferative activity can be an anti-tumor or anti-neoplastic activity of a compound. Such molecules will be useful to inhibit or prevent or reduce cellular proliferation or growth, e.g., in a tumor, such as a malignant neoplasm. Aryl: The term "aryl" refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl or anthryl), which are optionally unsubstituted or substituted with, e.g., halogen, alkyl, alkoxy, mercapto (-SH), alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-l -yl, or other functionality.

Carboxyl: This term refers to the radical -COOH, and substituted carboxyl refers to -COR where R is alkyl, lower alkyl or a carboxylic acid or ester.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single-strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. Halogen: The term "halogen" refers to fluoro, bromo, chloro, and iodo substituents.

Heterocycle: The term "heterocycle" refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g. benzyl, morpholino, pyridyl or furyl) or multiple condensed rings (e.g. naphthyl, quinolinyl, indolizinyl or benzo[b]thienyl) and having at least one heteroatom, defined as N, O, P, or S, within the ring, which can optionally be unsubstituted or substituted with, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-l-yl, or other functionality. Histone methyltransferase: Histone methyltransferase (HMTase) is defined as an enzyme that adds one or more methyl groups to one or more positions of a histone. DIM-5 is a representative example of a HMTase; it can add one or more methyl groups to lysine 9 of histone H3 or to fragments thereof. Hydroxyl: This term refers to the chemical group -OH.

Hyper-proliferative disorder: A disorder characterized by abnormal proliferation of cells, and generically includes skin disorders such as psoriasis as well as benign and malignant tumors of all organ systems. lnjectable composition: A pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g., a compound that binds to and or inhibits a HMTase. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like. Such injectable compositions that are useful for use with the compounds and peptides of this invention are conventional; formulations are well known in the art.

In vitro amplification: Techniques that increases the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization ofthe primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies ofthe nucleic acid. The product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134). Methylation: A chemical or biochemical process of introducing a methyl group into an organic molecule. DNA methylation, the addition of a methyl group onto a nucleotide, is a postreplicative covalent modification of DNA that is catalyzed by the DNA methyltransferase enzyme (DMeTase) (Koomar et al, Nucl. Acids Res. 22: 1-10, 1994; and Bestor et al, J. Mol. Biol. 203:971-983, 1988). Proteins also can be methylated, as described herein for histone methylation. In biological systems, DNA methylation can serve as a mechanism for changing the structure of DNA without altering its coding function or its sequence. DNA methylation is a heritable, reversible and epigenetic change. It can alter gene expression, particularly by suppressing or inactivating genes, which has profound developmental and disease consequences. Methylation of CpG islands that are associated with tumor suppressor genes can cause decreased gene expression. Increased methylation of such regions often leads to reduction of normal gene expression, which may cause the selection of a population of cells having a selective growth advantage and thus are or become malignant. As used herein, the term "DNA hypermethylation" refers to an increased or high level

(above a reference level, such as wild-type or other basal level) of DNA methylation at a specific site on a nucleic acid molecule (e.g., a CpG island), or more generally in a genome or region of a genome (e.g., a promoter region).

As used herein, the term "DNA hypomethylation" refers to a decreased or low level (below a reference level, such as wild-type or other basal level) of DNA methylation at a specific site on a nucleic acid molecule (e.g., a CpG island), or more generally in a genome or region of a genome (e.g., a promoter region).

As used herein, the term "DNA hypomethylating agent" refers to an agent that reduces or reverses DNA methylation, either at a specific site (e.g., a specific CpG island) or generally throughout a genome. Hypomethylating agents can be referred to as possessing "hypomethylating activity." By way of example, such activity is measured by determining the methylation state and/or level of a specific DNA molecule or site therein, or the general methylation state of a cell, on parallel samples that have and have not been treated with the hypomethylating agent (or putative hypomethylation agent). A reduction in methylation in the treated (versus the untreated) sample indicates that the agent has hypomethylating activity.

Different hypomethylating agents, or different treatments with the same agent, or different systems that are treated, or methyltransferase mutants, will yield different levels of methylation reduction. In some embodiments, the methylation level of a target biological molecule (e.g., a particular target DNA sequence, a particular residue of a protein) is reduced by at least 5% upon treatment with a hypomethylating agent; in other embodiments it is reduced by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, or by at least 50% compared to an untreated sample. Particularly effective hypomethylating agents, or agents used in particularly susceptible systems, will yield even greater reduction levels, for instance at least 60%, 70%, 80%, 90%, or in some examples 95% or more. Methylation-mediated or -related condition/disease/disorder: A biological condition, disease or disorder of a subject that is associated with, caused by, or influenced by the methylation state (e.g., the extent of methylation) of a DNA sequence, the level of methylation throughout the genome ofthe subject, and/or the level of methylation of a protein or residue within a protein or proteins. Some hypermethylation-associated diseases, disorders, and conditions are characterized by exhibiting hypermethylation of one or more target biological molecules. Such diseases, disorders, and conditions therefore can be identified by examining the methylation state (or level) of target molecules in a subject known to or suspected of suffering therefrom; a high level of specific or general methylation indicates that the disease/disorder/condition is hypermethylation-associated. It is beneficial to treat (or prevent) such diseases, disorders, and conditions with HMTase-activity altering compositions, for instance compositions identified using the methods described herein.

Hypomethylation-associated diseases, disorders, and conditions are characterized by exhibiting hypomethylation of one or more target biological molecules. As with hypermethylation, hypomethylation-associated diseases/disorders/conditions can be identified by examining the methylation state (or level) of target molecules in the subject known to or suspected of suffering therefrom.

Nucleoside: "Nucleoside" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine, or synthetic analogs thereof.

Nucleotide: A nucleotide is a nucleoside plus a phosphate, and forms one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA.

Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.

Ortholog: Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.

Parenteral: Administered outside ofthe intestine, e.g., not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.

Pharmaceutical agent or drug: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful with the compounds described herein are conventional. See, for instance, Remingto 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), which describes compositions and formulations suitable for pharmaceutical delivery.

In general, the nature ofthe carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pre-caπcerous lesion: This term includes syndromes represented by abnormal neoplastic, including dysplastic, tissue changes. Examples include dysplastic growths in colonic, breast, prostate, or lung tissues, or conditions such as dysplastic nevus syndrome (a precursor to malignant melanoma ofthe skin), polyposis syndromes, colonic polyps, precancerous lesions ofthe cervix (such as cervical dysplasia), esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breast and/or skin and related conditions (e.g , actinic keraosis), whether the lesions are clinically identifiable or not.

Prodrug: Any molecule that undergoes in vivo metabolic conversion to one or more pharmacologically active compound(s).

Quelling: A form of post-transcriptional gene silencing. Quelling is induced by a transgene containing sequences homologous to the transcribed region ofthe native silenced gene.

Tumor: A neoplasm that may be either malignant or non-malignant. "Tumors ofthe same tissue type" refers to primary tumors originating in a particular organ (such as breast, prostate, bladder, or lung). Tumors ofthe same tissue type may be divided into tumor of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor).

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incoφorated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. ///. Overview of Several Embodiments

Provided herein in a first embodiment are methods for identifying a compound with the potential for treating a methylation-related disease or condition (such as neoplasia). Such methods include determining a histone methyltransferase (HMTase) inhibitory activity ofthe compound, wherein high HMTase inhibition activity identifies that the compound has potential for treating a methylation-related disease or condition. In some examples of these methods, the HMTase inhibitory activity includes a histone H3 methyltransferase activity.

Some specific examples ofthe provided methods further include determining a DIM-5 inhibitory activity ofthe compound, wherein high DIM-5 inhibitory activity identifies that the compound has potential for treating a methylation-related disease or condition.

Still other examples ofthe methods include determining whether the compound inhibits tumor cell growth in a culture, wherein inhibition of tumor cell growth further identifies that the compound has potential for treating a methylation-related disease or condition, and/or determining whether the compound inhibits or reverses DNA methylation in a cell, wherein inhibition or reversal of DNA methylation in the cell further identifies that the compound has potential for treating a methylation-related disease or condition, and/or determining whether the compound induces apoptosis of a tumor cell, wherein induction of apoptosis further identifies that the compound has potential for treating a methylation-related disease or condition.

Other examples ofthe provided methods also include determining whether the compound being tested inhibits tumor cell growth (e.g., the growth of a mammalian tumor) in a sample (either in vivo or in vitro), wherein inhibition of tumor cell growth further identifies that the compound is useful for treating a methylation-related disease or condition.

Still further embodiments provided herein include methods of selecting a compound for inhibition of a methylation-related disease or condition, which method involves determining neoplastic cell growth inhibitory activity ofthe compound; determining HMTase inhibitory activity; and selecting a compound that exhibits neoplastic cell growth inhibitory activity and high HMTase inhibition activity as a compound to inhibit the methylation-related disease or condition. In specific examples of such methods, the methylation-related disease or condition involves disregulated cell growth, morphology, or division, and for instance in some instances involves a methylation-related disease or condition (e.g. a neoplasia or a neoplastic growth type).

Still further examples of methods provided herein further involve determining whether the test compound induces apoptosis in a cell; and selecting compounds that induce apoptosis for use and/or further testing.

Also provided herein are methods for identifying compounds for treatment of a methylation- related disease or condition, which methods involve determining HMTase inhibitory activity ofthe compounds; and identifying those compounds for treating a methylation-related disease or condition if the compounds exhibit high HMTase inhibition activity. Also provided are methods of reducing, preventing or reversing DNA methylation in a cell, which methods involve administering a hypomethylating effective amount of a HMTase inhibitory compound to the cell (e.g., a bacterial cell, a protist cell, a fungal cell, a plant cell, or an animal cell), thereby reducing, preventing or reversing DNA methylation in the cell. In examples of such methods, a nucleic acid in the cell is known to be or suspected of being hypermethylated. In some examples, the cell is a hyper-proliferative cell (e.g., a mammalian tumor cell).

This disclosure further provides methods of treating or ameliorating a hypermethylation- related disease, condition, or disorder (e.g., a hyper-proliferative disease) in a subject, which methods involve administering to the subject a hypomethylating effective amount of a HMTase inhibitory compound, which compound is optionally administered in the form of a pharmaceutical composition.

Another provided embodiment is a method of ameliorating a tumorigenic state of a cell, comprising administering a hypomethylating effective amount of a HMTase inhibitory compound (optionally administered in the form of a pharmaceutical composition) to the cell to reduce methylation of cytosine in a CpG dinucleotide in the cell, thereby ameliorating the tumorigenic state ofthe cell, in specific examples of this method, the method further involves administering an anti- cancer agent to the cell.

It is contemplated that these methods can be used to ameliorate the tumorigenic state of a cell in a subject.

Further provided embodiments include kits, which kits may optionally include instructions for carrying out a method with one or more components ofthe kit. Such kits include kits for inhibiting a DNA methyltransferase, which comprise an amount of a HMTase inhibitory compound effective to inhibit methylation of at least one DNA target. Other provided kits are kits for treating a hyper-methylation mediated disease or disorder in a subject suspected of needing such inhibition.

In some ofthe provided kits, included instructions include directions for administering at least one dose ofthe therapeutic substance to the subject in need of such treatment, for instance a methylation-related disease or condition ameliorating substance administered to a patient known or suspected of suffering from a methylation-related disease or condition.

Compositions provided in the kits disclosed herein optionally can be provided in the form of a pharmaceutical composition. Additional embodiments are purified proteins, which proteins have an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, or conservative substitutions thereof. In certain embodiments, these proteins are functional DNA methyltransferases, one of which is DIM-5. Also provided herein are nucleic acid molecules encoding such proteins (e.g., the nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4), recombinant nucleic acid molecules that include a promoter sequence operably linked to such a nucleic acid molecule, and transgenic cells containing one of these recombinant nucleic acid molecules. IV. Isolation of a Novel DNA Methyl Transferase, dim-5

DNA methylation is involved in epigenetic processes such as X-inactivation, imprinting and silencing of transposons. It has been demonstrated previously that dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora crassa. Here we describe and disclose that another Neurospora gene, dim-5, is required for DNA methylation as well as for normal growth and full fertility. We mapped dim-5 and identified it by transformation with a candidate gene. The mutant has a nonsense mutation in a SET domain of a gene related to histone methyltransferases involved in heterochromatin formation in other organisms. Transformation of a wildtype strain with a segment of dim-5 reactivated a silenced hph gene, apparently by "quelling" of dim-5*. We demonstrate that recombinant DIM-5 protein specifically methylates histone H3 and that replacement of lysine 9 in histone H3 with either a leucine or an arginine phenocopies the dim-5 mutation. Based on these findings, it is believed that DNA methylation depends on histone methylation.

Cytosine methylation is essential for normal development of mammals and plants. Mutations in any ofthe three known DNA methyltransferase (DMTase) genes ofthe mouse (Dnmtl, Dnmt3a and Dnmt3b) are lethal, either during embryogenesis or soon thereafter (Li et al, Cell 69:915-926, 1992; Okano e/ al, Cell 99:247-257, 1999). In humans, a syndrome characterized by immunodeficiency, centromere instability, and facial anomalies, results from mutations in the DNMT3B gene (Xu, Nature 402: 187-191, 1999). Reduction in methylation in the plant Arabidopsis thaliana, caused by expression of an antisense construct against the DMTase MET 1, or by mutation of a putative chromatin remodeling factor (ddml), results in developmental abnormalities and partial female sterility (Mittelsten Scheid & Paszkowski, Plant Mol. Biol. 43:235-241, 2000).

In contrast to the situation in plants and animals, DNA methylation is not essential in the filamentous fungus Neurospora crassa, facilitating investigations of DNA methylation in this organism. As a step to explore the control and mechanism of cytosine methylation, which remain largely unknown in eukaryotes, we searched for methylation mutants in Neurospora. A screen of strains surviving a chemical mutagenesis yielded one mutant completely defective in methylation (dim-2) and another with an approximately 50% reduction in total DNA methylation (dim-3) (Foss et al, Science 262:1737-1741, 1993). The dim-2 gene has recently been isolated and demonstrated to encode a DMTase responsible for both de novo and maintenance methylation at both symmetrical and non-symmetrical sites (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001). Mutations in dim-2 relieve silencing of methylated genes (Rountree & Selker, Genes Dev. 1 1 :2383-2395, 1997; Cambareri et al, Genetics 143: 137-146, 1996), but do not noticeably affect growth or development (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001). Mutants with defects in an unidentified Neurospora gene affecting methylation, dim-1, were identified among 5-azacytidine resistant derivatives of a DNA repair mutant, mus-20 (Foss el al, Mol. Gen. Genet. 259:60-71, 1998). The current study arose from an attempt to tag the dim-1 gene by insertional mutagenesis in a mus-20 strain. Unexpectedly, we generated a mutation in a previously unknown gene required for DNA methylation, dim-5, and mapped the mutation to an 80 kb region including a gene homologous to histone methyltransferases required for heterochromatin formation in fission yeast, Drosophila and mammals. Transformation experiments confirmed that the candidate gene is dim-5 and biochemical tests on recombinant DIM-5 demonstrated that this protein methylates histone H3. The implication that histone methylation controls DNA methylation was supported by demonstrating that replacements of lysine 9 in histone H3 cause loss of DNA methylation in vivo. This is explained in additional detail in the Examples, below.

Interestingly, the heterochromatic state ofthe pericentric heterochromatin in mammals (Rea et al, Nature 406:593-9, 2000; Melcher et al, Mol Cell Biol 20:3728-41 , 2000; Peters et al, Cell 107:323-37, 2001), the silent mating type region and centromeres in Schizosaccharomyces pombe (Nakayama et al, Science 292: 1 10-3, 2001 ; Noma et al, Science 293: 1 150-5, 2001), the inactive X chromosome (Peters et al, Nat Genet 30:77-80, 2001 ; Heard et al, Cell 107:727-38, 2001), and at least some DNA methylation in Arabidopsis thaliana (Jackson et al, Nature 416:556-60, 2002; Johnson et al, Curr Biol 12: 1360, 2002) depends on methylation of histone H3 lysine 9.

The activities of histone methyltransferases (e.g., DIM-5, Clr-4 and Su(var)3-9) can be strongly influenced by preexisting modifications to the N-terminal tail ofthe target histone, such as (but not limited to) acetylation, methylation, and phosphorylation of particular residues (e.g., lysines 4, 9, and 14, serine 10 and probably other sites such as lysines 18, 23, 27, and 36). We now propose that H3 histone methylases function at least in part to "integrate" the information provided in the form of modifications to H3 and quite possibly similar information on other molecules (e.g., histone H4, histone H2A and histone H2B). Because DNA methylation is controlled, at least in part, by histone methylation, DNA methylation should be affected by a variety of signals (e.g., other histone modifications) that influence H3 histone methyltransferases. Based on the work described herein, it is now apparent that procedures and drugs that influence (inhibit or stimulate) these underlying modifications also could be useful to influence DNA methylation, and therefore could be used (among other things) to clinically to treat conditions associated with hyper- or hypo-methylation of DNA.

V. Methods of Making DIM-5 Encoding Sequences

The foregoing discussion and several ofthe Examples describe the original means by which the DIM-5 encoding sequence was obtained and also provides the nucleotide sequence of this clone. With the provision of this sequence information, the polymerase chain reaction (PCR) or other in vitro amplification techniques may now be utilized in a more direct and simple method for producing DIM-5-encoding sequences.

Total RNA is extracted from cells by any one of a variety of methods well known to those of ordinary skill in the art. Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998) provide descriptions of methods for RNA isolation. Any cell line derived from a non-DIM-5 deleted subject would be suitable. For extraction from a human cell line it is believed that the widely used HeLa cell line, or the WI-38 human skin fibroblast cell line available from the American Type Culture Collection, Manassas, VA USA, could be used. The extracted RNA is then used as a template for performing the reverse transcription- polymerase chain reaction (RT-PCR) amplification of cDN A. Methods and conditions for RT-PCR are described in Kawasaki et al, In PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, California, 1990. The selection of PCR primers will be made according to the portions ofthe cDNA which are to be amplified. Primers may be chosen to amplify small segments of a cDNA or the entire cDNA molecule. Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). One skilled in the art will appreciate that many different primers may be derived from the provided DIM-5-encoding sequence in order to amplify particular regions ofthe molecule.

Re-sequencing of PCR products obtained by these amplification procedures is recommended; this will facilitate confirmation o the amplified sequence and will also provide information on natural variation on this sequence in different populations or species. Oligonucleotides derived from the provided DIM-5 sequences provided may be used in such sequencing methods.

Orthologs of DIM-5 can be cloned in a similar manner, where the starting material consists of cells taken from a non-human species. Orthologs will generally share at least 50% sequence homology with one or more ofthe disclosed DIM-5 encoding sequences. Where the species is more closely related to Neurospora, the sequence homology will in general be greater. Closely related orthologous DIM-5 molecules may share at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% sequence homology with the disclosed sequences (e.g., SEQ ID NO: 1, 2, and/or 4).

Oligonucleotides derived from the DIM-5 encoding sequences (SEQ ID NO: 1 , 2, and/or 4), are encompassed within the scope ofthe present invention. Oligonucleotide primers may comprise a sequence of at least 10 consecutive nucleotides ofthe DIM-5 nucleic acid sequence. To enhance amplification specificity, oligonucleotide primers comprising at least 15, 25, 30, 35, 40, 45, 50, or 100 or more consecutive nucleotides of these sequences may also be used. These primers for instance may be obtained from any region ofthe disclosed sequences. By way of example, the D1M- 5 cDNA, ORF and gene sequences may be apportioned into about halves or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides) may be derived from the first or second halves ofthe molecules, or any ofthe four quarters. The DIM-5 cDNA, shown in SEQ ID NO: 1, can be used to illustrate this. The portion of a prototypical DIM-5 encoding sequence shown in SEQ ID NO: 1 is 1081 nucleotides in length and so may be hypothetically divided into about halves (nucleotides 1-540 and 541-1081) or about quarters (nucleotides 1-270, 271-540, 541- 81 1 and 812-1081).

Nucleic acid molecules may be selected that comprise at least 10, 15, 20, 25, 30, 35, 40, 50, or 100 or more consecutive nucleotides of any of these or other portions of a DIM-5 encoding sequence, or ofthe 5' or 3' flanking regions.

VI. Nucleotide and Amino Acid Sequence Variants of DIM-5

With the provision ofthe DIM-5 protein and corresponding nucleic acid sequences herein, the creation of variants of these sequences is now enabled.

Variant DIM-5 proteins include proteins that differ in amino acid sequence from the DIM-5 sequences disclosed but that share at least 50% amino acid sequence homology with the provided DIM-5 protein. Other variants will share at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% amino acid sequence homology. Manipulation ofthe nucleotide sequence of DIM-5 using standard procedures, including for instance site-directed mutagenesis or PCR, can be used to produce such variants. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These so-called conservative substitutions are likely to have minimal impact on the activity ofthe resultant protein, so long as they do not affect amino acids in any active sites and/or binding pockets. Table 1 shows amino acids that may be substituted for an original amino acid in a protein, and which are regarded as conservative substitutions.

Table 1

Original Residue Conservative Substitutions

Ala ser

Arg lys

Asn gin; his

Asp glu

Cys ser

Gin asn

Glu asp

Gly pro

His asn; gin

Ile leu; val

Leu ile; val

Lys arg; gin; glu

Met leu; ile

Phe met; leu; tyr

Ser thr

Thr ser

Trp tyr

Tyr trp; phe

Val ile; leu More substantial changes in enzymatic function or other protein features may be obtained by selecting amino acid substitutions that are less conservative than those listed in Table 1. Such changes include changing residues that differ more significantly in their effect on maintaining polypeptide backbone structure (e'.g, sheet or helical conformation) near the substitution, charge, or hydrophobicity ofthe molecule at the target site, or bulk of a specific side chain. The following substitutions are generally expected to produce the greatest changes in protein properties: (a) a hydrophilic residue (e.g., seryl or threonyl) is substituted for (or by) a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain (e.g., lysyl, arginyl, or histadyl) is substituted for (or by) an electronegative residue (e.g., glutamyl or aspartyl); or (d) a residue having a bulky side chain (e.g., phenylalanine) is substituted for (or by) one lacking a side chain (e.g., glycine).

Variant DIM-5-encoding sequences may be produced by standard DNA mutagenesis techniques, for example, Ml 3 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ch. 15. By the use of such techniques, variants may be created which differ in minor ways from the DIM-5 sequences disclosed. DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein, and which differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein that has at least 70% sequence identity with the DIM-5 sequence disclosed (SEQ ID NO: 1, 2, and/or 4), are comprehended by this invention. Also comprehended are more closely related nucleic acid molecules that share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% or more nucleotide sequence homology with the disclosed DIM-5 sequences. In their most simple form, such variants may differ from the disclosed sequences by alteration ofthe coding region to fit the codon usage bias ofthe particular organism into which the molecule is to be introduced.

Alternatively, the coding region may be altered by taking advantage ofthe degeneracy ofthe genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to the disclosed DIM-5 protein sequences (SEQ ID NOs: 3 and 5). Based upon the degeneracy ofthe genetic code, variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. Thus, this invention also encompasses nucleic acid sequences which encode a DIM-5 protein, but which vary from the disclosed nucleic acid sequences by virtue ofthe degeneracy ofthe genetic code. Variants ofthe DIM-5 protein may also be defined in terms of their sequence identity with the prototype DIM-5 protein shown in SEQ ID NOs: 3 and 5. For instance, DIM-5 proteins share at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% or more amino acid sequence identity with a DIM-5 protein disclosed herein. Nucleic acid /035844

-23-

sequences that encode such proteins may readily be determined simply by applying the genetic code to the amino acid sequence of a DIM-5 protein, and such nucleic acid molecules may readily be produced by assembling oligonucleotides corresponding to portions ofthe sequence.

Nucleic acid molecules that are derived from the human DIM-5 encoding nucleic acid sequences disclosed include molecules that hybridize under stringent conditions to the disclosed prototypical DIM-5 nucleic acid molecules, or fragments thereof. Stringent conditions are hybridization at 65° C in 6 x SSC, 5 x Denhardt's solution, 0.5% SDS and 100 μg sheared salmon testes DNA, followed by 15-30 minute sequential washes at 65° C in 2 x SSC, 0.5% SDS, followed by 1 x SSC, 0.5% SDS and finally 0.2 x SSC, 0.5% SDS. Low stringency hybridization conditions (to detect less closely related homologs) are performed as described above but at 50° C (both hybridization and wash conditions); however, depending on the strength ofthe detected signal, the wash steps may be terminated after the first 2 x SSC wash.

DIM-5 encoding molecules (including SEQ ID NOs: 1 , 2, and 4), and orthologs and homologs of these sequences may be incoφorated into transformation or expression vectors.

VII Expression of DIM-5 Polypeptides

With the provision of Neurospora DIM-5 encoding sequences, the expression and purification ofthe DIM-5 protein by standard laboratory techniques is now enabled. In addition, proteins or polypeptides encoded by the antisense strand ofthe DIM-5 cDNA can likewise be expressed. After expression, the purified DIM-5 protein or polypeptide may be used for functional analyses, antibody production, diagnostics, and patient therapy. Furthermore, the DNA sequence of the DIM-5 cDNA and its antisense strand can be manipulated in studies to understand the expression ofthe gene and the function of its product. Mutant forms of DIM-5 or homologous proteins from other species (for instance from mammals, such as humans) may be isolated based upon information contained herein, and may be studied in order to detect alteration in expression patterns in terms of relative quantities, tissue specificity and functional properties ofthe encoded mutant DIM-5 protein. Partial or full-length cDNA sequences, which encode for the subject protein, may be ligated into bacterial expression vectors. Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli (E. coli) may be utilized for the purification, localization and functional analysis of proteins. For example, fusion proteins consisting of amino terminal peptides encoded by a portion ofthe E. coli lacZ or trpE gene linked to DIM-5 proteins may be used to prepare polyclonal and monoclonal antibodies against these proteins. Thereafter, these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by immunofluorescence. Intact native protein may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al. (Sambrook et al, In Molecular Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989) Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome-binding site upstream ofthe cloned gene If low levels of protein are produced, additional steps may be taken to increase protein production, if high levels of protein are produced, purification is relatively easy Suitable methods are presented in Sambrook et al (In Molecular Cloning A Laboratory Manual, CSHL, New York, 1989) and are well known in the art Often, proteins expressed at high levels are found in insoluble inclusion bodies Methods for extracting proteins from these aggregates are described by Sambrook et al (In Molecular Cloning A Laboratory Manual, Oh 17, CSHL, New York, 1989) Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller- Hill, EMBO J 2 1791, 1983), pEXl-3 (Stanley and Luzio, EMBOJ 3 1429, 1984) and pMR 100 (Gray et al , Proc Natl Acad Sci USA 79 6598, 1982) Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, Nature 292 128, 1981), pKK177-3 (Amann and Brosius, Gene 40 183, 1985) and pET-3 (Studiar and Moffatt, J Mol Biol 189 113, 1986) DIM-5 fusion proteins may be isolated from protein gels, lyophilized, ground into a powder and used as an antigen The DNA sequence can also be transferred from its existing context to other cloning vehicles, such as other plasmids, bacteπophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al , Science 236 806-812, 1987) These vectors may then be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, Science 244 1313-1317, 1989), invertebrates, plants (Gasser and Fraley, Science 244 1293, 1989), and animals (Pursel et al , Scιence 244 1281-1288, 1989), which cell or organisms are rendered transgenic by the introduction ofthe heterologous DIM-5 cDNA

For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc Natl Acad Sci USA 78 2072-2076, 1981), and introduced into cells, such as monkey COS-1 cells (Gluzman, Cell 23 175-182, 1981), to achieve transient or long-term expression The stable integration ofthe chimeπc gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, J Mol Appl Genet 1 327-341, 1982) and mycophenolic acid (Mulligan and Berg, Proc Natl Acad Sci USA 78 2072-2076, 1981)

DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, hgation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteπophage intermediate or with the use of specific oligonucleotides in combination with PCR

The cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques These vectors are designed to permit the transcription ofthe cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription ofthe cDNA and ensure its proper splicing and polyadenylation. Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) ofthe Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al, Proc. Natl Acad. Sci. USA 78: 1078-2076, 1981 ; Gorman et al, Proc. Natl Acad. Sci USA 78:6777-6781, 1982). The level of expression ofthe cDNA can be manipulated with this type of vector, either by using promoters that have different activities (for example, the baculovirus pAC373 can express cDNAs at high levels in S. frugiperda cells (Summers and Smith, In Genetically Altered Viruses and the Environment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold Spring Harbor, New York, 1985) or by using vectors that contain promoters amenable to modulation, for example, the glucocorticoid-responsive promoter from the mouse mammary tumor virus (Lee et al, Nature 294:228, 1982). The expression ofthe cDNA can be monitored in the recipient cells 24 to 72 hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) or neo (Southern and Berg J. Mol. Appl Genet. 1 :327-341, 1982) bacterial genes. These selectable markers permit selection of transfected cells that exhibit stable, long-term expression ofthe vectors (and therefore the cDNA). The vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al, Mol. Cell Biol. 1 :486, 1981) or Epstein-Barr (Sugden et al, Mol. Cell Biol. 5:410, 1985). Alternatively, one can also produce cell lines that have integrated the vector into genomic DNA. Both of these types of cell lines produce the gene product on a continuous basis. One can also produce cell lines that have amplified the number of copies ofthe vector (and therefore ofthe cDNA as well) to create cell lines that can produce high levels ofthe gene product (Alt et al, J. Biol. Chem. 253:1357, 1978).

The transfer of DNA into eukaryotic, in particular human or other mammalian cells, is now a conventional technique. The vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, Virology 52:466, 1973) or strontium phosphate (Brash et al, Mol. Cell Biol. 7:2013, 1987), electroporation (Neumann et al, EMBOJ 1:841, 1982), lipofection (Feigner et al, Proc. Natl. Acad. Sci USA 84:7413, 1987), DEAE dextran (McCuthan et al, J. Natl. Cancer lnst. 41 :351, 1968), microinjection (Mueller et al, Cell 15:579, 1978), protoplast fusion (Schafi er, Proc. Natl. Acad. Sci. USA 77:2163-2167, 1980), or pellet guns (Klein et al, Nature 327:70, 1987). Alternatively, the cDNA, or fragments thereof, can be introduced by infection with virus vectors. Systems are developed that use, for example, retroviruses (Bernstein et al, Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al, J. Virol. 57:267, 1986), or Heφes virus (Spaete et al, Cell 30:295, 1982). DIM-5-encoding sequences can also be delivered to target cells in vitro via non-infectious systems, for instance liposomes.

These eukaryotic expression systems can be used for studies of DIM-5 encoding nucleic acids and mutant forms of these molecules, the DIM-5 protein and mutant forms of this protein. Such uses include, for example, the identification of regulatory elements located in the 5' region ofthe DIM-5 gene on genomic clones that can be isolated from human genomic DNA libraries using the information contained in the present invention. The eukaryotic expression systems may also be used to study the function ofthe normal complete protein, specific portions ofthe protein, or of naturally occurring or artificially produced mutant proteins.

Using the above techniques, the expression vectors containing the DIM-5 gene sequence or cDNA, or fragments or variants or mutants thereof, can be introduced into human cells, mammalian cells from other species or non-mammalian cells as desired. The choice of cell is determined by the puφose ofthe treatment. For example, monkey COS cells (Gluzman, Cell 23: 175-182, 1981) that produce high levels ofthe SV40 T antigen and permit the replication of vectors containing the SV40 origin of replication may be used. Similarly, Chinese hamster ovary (CHO), mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.

The present disclosure thus encompasses recombinant vectors that comprise all or part ofthe DIM-5 gene or cDNA sequences for expression in a suitable host. The DIM-5 DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that the DIM-5 polypeptide can be expressed. The expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof. The expression control sequence may be specifically selected from the group consisting ofthe lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter ofthe yeast alpha-mating factors and combinations thereof.

The host cell, which may be transfected with the vector of this invention, may be selected from the group consisting of E. coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or other bacilli; other bacteria; yeast; fungi; insect; mouse or other animal; or plant hosts; or human tissue cells.

It is appreciated that for mutant or variant DIM-5 DNA sequences, similar systems are employed to express and produce the mutant product. In addition, fragments ofthe DIM-5 protein can be expressed essentially as detailed above. Such fragments include individual DIM-5 protein domains or sub-domains (such as all or a portion of a SET domain), as well as shorter fragments such as peptides. DIM-5 protein fragments having therapeutic properties may be expressed in this manner also.

VIII. Production of DIM-5 Protein Specific Binding Agents

Monoclonal or polyclonal antibodies may be produced to either the normal DIM-5 protein or mutant forms of this protein (including for instance the specific mutant isolated and discussed herein), as well as to proteins or peptides encoded for by the reverse complement ofthe disclosed DIM-5 sequences. Optimally, antibodies raised against these proteins or peptides would specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the DIM-5 protein or a fragment thereof would recognize and bind the DIM-5 protein and would not substantially recognize or bind to other proteins found in target cells. The determination that an antibody specifically detects the DIM-5 protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989). To determine that a given antibody preparation (such as one produced in a mouse) specifically detects the DIM-5 protein by Western blotting, total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non- specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase. Application of an alkaline phosphatase substrate 5-bromo-4-chloro-3-indoIyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase. Antibodies that specifically detect the DIM-5 protein will, by this technique, be shown to bind to the DIM-5 protein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding ofthe antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-DIM-5 protein binding.

Substantially pure DIM-5 protein or protein fragment (peptide) suitable for use as an immunogen may be isolated from the transfected or transformed cells as described above. Concentration of protein or peptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes ofthe DIM-5 protein identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms ofthe selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells ofthe spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess un-fused cells destroyed by growth ofthe system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots ofthe dilution placed in wells of a microtiter plate where growth ofthe culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid ofthe wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth Enzymol 70 419-439, 1980), and derivative methods thereof Selected positive clones can be expanded and their monoclonal antibody product harvested for use Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protem can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance lmmunogenicity Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species For example, small molecules tend to be less lmmunogenic than others and may require the use of carriers and adjuvant Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable An effective immunization protocol for rabbits can be found in Vaitukaitis et al (J Clin Endocnnol Metab 33 988-991, 1971)

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations ofthe antigen, begins to fall See, for example, Ouchterlony et al (In Handbook of Experimental Immunology, Wier, D (ed ) chapter 19 Blackwell, 1973) Plateau concentration of antibody is usually in the range of about 0 1 to 02 mg/ml of serum (about 12 μM) Affinity ofthe antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch 42, 1980) C.N Antibodies Raised against Synthetic Peptides

A third approach to raising antibodies against DIM-5 encoded proteins or peptides is to use one or more synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence ofthe DIM-5 encoded protein or peptide

By way of example only, polyclonal antibodies to specific peptides within DIM-5 are generated using well-known peptide-based injection techniques Briefly, polyclonal antibodies are generated by injecting DIM-5 peptides into rabbits D. Antibodies Raised by Injection of DIM-5-Encoding Sequence

Antibodies may be raised against proteins and peptides of DIM-5 by subcutaneous injection of a DNA vector that expresses the desired protein or peptide, or a fragment thereof, into laboratory animals, such as mice Delivery ofthe recombinant vector into the animals may be achieved using a hand-held form ofthe Biohstic system (Sanford et al , Paniculate Sci Technol 5 27-37, 1987) as described by Tang et al (Nature 356 152-154, 1992) Expression vectors suitable for this puφose may include those that express the DIM-5 encoding sequence under the transcriptional control of either the human β-actin promoter or the cytomegalovirus (CMV) promoter Antibody preparations prepared according to any one of these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample; or for immunolocalization ofthe DIM-5 protein, for instance for studies of chromatin structure and regulation.

For administration to human patients, antibodies, e.g., DIM-5-specific monoclonal antibodies, can be humanized by methods known in the art. Antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, CA).

IX. Methods of Modifying DNA Methylation

With the provision herein ofthe connection between histone methylation and DNA methylation, methods of modifying DNA methylation by altering histone methylation are now enabled. It is demonstrated herein that methylation ofthe lysine 9 residue of histone H3 is necessary in order for DNA methylation in Neurospora; it is believed that methylation of histones will play an important role in influencing and controlling DNA methylation in other cells, including cells of protists, animals, and plants. Therefore, inhibiting methylation of this and possibly other histones can be used to influence the methylation of DNA, and thereby influence the expression of (or silencing of) genes. This disclosure includes methods of modifying DNA methylation of one or more target genes, or target regions within a genome, by influencing the activity of a histone methyltransferase. Thus, in some embodiments inhibition of histone methylation is used to reduce the level of DNA methylation, thereby increasing the expression of one or more otherwise silenced genes. In other embodiments, enhancement of histone methylation is used to increase the level of DNA methylation, thereby decreasing the expression of one or more target genes.

The activity of a HMTase in a cell can be influenced in any one of myriad ways, including increasing or decreasing the expression of a native HMTase; providing an additional copy of a native or heterologous HMTase (under control of, for instance, a constitutive or regulatable promoter) to a cell to increase the amount of HMTase expressed therein; providing a HMTase antisense or other suppressive construct (e.g., siRNA, dominant negative constructs, and so forth) to the cell to reduce the amount of HMTase expressed therein; applying recombinant purified HMTase to the cell; applying one or more agents that inhibit the activity of a HMTase to the cell (for instance, by competitive binding), thereby reducing the activity ofthe HMTase in the cell; applying one or more agents that increase the activity of a HMTase (for instance, by increasing the affinity ofthe HMTase for its target histone residue(s)); and so forth. Methods are provided herein for identifying and assaying such agents, and for determining the amount of change in the HMTase activity of a target methyltransferase after administration of an activity-influencing agent. In particular embodiments, the histone methylation that is influenced is a methylation ofthe lysine 9 position of histone H3. It is believed that, as with other amino acid residues in target proteins, up to three methyl groups can modify this one amino acid residue. Therefore, it is contemplated that the methylation which is influenced or modified (e.g., inhibited) as described herein may be a first methylation, a second methylation, or a third methylation, or any combination thereof.

In Neurospora, the DIM-5 histone methyltransferase, which is essential for DNA methylation, trimethylates H3 lysine 9. In vitro studies demonstrated that DIM-5 can generate mono- di-, and, especially, trimethylated species. Chromatin immunoprecipitation experiments revealed trimethyl-lysine 9, but not dimethyl-lysine 9, associated with methylated DNA in Neurospora and dimethyl-lysine 4 preferentially associated with active genes. Elimination of DNA methylation by mutation ofthe DNA methyltransferase gene, dim-2, did not prevent trimethylation of lysine 9 but mutation of dim-5 did, suggesting that trimethylation of histone H3 lysine 9 directs DNA methylation in Neurospora.

X. Methods of Disease Detection

The identification that histone methylation influences and in some circumstances controls DNA methylation suggests that the level of histone methylation may provide a convenient indicator of disease state, or tendency to development of a disease, in a subject. Lysine residues such as lysine 9 in histone H3 may be modified in various ways, e.g., by the addition of acetyl groups (by histone acetytransferases, also known as HDACs) or by the addition of one, two, or three methyl groups. This disclosure describes for the first time, a histone methytransferase (DIM-5) that efficiently trimethylates a lysine residue in a histone, which is illustrated by the demonstration that a dimethyl-K9 peptide (based on the sequence of histone H3 tails) is an excellent substrate for DIM-5.

It is believed that the dimethyl and trimethyl (and perhaps monomethyl) K9 H3 histone forms constitute alternative signals, along the line ofthe "histone code" hypothesis (Strahl & Allis, Nature 403:41-45, 2000). In particular, the trimethyl-K9 H3 may be a specific signal to methylate DNA in organisms such as Neurospora, plants, and animals. Thus, DNA methylation-related diseases may be associated with alterations in the degree of methylation (0, 1 , 2, or 3 methyl groups) on K 9 of histone H3, or on one or more other residues in histone H3 or another histone molecule. To characterize such diseases, this disclosure provides methods to assay the degree of methylation. Also provided are methods to treat DNA methylation-related diseases by specifically affecting histone MTases specific for a particular methylation degree (e.g., trimethyl). With respect to characterization ofthe extent of methylation of a particular residue such as

K9), antibodies can distinguish between the dimethyl and the trimethyl forms. Thus, methylation- degree specific antibodies can be used as reagents to recognize chromatin defects that lead to defects in DNA methylation. This disclosure therefore further includes methods of determining whether a subject is suffering from, or is likely to develop, a DNA methylation-related disease or condition, which methods involve determining the amount, extent, number (e.g., first, second, or third methylation on a single residue) or position (e.g., on which residue) of histone methylation in a cell ofthe subject. One of ordinary skill in the art will know methods for assessing these qualities regarding the methylation of specific target proteins. Specific examples of certain methylation detection methods are provided herein, for instance in Example 1.

In some embodiments, tri-methylation specifically is assessed, using for instance an antibody specific for the tri-methyl form of a specific target methylated residue in a histone. By way of example, an antibody specific for the tri-methyl modified residue at position lysine 9 of histone H3 can be used to determine the extent of tri-methylation of this residue in a cell. In certain examples, such antibodies can be used to perform a chromatin immunoprecipitation, and the precipitated material analyzed for instance to determine what regions ofthe genome are associated with methylated histones. In other embodiments, a HMTase (such as DIM-5) is used in a method to assess the potential for a cell to accept histone methylation that is correlated with the potential for developing a methylation-related condition or disease. In such methods, the HMTase is contacted with a cell, or a nuclear or chromatin preparation ofthe cell, in the presence of a detectable methyl group donor (for instance, labeled with an isotope or fluorescent tag) under conditions in which the HMTase can methylate appropriate available targets. After incubation for a period of time (e.g., minutes to hours), the sample is analyzed to determine where and/or to what extent the HMTase has methylated one or more molecules in the sample. In some embodiments, the extent of labeled methylation is determined for the entire sample (after the sample is washed to remove unincoφorated label). In other embodiments, the sample is analyzed to see if label is incoφorated at one or more specific sites, for instance one or more specific residues on a histone molecule. In specific embodiments, the reaction is run in the presence of at least one DMTase, and the resultant sample is further analyzed for the amount and/or location of incoφoration of labeled methyl groups into one or more DNA target sequences.

It is particularly contemplated that cancer cells, or potentially cancerous or precancerous cells, can be analyzed to determine the level, extent, location, and form of histone methylation. The data gathered from such analyses is then used to predict the likelihood of cancer development or progression, efficacy of treatment, to aid in the selection of treatment, and/or to diagnose whether cancer is present.

XL Methods of Screening for a Compound

This disclosure further relates in some embodiments to novel methods for screening test compounds for their ability to treat, detect, analyze, ameliorate, reverse, and/or prevent a methylation-related disease or condition, especially neoplasia and pre-cancerous lesions. In particular, the present disclosure provides methods for identifying test compounds that can be used to treat, ameliorate, reverse, and/or prevent neoplasia, including pre-cancerous lesions. The compounds of interest can be tested by exposing the novel HMTase described herein to the compounds, and if a compound inhibits this novel HMTase, the compound is then further evaluated for its anti-neoplastic properties.

One aspect involves a screening method to identify a compound effective for treating, preventing, or ameliorating a methylation-related disease or condition, especially neoplasia, which method includes ascertaining the compound's inhibition of this novel HMTase or another HMTase. In some embodiments, the screening method further includes determining whether the compound inhibits the growth of tumor cells in a cell culture.

By screening compounds in this fashion, potentially beneficial and improved compounds for treating a methylation-related disease or condition can be identified more rapidly and with greater precision than possible in the past.

A. In General

Histone methyltransferases, for instance the novel HMTase DIM-5 and homologs and orthologs of this molecule, are useful to identify compounds that can be used to treat, ameliorate, or prevent a methylation-related disease or condition, such as neoplasms.

The screening or creation, identification and selection of appropriate high affinity inhibitors of histone methyltransferases can be accomplished by a variety of methods. Broadly speaking these may include, but are not limited to two general approaches. One approach is to use structural knowledge about the target enzyme to design a candidate molecule with which it will precisely interact. Examples include computer assisted molecular design and protein crystallographic studies. Specific examples of certain protein crystallographic studies are provided herein, for instance in Example 11. A second approach is to use combinatorial or other libraries of molecules, whereby a large library of molecules is screened for affinity with regard to the target enzyme.

Cancer and precancer may be thought of as diseases that involve unregulated cell growth. Cell growth involves a number of different factors. One factor is how rapidly cells proliferate, and another involves how rapidly cells die. Cells can die either by necrosis or apoptosis depending on the type of environmental stimuli. Cell differentiation is yet another factor that influences tumor growth kinetics. Resolving which ofthe many aspects of cell growth a test compound affects can be important to the discovery of a relevant target for pharmaceutical therapy. Screening assays based on this technology can be combined with other tests to determine which compounds have growth inhibiting and pro-apoptotic activity. B. Inhibitor Screening

Some embodiments provided herein involve determining the histone methyltransferase inhibition activity of a given compound, for instance an H3 histone methyltransferase inhibition activity. Test compounds can be assessed for their probable ability to treat neoplastic lesions either directly, or indirectly by comparing their activities against compounds known to be useful for treating neoplasia.

Methods are provided herein for determining the methylation level of a target protein, such as histone H3. These methods can be used to determine the effectiveness of test compounds for inhibiting methylation. Other methods for determining methylation of proteins or specific residues within proteins will be known to those of ordinary skill in the art.

C. Determining Histone Methylation Influencing Activity

Compounds can be screened for inhibitory or other effects on the activity ofthe novel histone methyltransferase DIM-5 described herein (or on another H3 histone methyltransferase such as clr4 or su(var)3-9 or another homolog) using an expressed recombinant version ofthe enzyme, or a homolog or ortholog isolated from another species, for instance a mammal such as a human. Alternatively, cells expressing one of these HMTases can be treated with a test compound and the effect ofthe test compound on methylation of a specific methylation target (e.g., K9 of histone H3) can be determined, for instance using one ofthe techniques described herein. Additional detail regarding methods for determining histone methylation influencing activity (e.g., inhibition) is provided herein.

D. Determining Whether a Compound Reduces the Number of Tumor Cells

In an alternate embodiment, provided screening methods involve further determining whether the compound reduces the growth of tumor cells. Various cell lines can be used, which may be selected based on the tissue to be tested. For example, these cell lines include: SW-480 - colonic adenocarcinoma; HT-29 - colonic adenocarcinoma, A-427 - lung adenocarcinoma carcinoma; MCF- 7 - breast adenocarcinoma; and UACC-375 - melanoma line; and DU145 - prostrate carcinoma. Cytotoxicity data obtained using these cell lines are indicative of an inhibitory effect on neoplastic lesions. These cell lines are well characterized, and are used for instance by the United States National Cancer Institute (NCI) in their screening program for new anti-cancer drugs.

By way of example, a test compound's ability to inhibit tumor cell growth in vitro can be measured using the HT-29 human colon carcinoma cell line obtained from ATCC (Bethesda, MD). HT-29 cells have previously been characterized as a relevant colon tumor cell culture model (Fogh & Tre pe, In: Human Tumor Cells in Vitro, Fogh (ed.), Plenum Press, N.Y., pp. 1 15-159, 1975). HT- 29 cells are maintained in RPMI media supplemented with 5% fetal bovine calf serum (Gemini Bioproducts, Inc., Carlsbad, Calif.) and 2 mM glutamine, and 1% antibiotic-antimycotic, in a humidified atmosphere of 95% air and 5% C0₂ at 37° C. Briefly, HT-29 cells are plated at a density of 500 cells/well in 96 well microtiter plates and incubated for 24 hours at 37° C. prior to the addition of test compound. Each determination of cell number involved six replicates. After six days in culture, the cells are fixed by the addition of cold trichloroacetic acid (TCA) to a final concentration of 10% and protein levels are measured, for instance using the sulforhodamine B (SRB) colorimetric protein stain assay as previously described by Skehan et al. (J. Natl. Cancer Inst. 82: 1107-112, 1990). In addition to the SRB assay, a number of other methods are available to measure growth inhibition and could be substituted for the SRB assay. These methods include counting viable cells following trypan blue staining, labeling cells capable of DNA synthesis with BrdU or radiolabeled thymidine, neutral red staining of viable cells, or MTT staining of viable cells.

Significant tumor cell growth inhibition greater than about 30% at a dose of 100 μM or below is further indicative that the compound is useful for treating neoplastic lesions. An IC₅₀ value may be determined and used for comparative purposes. This value is the concentration of drug needed to inhibit tumor cell growth by 50% relative to the control. In some embodiments, the IC₅₀ value is less than 100 μM in order for the compound to be considered further for potential use for treating, ameliorating, or preventing neoplastic lesions. E. Determining Whether a Test Compound Induces Apoptosis

In other embodiments, screening methods provided herein further involve determining whether the test compound induces apoptosis in cultures of tumor cells.

Two distinct forms of cell death may be described by moφhological and biochemical criteria: necrosis and apoptosis. Necrosis is accompanied by increased permeability ofthe plasma membrane, whereby the cells swell and the plasma membrane ruptures within minutes. Apoptosis is characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases.

Apoptosis occurs naturally during normal tissue turnover and during embryonic development of organs and limbs. Apoptosis also can be induced by various stimuli, including cytotoxic T-lymphocytes and natural killer cells, by ionizing radiation and by certain chemotherapeutic drugs. Inappropriate regulation of apoptosis is thought to play an important role in many pathological conditions including cancer, AIDS, or Alzheimer's disease, etc.

Test compounds can be screened for induction of apoptosis using cultures of tumor cells maintained under conditions as described above. In some examples of such screening methods, treatment of cells with test compounds involves either pre- or post-confluent cultures and treatment for two to seven days at various concentrations ofthe test compounds. Apoptotic cells can be measured in both the attached and "floating" portions ofthe cultures. Both are collected by removing the supernatant, trypsinizing the attached cells, and combining both preparations following a centrifugation wash step (10 minutes, 2000 rpm). The protocol for treating tumor cell cultures with sulindac and related compounds to obtain a significant amount of apoptosis has been described in the literature (e.g., Piazza et al, Cancer Res., 55:3110-16, 1995). Particular features include collecting both floating and attached cells, identification ofthe optimal treatment times and dose range for observing apoptosis, and identification of optimal cell culture conditions.

Following treatment with a test compound, cultures can be assayed for apoptosis and necrosis, for instance by florescent microscopy following labeling with acridine orange and ethidium bromide. Many methods for measuring apoptotic cells are known to those of ordinary skill in the art; for instance, one method for measuring apoptotic cell number has been described by Duke & Cohen (Curr. Prot. lmmuno., Coligan et al, eds., 3.17.1-3.17.1, 1992). For example, floating and attached cells are collected by trypsinization and washed three times in PBS. Aliquots of cells are then centrifuged. The pellet is resuspended in media and a dye mixture containing acridine orange and ethidium bromide prepared in PBS and mixed gently. The mixture then can be placed on a microscope slide and examined for moφhological features of apoptosis.

Apoptosis also can be quantified by measuring an increase in DNA fragmentation in cells that have been treated with test compounds. Commercial photometric EIA for the quantitative in vitro determination of cytoplas ic histone-associated-DNA-fragments (mono- and oligo- nucleosomes) are available (e.g., Cell Death Detection ELISA, Boehringer Mannheim). The Boehringer Mannheim assay is based on a sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively. This allows the specific determination of mono- and oligo-nucleosomes in the cytoplasmic fraction of cell lysates. According to the vendor, apoptosis is measured as follows: The sample (cell-lysate) is placed into a streptavidin-coated microtiter plate ("MTP"). Subsequently, a mixture of anti-histone-biotin and anti-DNA peroxidase conjugates is added and incubated for two hours. During the incubation period, the anti-histone antibody binds to the histone-component ofthe nucleosomes and simultaneously fixes the immunocomplex to the streptavidin-coated MTP via its biotinylation. Additionally, the anti- DNA peroxidase antibody reacts with the DNA component ofthe nucleosomes. After removal of unbound antibodies by a washing step, the amount of nucleosomes is quantified by the peroxidase retained in the immunocomplex. Peroxidase is determined photometrically with ABTS7 (2,2'-Azido- [3-ethylbenzthiazolin-sulfonate]) as substrate.

By way of example, SW-480 colon adenocarcinoma cells are plated in a 96-well MTP at a density of 10,000 cells per well. Cells are then treated with test compound, and allowed to incubate for 48 hours at 37° C. After the incubation, the MTP is centrifuged and the supernatant is removed. The cell pellet in each well is then resuspended in lysis buffer for 30 minutes. The lysates are then centrifuged and aliquots ofthe supernatant (i.e., cytoplasmic fraction) are transferred into a streptavidin-coated MTP. Care is taken not to shake the lysed pellets (i.e., cell nuclei containing high molecular weight, un-fragmented DNA) in the MTP. Samples are then analyzed. Fold stimulation (FS = OD_max /OD_veh), an indicator of apoptotic response, is determined for each compound tested at a given concentration. EC₅₀ values may also be determined by evaluating a series of concentrations of the test compound.

Statistically significant increases of apoptosis (i.e., greater than two fold stimulation at a test compound concentration of 100 μM) are further indicative that the compound is useful for treating neoplastic lesions. Preferably, the EC₅₀ value for apoptotic activity should be less than 100 μM for the compound to be further considered for potential use for treating neoplastic lesions. EC₅₀ is understood herein to be the concentration that causes 50% induction of apoptosis relative to vehicle treatment. /035844

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F. Organ Culture Model Tests

Test compounds identified by the methods described herein can be tested for antineoplastic activity by their ability to inhibit the incidence of preneoplastic lesions in an organ culture system, such as a mammary gland organ culture system. The mouse mammary gland organ culture technique has been successfully used by other investigators to study the effects of known antineoplastic agents such as NSAIDs, retinoids, tamoxifen, selenium, and certain natural products, and is useful for validation ofthe screening methods provided herein.

By way of example, female BALB/c mice can be treated with a combination of estradiol and progesterone daily, in order to prime the glands to be responsive to hormones in vitro. The animals are sacrificed, and thoracic mammary glands are excised aseptically and incubated for ten days in growth media supplemented with insulin, prolactin, hydrocortisone, and aldosterone. DMBA (7,12- dimethylbenz(a)anthracene) is added to medium to induce the formation of premalignant lesions. Fully developed glands are then deprived of prolactin, hydrocortisone, and aldosterone, resulting in the regression ofthe glands but not the premalignant lesions. The test compound is dissolved in, for instance, DMSO and added to the culture media for the duration ofthe culture period. At the end ofthe culture period, the glands are fixed in 10% formalin, stained with alum carmine, and mounted on glass slides. The incidence of forming mammary lesions is the ratio ofthe glands with mammary lesions to glands without lesions. The incidence of mammary lesions in test compound treated glands is compared with that ofthe untreated glands.

The extent ofthe area occupied by the mammary lesions can be quantitated by projecting an image ofthe gland onto a digitation pad. The area covered by the gland is traced on the pad and considered as 100% ofthe area. The space covered by each ofthe unregressed structures is also outlined on the digitization pad and quantitated by the computer.

XII. Use of Identified Compounds to Treat, Detect, Analyze, Cure, Ameliorate, or Prevent a Methylation-Linked Disease, Disorder or Condition

With the provision herein of methods for identifying compounds that influence (e.g., inhibit) the activity of an HMTase, particularly a histone H3 methyltransferase, and more particularly a methyltransferase that methylates the lysine 9 residue of histone H3, and by such methylation influences the methylation state of DNA, the benefits of using these compound to cure, detect, analyze, ameliorate, prevent, or treat diseases and conditions that involve methylation of DNA (for instance, hypermethylation of DNA) are now made clear.

Hypermethylation-associated diseases, disorders, and conditions are characterized by exhibiting hypermethylation of one or more DNA sequences. Such diseases, disorders, and conditions therefore can be identified by examining the methylation state (or level) of nucleic acids in a subject known to or suspected of suffering therefrom; a high level of specific or general DNA methylation indicates that the disease/disorder/condition is hypermethylation-associated. It is beneficial to treat (or prevent) such diseases, disorders, and conditions with compounds that influence (e.g., inhibit) an activity of a HMTase. In some embodiments, the compound is provided in the form of a pharmaceutical composition.

In certain embodiments therefore, prior to administration of a HMTase-inhibiting compound, subjects will be screened to find those whose condition involves hypermethylation of one or more DNA sequences, and thus are most likely to be susceptible to treatment with an HMTase inhibitor. Such screening in some embodiments involves examining the methylation level ofthe genome of cell or tissue sample from the subject, or of a specific target sequence from such genome, or of a specific target protein such as H3 histone, or a specific amino acid residue of a target protein, or some combination of two or more of these. Methods for testing methylation state of various biological target molecules are provided herein.

Many processes are mediated by methylation of DNA, and inhibitors of HMTase methylation presumably can be used to influence these processes by altering the DNA methylation state ofthe system. In particular, it is contemplated that the hypomethylation activity of HMTase inhibitors can be used to reduce antimicrobial resistance, similarly to the system described in United States Patent No. 5,872,104 (entitled "Combinations and Methods for Reducing Antimicrobial Resistance"). Examples of such methods work by reducing the methylation-mediated binding inhibition of an antibiotic agent, for instance on an rRNA molecule, thereby increasing the susceptibility ofthe treated microbes to that antibiotic agent.

XIII Detecting/Measuring DNA Methylation

Because the HMTase described herein influences the methylation of DNA, it can be useful to be able to detect and/or quantify DNA methylation for use with one or more aspects ofthe methods and compositions disclosed herein. Though specific examples of detection and quantification methods are provided, those of ordinary skill in the art are familiar with other methods that could be used.

One class of methods used for determining and/or measuring the 5-methyl state of a cytosine in a nucleotide relies on using methylation-sensitive restriction endonucleases (RE). Each RE can "cut" DNA at a certain short (e.g., 4-8 nucleotide) recognition sequence. The position of such cuts can be determined based on the length of fragments produced after a digestion reaction, which fragments are detected, for instance, by gel electrophoresis, transfer to a membrane and hybridization. Certain REs are "methylation-sensitive" in that certain bases within the recognition sequence must be unmethylated for digestion to occur. Examples of methylation-sensitive REs include Sαw3AI and Dpnll. The band pattern after digestion with a methylation-sensitive RE changes depending on the methylation pattern ofthe DNA. Techniques based on methylation-sensitive REs can be somewhat limited, because many CpG's that might be methylated are outside the recognition sequences of REs, and thus cannot be examined using these methods. Methods also are available to examine individual potential methylation sites. See, for instance, S emer et al. (PNAS 93:6371-6376, 1996) and Kafri et al. (Genes Dev. 6:705-714, 1992), which describe a PCR-based method to detect methylation in a specific target sequence.

Other methods for determining/measuring the presence of 5-methylcytosine are based on specific reaction of bisulfite with cytosine. When cytosine is reacted with bisulfite it forms uracil; 5- methylcytosine is not modified. This makes cytosine and 5-methylcytosine chemically distinguishable, due to base pairing ofthe reacted cytosine (now uracil) with adenine in nucleic acid hybridization reactions. For examples of such methods, see, Frommer et al, Proc. Natl. Acad. Sci. USA 89: 1827-1831 , 1992; Sadri and Hornsby, Nuc. Acids Res. 24:5058-5059, 1996; Warnecke et al, Nuc. Acids Res. 25:4422-4426, 1997; Ziong and Laird, Nuc. Acids Res. 25:2532-2534, 1997; Selker et al, Science 262:1724-1728, 1993; and Gonzalgo and Jones, Nuc. Acids Res. 25:2529-2531, 1997.

Another method for quantitation of methylation is the Methylation-sensitive Single Nucleotide Primer Extension (Ms-SNuPE) assay, described in Gonzalgo and Jones (Nucleic Acids Res. 25:2529-2531, 1997) and United States Patent No. 6,251,594. This procedure provides a quantitative measurement of methylation levels of specific CpG sites in DNA. Briefly, genomic DNA is treated with bisulfite as discussed above. The DNA region of interest is then amplified by PCR, and primers are annealed to the PCR product and terminated immediately 5' to the original CpG site of interest. Quantitation ofthe relative ratios of methylated vs. unmethylated cytosines (C or T) is determined by incubating the annealed product with Taq polymerase and either (a-³²P) dCTP or (a-³²P) dTTP, followed by gel electrophoresis and Phosphorlmager analysis.

High-throughput methylation assays are also useful for measuring methylation. For instance, one such assay is the Methylight assay (Eads et al, Cancer Res. 61 :3410-3418, 2001; published international patent application PCT/US00/ 13029), a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology. The patent literature is also replete with methods for detecting and/or measuring methylation in a nucleic acid molecule. See, for instance:

United States Patent No. 5,786,146 (entitled "Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids"); United States Patent No. 5,871 ,917 (entitled "Identification of differentially methylated and mutated nucleic acids");

United States Patent No. 6,017,704 (entitled "Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids"); United States Patent No. 6,200,756 (entitled "Methods for identifying methylation patterns in a CpG containing nucleic acid");

United States Patent No. 6,214,556 (entitled "Method for producing complex DNA methylation fingeφrints"); and United States Patent No. 6,251 ,594 (entitled "Cancer diagnostic method based upon DNA methylation differences").

Specific examples of DNA methylation quantitation and detection methods are illustrated in the Examples, below.

XIV. Methods of Treatment

The present disclosure also includes methods of treatment for methylation-mediated disease, such as a hyper-proliferative disease or disorder, in a subject. The method includes administering an HMTase-inhibitory compound, or an analog, mimetic, prodrug, or derivative thereof that has similar hypomethylation function, or a combination of such compound and one or more other pharmaceutical agents, to the subject in a pharmaceutically compatible carrier and in an amount effective to inhibit the development or progression of a methylation-mediated disease. Although the treatment can be used prophylactically in any patient in a demographic group at significant risk for such diseases, subjects can also be selected using more specific criteria, such as a definitive diagnosis ofthe disease/condition or identification of one or more factors that increase the likelihood of developing such disease (e.g., a genetic, environmental, or lifestyle factor).

Because histone methylation is an example of a stable modification of histones and is demonstrated herein as influencing DNA methylation, limited treatments to cause DNA hypomethylation (e.g., treatment with 5-azacytidine or zebularine) could fail if the underlying- methylation of histone H3 is not changed. It is believed that in some circumstances it is more effective to target histone methylation in treatments that aim to change DNA methylation in a stable, or relatively stable, way, rather than attempting to alter (e.g., inhibit or block) DNA methylation directly.

The vehicle in which the drug is delivered can include pharmaceutically acceptable compositions ofthe compounds, using methods well known to those with skill in the art. Any ofthe common carriers, such as sterile saline or glucose solution, can be utilized. Routes of administration include but are not limited to oral and parenteral routes, such as intrathecal, intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal.

The compounds may be administered intravenously in any conventional medium for intravenous injection, such as an aqueous saline medium, or in blood plasma medium. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like. A more complete explanation of parenteral pharmaceutical carriers can be found in Remington: The Science and Practice of Pharmacy (19th Edition, 1995) in chapter 95. Embodiments of other pharmaceutical compositions can be prepared with conventional pharmaceutically acceptable carriers, adjuvants, and counter-ions as would be known to those of skill in the art. The compositions in some embodiments are in the form of a unit dose in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions.

The compounds ofthe present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition ofthe subject being treated, the severity ofthe disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with the drug is contemplated, for instance in order to reduce the occurrence of remethylation of a tumor suppressor gene.

In some embodiments, sustained intra-tumoral (or near-tumoral) release ofthe pharmaceutical preparation that comprises a hypomethylation effective amount of a HMTase inhibitor may be beneficial. Those of ordinary skill in the art are familiar with slow-release formulations. By way of example, polymers such as bis(p-carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained intra-tumoral release.

It is specifically contemplated in some embodiments that delivery is via an injected and/or implanted drug depot, for instance comprising multi-vesicular liposomes such as in DepoFoam (SkyePharma, Inc, San Diego, CA) (see, for instance, Chamberlain et al, Arch. Neuro. 50:261-264, 1993 ; Katri et al. , J. Pharm. Sci. 87: 1341 - 1346, 1998; Ye et al , J. Control Release 64: 155- 166, 2000; and Howell, Cancer J. 7:219-227, 2001).

In other embodiments, perfusion of a tumor with a pharmaceutical composition that contains a hypomethylation effective amount of a HMTase-inhibitory compound is contemplated.

Therapeutically effective doses ofthe compounds ofthe present disclosure can be determined by one of skill in the art. Low toxicity of certain identified compounds makes it possible to administer high doses, for example 100 mg/kg, although doses of 10 mg/kg, 20 mg/kg, 30 mg/kg or more are contemplated, though lower dosages are also contemplated. An example of a dosage range is 0.1 to 200 mg/kg body weight orally in single or divided doses. Another example of a dosage range is 1.0 to 100 mg/kg body weight orally in single or divided doses. For oral administration, the compositions are, for example, provided in the form of a tablet containing 0.01 to 1000 mg ofthe active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 100, 200, 400, 500, 600, 800, and 1000 mg ofthe active ingredient for the symptomatic adjustment ofthe dosage to the subject being treated.

The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity ofthe specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity ofthe condition ofthe host undergoing therapy. The pharmaceutical compositions comprising a hypomethylation effective amount of at least one HMTase inhibitor can be used in the treatment or prevention of a variety of diseases and conditions that are associated with and/or caused by hypermethylation of one or more specific gene sequences. Examples of such diseases include cancers, in particular tumors that are characterized by having one or more hypermethylated sequences such as a tumor suppressor gene, particularly where the hypermethylation has resulted in the inactivation (silencing) of that gene. Many of such inactivated genes and associated cancers have now been identified, including for instance: cadherin (inactivation of which is often associated with breast or prostate tumors and squamous cell lung carcinoma); estrogen receptor (inactivation of which is often associated with estrogen receptor negative breast tumors); VHL (inactivation of which is associated with renal cancer); HI 9 (a tumor suppressor gene located on 1 lp, the inactivation of which is implicated in many tumors); 14-3-3 σ (silenced in some breast cancers); Apaf-1 (inactivated in metastatic melanomas, though it appears that the methylation inactivation related to this gene may be indirect or through a genetic region other than the Apaf-1 promoter); and p53 (a tumor suppressor gene, the inactivation of which is implicated in many tumors, particularly unstable tumors). In addition, hypermethylation at CpG islands which are not or have not yet been associated with a specific gene, such as the one identified at 17pl 3.3, can contribute to cancer formation.

It is believed that several other genes show activities that help to inhibit tumor growth, aggressiveness, and/or metastasis. Methylation-mediated inactivation of any of these genes may lead to increased tumorigenesis, metastasis, and/or more highly aggressive tumors, and thus inhibition or reversal of methylation-mediated inactivation of these genes using a HMTase inhibitor can be beneficial in controlling cancers. Examples of such genes include glutathione-S-transferase (GST), methyl guanine methyltransferase, and TIMP-3 (tissue inhibitor of metalloproteinase-3).

The presence of methylcytidine in the genome can lead to mutation. In addition, some cancers arise from or are enhanced by mutations in genes where the mutation is thought to have been caused by methylation of a cytidine residue, followed by the subsequent conversion ofthe methylated cytidine to a guanidine. This can result in tumor gene destabilization, tumor metastasis, tumor progression, tumor recurrence, and resistance ofthe tumor to therapy by cytotoxic agents. Sub- clones ofthe tumor containing the mutated gene(s) may be more aggressive, metastatic, and therapy resistant. It is believed that a HMTase inhibitor DNA hypomethylation activity may be used to prevent or reduce the likelihood of such mutations.

XV. Combination Therapy

The present disclosure also contemplates combinations of one or more HMTase inhibitory compounds with one or more other agents useful in the treatment of hypermethylation-related disease. For example, the compounds of this disclosure may be administered in combination with effective doses of other medicinal and pharmaceutical agents. In some embodiments, one or more known anti-cancer drugs are included with the HMTase inhibitor. The term "administration in combination with" refers to both concurrent and sequential administration ofthe active agents.

In addition, the compounds and/or peptides of this invention may be administered in combination with effective doses of radiation, anti-proliferative agents, anti-cancer agents, direct DNA methylation inhibitors, immunomodulators, anti-inflammatories, anti-infectives, hypomethylation agents, nucleosides and analogs thereof, and/or vaccines.

Examples of anti-proliferative agents that can be used in combination with a HMTase inhibitor as provided herein include, but are not limited to, the following: ifosamide, cisplatin, methotrexate, procarizine, etoposide, BCNU, vincristine, vinblastine, cyclophosphamide, gencitabine, 5-fluorouraciI, paclitaxel, or doxorubicin.

Non-limiting examples of immuno-modulators that can be used in combination with a HMTase inhibitor as provided herein are AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Specific examples of other compounds that in some embodiments are used in combination with a HMTase inhibitor are 5-azacytidine, Zebularine, 2'-deoxy-4-azacytidine, ara-C, and tricostatin A. It is believed that such agents may be additive and/or synergistic with the HMTase inhibitor in inhibiting DNA methylation.

The combination therapies are of course not limited to the lists provided in these examples, but includes any composition for the treatment of diseases or conditions associated with hypermethylation of one or more gene sequences.

XVI. Kits

The HMTase inhibitors and related compounds disclosed herein can be supplied in the form of kits for use in inhibiting a DNA methylation, kits for use in reducing the methylation of a histone or a nucleic acid, and kits for prevention and/or treatment of a disorder, condition or diseases (e.g., a hyper-proliferative disorder, such as neoplasm, in particular a hyper-proliferative disorder that is mediated by methylation of one or more gene sequences). In such a kit, a hypomethylating effective amount of one or more ofthe compounds is provided in one or more containers. The compounds may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. In certain embodiments, the compounds will be provided in the form of a pharmaceutical composition. Kits can also include instructions, usually written instructions, to assist the user in treating or preventing a disorder, condition or disease (e.g., a methylation-mediated hyper-proliferative disorder) with a HMTase-activity modifying compound and/or binding peptide. Such instructions can optionally be provided on a computer readable medium. The container(s) in which the compound(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, the therapeutic compound may be provided in pre-measured single use amounts in individual, typically disposable, tubes, or other such containers. The amount of a compound supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit were adapted for research or clinical use, the amount of each HMTase-activity modifying compound provided likely would be an amount sufficient for several treatments.

Certain kits will also include one or more other agents useful treating or preventing a disease or condition, for instance an agent useful in directly inhibiting DNA methylation, or another agent useful in inhibiting cell proliferation that is mediated by or influenced by hypermethylation of a gene sequence, e.g. in treating hyper-proliferation of a methylation-associated tumor. For example, such kits may include one or more effective doses of anti-proliferative or anti-cancer drugs.

Aspects ofthe invention are further illustrated by the following non-limiting Examples.

EXAMPLES

Example 1 : General Methods

Analysis of DNA methylation

Genomic DNA was isolated from liquid cultures grown two days at 32° C and analyzed for DNA methylation by Southern hybridization as previously described (Foss et al, Science 262:1737- 1741 , 1993). The probe for the Ψ63 region was a 0.9 kb Banll-EcoO\09 fragment isolated from pPG22 (Margolin et al. , Genetics 149: 1787-1797, 1998). The 0.8 kb BamHλ fragment was used to probe for the ζ-η region and a 9.2 kb Kpnl fragment representing one repeat unit ofthe rDNA was used was used to probe for rDNA. The 1D21 and 9a20 probes were generated by PCR from the wild- type strain 74-OR23-IVA. Strains used for the methylation analysis shown in FIG 2 were: 74-OR23- IVA (wt; all blots), dim-2 strains N1275 (Foss et al, Science 262:1737-1741, 1993) (dim-2 arg-10 mat A; Ψ63, ζ-η and rDNA blots) and N1877 (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001 ) (dim-2::hph his-3 mat a; 1 D21 and 9A20 blots) and dim-5 strains N2144 (dim- 5; Ψ63 , ζ-η and rDNA blots); N2145 (dim-5 leu-2 pan-2 mat a; 1D21 blot) and N2140 (dim-5 leu-2 pan-2 mat A; 9A20 blot). Complementation tests and quelling experiments Complementation of dim-5 was accomplished with either a 4.3 kb hibDldim-5 PCR fragment amplified from wildtype strain 74-OR8-la with Pfu DNA polymerase (Promega) or restriction fragments of this PCR fragment (FIG 3 A). Strain N2145 (dim-5 leu-2 pan-2 mat a) was co-transformed by electroporation (Margolin et al, Fungal Genetics Newsl. 44:34-36, 1997) using 100 μg pBT6, which confers bml^R (Orbach et al , Mol Cell Biol 6 2452-2461, 1986), and 300 μg of the test DNA

For quelling, a PCR fragment containing the presumed dim-5 ORF was generated, cut within the SET domain ofthe gene with EcoRV, and introduced into strain N644 (Irelan & Selker, Genetics 146 509-523, 1997, dιm-5⁺ am ^pIhph/am^R"'am'³² ml mat a) by co-transformation with pBT6

Approximately 10⁴ conidia of representative bml^R transformants were spot-tested on Vogel's sorbose medium in the presence or absence hygromycin (200 μg/ml, Calbiochem) Genomic DNA was isolated from liquid cultures grown two days at 32° C and analyzed for DNA methylation (Foss et al , Science 262 1737-1741, 1993) Identification of mutation in dιm-5 allele HTl

To identify the mutation in dim-5 allele HTl of strain N2140, pooled PCR products from six independent reactions produced with Pfu DNA polymerase and the dιm-5 ORF primers were gel- puπfied and sequenced directly on both strands The wildtype (FGSC#988) allele was isolated and sequenced in the same way (accession AF419248) Generation and purification of GST-DIM-5 fusion protein

A segment ofthe dιm-5 ORF, including amino acid residues 19-318, was amplified from Neurospora wild-type strain 74-OR8-la (FGSC#988) with Pfu DNA polymerase and GST-DIM-5 primers (PCR primers are listed in the Brief Description ofthe Sequences Listing as SEQ ID NOs 6-21) The PCR product was digested with BamHλ and EcoPΛ, gel-purified and cloned into the GST- fusion expression vector pGEX-5X-3 (Pharmacia) using E coli strain DH5αF' Recombinant protein was prepared from a 800 ml culture of E coli cells grown three hours at 37° C in LB medium with ampicillin (400 μg/ml), shifted to 30° C for 40 minutes, induced with IPTG (0 1 M) and collected one hour later Cells were lysed by somcation on ice in 8 ml of RIPA buffer [20 mM Tπs (pH 7 5), 500 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630 (Sigma), 0 5% deoxycholate] (Rea et al , Nature 406 593-599, 2000) with 5 mg/ml lysozyme and a proteinase inhibitor cocktail (Complete™,

Boehringer Mannheim) The extract was clarified by centrifugation and the soluble proteins were incubated at 4° C for 15 minutes with 700 μl glutathione-agarose (Sigma) equilibrated in RIPA GST-DIM-5 protein was purified by washing three times with 30 ml RIPA buffer, followed by elution from glutathione-agarose using 75 mM HEPES (pH7 9), 150 mM NaCl, 5 mM DTT and 10 mM reduced glutathione (Sigma) The eluate was concentrated with a Centπcon-30 filter (Millipore) Histone methyltransferase assay

HMTase assays were carried out on a natural mixture of calf thymus histones as described (Rea et al , Nature 406 593-599, 2000) except that the reaction was carried out at 20° C for 6 hours with 2 75 μCi S-adenosyl-[methyl-³H]-L-methιomne (0 55 mCi/ml, NEN) Products were fractionated on SDS-polyacrylamide (16 5%, 29 1) gels and fluorographed (4-12 hours) using ENTENSIFY™ (DuPont) Example 2: Isolation and Genetic Mapping of the dim-5 Mutation

One hundred fifty 5-azacytidine-resistant strains, selected from approximately 12,000 pRALl (qa-2⁺) transformants of an aro-9 qa-2 mus-20 strain (N2141 ) were tested for methylation defects by Southern hybridization using probes for the ζ-η (Foss et al, Science 262:1737-1741, 1993) and Ψ63 (Margolin et al, Genetics 149:1787-1797, 1998) methylated regions. One strain showed greatly reduced methylation in both regions. The methylation defect segregated in genetic crosses as expected of a normal Mendelian allele but, suφrisingly, it did not co-segregate with either 5-azacytidine resistance or a pRALl insertion. Moreover, complementation tests between the new dim strain and previously identified mutants (dim-1, dim-2 and dim-3) demonstrated that the mutant represents a new complementation group, which we designated dim-5. Prior to detailed characterization, the dim-5 mutation was purified from the mutagenized mus-20 background by five backcrosses to a wildtype strain. Dim-5 strains showed slow, irregular growth (FIG 1) unlike other methylation mutants, including dim-2 (DMTase) null mutants (see Supplementary Information). In addition, homozygous dim-5 crosses revealed a partial barren phenotype; few spores were produced, and most of those produced were inviable.

About 1.5% ofthe cytosines in N. crassa DNA are methylated (Foss et al, Science 262: 1737-1741, 1993) and all known methylation is in relics of repeat-induced point mutation (RIP) and rDNA (Selker, Annu. Rev. Genet. 24:579-613, 1990). We therefore analyzed four relics of RIP (ζ-η (Selker, Annu. Rev. Genet. 24:579-613, 1990), Ψ63 (Margolin et al. , Genetics 149: 1787-1797, 1998), 1D21 and 9A20) and rDNA for methylation in dim-5 strains. Southern hybridizations using the isoschizomers Dpnll and Sau3Al, which both recognize GATC but differ in that only Sau3 AI is inhibited by cytosine methylation, revealed no methylation in dim-5 strains at any ofthe five regions tested (FIG 2). Inspection of total genomic DNA digested with Dpnll or Sau3Al and stained with ethidium bromide indicated that dim-5, like dim-2, eliminates all, or nearly all, DNA methylation (FIG 2).

To identify the dim-5 gene, we first mapped it by conventional genetics, scoring the mutation by Southern hybridization. Results of initial crosses localized the gene to linkage group IV. Analysis of three-point linkage data from a cross between a dim-5 pan- 1 pyr-2 strain (N2142) and a trp-4 strain (N185) placed dim-5 ~2 map units centromere-distal of trp-4 (see Supplementary Information). To determine whether dim-5 is in the 2-4 map unit region between trp-4 and leu-2, or centromere-distal of leu-2, we tested methylation of DNA from trp-4* leu-2* recombinant progeny from a cross ofthe trp-4 strain with a dim-5 leu-2 strain (N2140). Fifteen of 42 recombinants were defective in methylation, establishing that dim-5 is between trp-4 and leu-2, a region in which no mutation had been previously mapped (Perkins et al, The Neurospora Compendium; chromosomal loci, Academic Press, San Diego, CA., 2001). /035844

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Exainple 3: Identification of the dim-5 ORF

As a step to identify the dim-5 gene, we searched N. crassa genomic sequence data (Assembly version 1. Neurospora Sequencing Project, Whitehead Institute/MIT Center for Genome Research, 2001 ) for leu-2 and trp-4 based on their expected homology to Saccharomyces cerevisiae LEU1 and TRP 4, respectively (Perkins et al, The Neurospora Compendium; chromosomal loci,

Academic Press, San Diego, CA., 2001). Candidates for both genes were found separated by -80 kb, consistent with the genetic distance between these genes.

The interval between the putative leu-2 and trp-4 genes was scrutinized for dim-5 candidates. One of 15 candidates in this region identified using BLASTx is predicted to encode a protein related to chromatin-associated proteins involved in gene silencing in fission yeast and fruit flies, namely Schizosaccharomyces pombe Clr4 (Ivanova et al, Nat Genet 19:192-195, 1998) and Drosophila melanogaster Su(var)3-9 (Tschiersch et al, EMBO J. 13:3822-3831, 1994). Mutations in clr4 de-repress silent mating type genes and genes inserted in other repressive domains and cause aberrant chromosome behavior (Allshire et al, Genes Dev 9:218-233, 1995) while mutations in su(var)3-9 suppress the gene silencing phenomenon known as position effect variegation in Drosophila (Tschiersch et al, EMBO J. 13:3822-3831 , 1994).

To test the possibility that the related Neurospora gene is required for DNA methylation in Neurospora, fragments were tested from the region for complementation ofthe dim-5 mutation. Because co-transformation is highly efficient in Neurospora, we simply transformed a dim-5 strain with a mixture of a plasmid (pBT6) conferring resistance to benomyl (bml^R) plus a test fragment and then assayed random bml^R transformants for de novo methylation of Ψ63. Southern blot analysis of 26 bml^R transformants generated with a 4.3kb PCR fragment containing the clr4/ su(var)3-9 homologue, plus a putative 3-hydroxyisobutyrate dehydrogenase (hibD) gene, showed that 24 had integrated the non-selected fragment and all of these displayed substantial de novo DNA methylation at the Ψ63 and ζ-η regions and in the genome overall. Equivalent results were obtained with smaller restriction fragments including just the clr4/su(var)3-9-related gene (FIG 3 A, B). We therefore tentatively concluded that this ORF is dim-5.

Example 4: De-repression of a Silenced Transgene by Quelling dim-5 It remained formally possible that ectopic insertions ofthe Neurospora clr4l su(var)3-9 homologue suppressed the dim-5 mutation as the result of a dosage effect but was not itself dim-5. We took advantage of "quelling," a post-transcriptional gene silencing mechanism of Neurospora that does not depend on DNA methylation (Cogoni et al. , Embo J 15:3153-3163, 1996) to carry out an independent test ofthe clr4/su(var)3-9 homologue. In particular, we tested whether introduction of fragments ofthe presumptive dim-5 coding region into a Dim⁺ Neurospora strain (N644) carrying a methylated, silent transgene (hph) would silence the wildtype dim-5 gene and thereby activate the transgene (FIG 4 A). Strain N644 was co-transformed with a mixture of pBT6 and an £coRV-digest of a PCR fragment containing the entire presumptive dim-5 ORF and tested bml^R transformants for resistance to hygromycin (hyg^R), which should result if the hph gene were demethylated.

Five of twelve bml^R transformants generated with the DNA mixture showed strong hyg^R and several others showed partial resistance (FIG 4B). In contrast, none ofthe control transformants generated with pBT6 alone were hyg^R. To test directly for loss of DNA methylation, DNA was isolated from these strains grown under selection for benomyl, but not hygromycin, and assayed methylation at Ψ63 (FIG 4C). Substantial hypomethylation was observed and was correlated with activation ofthe silenced hph gene. These results therefore confirmed the tentative identification of dim-5 and demonstrated that this gene is subject to quelling.

Example 5: The dim-5 Mutant has a Nonsense Mutation in the Evolutionarily

Conserved SET Domain

As a further test of whether the dim-5 gene was correctly identified, the ORF was PCR- amplified and sequenced from the mutant and from its wildtype parental strain. A single C to G mutation was found in the serine codon (TCA) at amino acid position 216 ofthe predicted 318 amino acid polypeptide. The mutation generated a stop codon in the middle of a distinctive ~130 amino acid sequence motif called the SET domain (FIG 5). DIM-5 is a SET domain protein homologous to genes required for heterochromatin formation. The SET domain was initially identified as a region of apparent homology in three nuclear proteins of Drosophila, Su(var)3-9, the polycomb group protein E(Z) and trithorax-group protein TRX (Tschiersch et al, EMBOJ. 13:3822-3831, 1994). Greater than 200 genes with SET domains are now known (Jenuwein et al, Trends Cell Biol 1 1 :266-273, 2001). Like clr4, su(var)3-9 , SUV39H1, Suv39hl and Suv39h2, dim-5 includes cysteine-rich sequences flanking a SET domain (FIG 5). Noting that some SET proteins are protein methyltransferases, Jenuwein and colleagues examined the possibility that chromatin-associated SET domain proteins are histone methyltransferases (HMTases) (Rea et al, Nature 406:593-599, 2000). Although not detected with all the chromatin-associated SET proteins, S. pombe Clr4 (Rea et al, Nature 406:593-599, 2000; Nakayama et al, Science 292:1 10-1 13, 2001) and the closely related proteins from humans (SUV39H1 ; Rea et al, Nature 406:593-599, 2000) and mouse (Suv39hl, Rea et al, Nature 406:593- 599, 2000; and Suv392h2, O'Carroll et al, Mol Cell Biol 20:9423-9433, 2000) did indeed show HMTase activity. Analyses of in v / O-generated variants indicated that both the SET domain and the associated cysteine-rich sequences are required for HMTase activity (Rea et al, Nature 406:593-599, 2000; Nakayama et al, Science 292: 1 10-1 13, 2001).

Example 6: Dim-5 Encodes a Histone H3 Methyltransferase

The possibility that DIM-5, like Clr4 and Suv39h2, is a histone methyltransferase was directly investigated. An inducible glutathione-S-transferase (GST) fusion construct was built, containing nearly the entire DIM-5 coding region, similar to constructs made to assay HMTase activity of Clr4 and Suv39h2 (FIG 5b) Recombinant DIM-5 fusion protein was purified from E coli, provided with S-adenosyl-[methyl-³H]-L-methιonιne as a potential methyl-group donor and incubated with a natural mixture of histones from calf thymus The proteins were then fractionated by SDS-polyacrylamide gel electrophoresis and assayed for incoφoration of methyl groups by fluorography and scintillation counting of gel slices Significant incoφoration of labeled methyl groups into histones was detected, indicating that DIM-5 is a bonafide HMTase Moreover, histone H3 was the acceptor of nearly all the methylation, as found with CIr4 and Suv39h2 (FIG 6) Much weaker incoφoration of label was detected in a protein tentatively identified as a histone HI

Example 7: Lysine 9 of Histone H3 Gene is Involved in DNA Methylation

The clear experimental demonstration that DIM-5 has HMTase activity suggested that methylation of histone H3 is necessary for DNA methylation in Neurospora It remained possible, however, that the HMTase activity was not relevant to its role in DNA methylation In vivo evidence was therefore sought that would demonstrate the involvement of histone H3 in DNA methylation The Clr4 and Suv39h2 HMTases are specific for lysine 9 (K9) of H3 (Rea et al , Nature 406 593-599, 2000, Nakayama et al , Science 292 1 10-113, 2001), a residue that can be either methylated or acetylated (Jenuwein et al , Trends Cell Biol 1 1 266-273, 2001) (FIG 7A)

If methylation of K9 of histone H3 were required for DNA methylation in Neurospora, then replacement of this residue with an amino acid that is not subject to this modification should interfere with DNA methylation To test this idea, the hH3 gene was mutated in vitro, replacing the lysine codon with codons for leucine (L) or arginine (R), and the modified genes were introduced into strain N644 using co-transformation Leucine and arginine were chosen because (1) they are structurally similar to lysine, (2) the neutral amino acid leucine can be regarded as a mimic of an acetylated lysine, (3) the positively charged amino acid arginine can be regarded as a mimic of an unacetylated lysine, and (4) leucine is known not to be a substrate for methylation of recombinant Suv39hl HMTase (Rea et al , Nature 406 593-599, 2000)

Lysine 9 of N crassa histone H3 (Woudt et al , Nucl Acids Res 1 1 5347-5361, 1983) was changed to leucine and arginine using the PCR-based QuickChange™ site-directed mutagenesis protocol (Stratagene) with a 4 9 kb plasmid carrying the wildtype H3 gene (hH3) and 1 161 bp of 5'- flanking sequences (pSH12) as template Primer pairs H3L9 and H3R9 were used to generate CTC and CGT codons in place ofthe AAG codon, respectively The resulting plasmids (pSH12L9 and pSH12R9, respectively), and the wildtype control were linearized using Xbal and cotransformed into N crassa strain N644 along with / /rtdlll-lineaπzed pBT6 Approximately 1000 conidia from transformants grown en masse on solidified Vogel's sucrose medium in flasks were plated on media containing no drug, benomyl (0 5 μg/ml) or hygromycin (200 μg/ml) Random hyg^R transformants, which were obtained with pSH12L9 and pSH12R9 but not with pSH12 (FIG 7), were grown in liquid medium in the absence of hygromycin to isolate DNA The presence of ectopic hH3 sequences with /035844

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the expected mutations was verified by Southern hybridization and direct sequencing of PCR products generated using H3-ORF primers.

N. crassa has only a single copy ofthe hH3 gene (Woudt et al, Nucl Acids Res. 1 1 :5347- 5361, 1983), but gene replacement by homologous recombination is inefficient in Neurospora. In addition, replacement ofthe wild-type hH3 with the mutated versions might be lethal. It seemed possible, however, that the mutations would prove dominant or semi-dominant. Advantage was taken ofthe methylated hph allele of N644 to test for loss of DNA methylation in random transformants generated with the mutated hH3 constructs. Transformants generated with mutant or wildtype hH3 genes together with the cotransformation marker, Bml, were selected en masse on benomyl medium, then tested for expression of hph. About 500 asexual spores from each pool, representing -30 bml^R transformants, were spread on hygromycin plates (FIG 7B).

Striking results were obtained: Hyg^R colonies were obtained with both mutants, but not with the wildtype control, in two independent experiments. Southern hybridization analyses of DNA isolated from representative hyg^R transformants demonstrated that the strains had drastically reduced DNA methylation at Ψ63 and confirmed that they contained modified hH3 alleles (FIG 7C).

Curiously, the hyg^R transformants in each experiment contained a single ectopic copy ofthe mutant allele, which is unusual for Neurospora. Perhaps additional copies ofthe mutant hH3 genes were toxic, either directly or because they caused quelling, reducing H3 levels beyond the point that the cells could survive. Direct DNA sequencing of hH 3 PCR products confirmed that the strains contained both the wildtype and mutant sequences (FIG 7C). These results strongly support the inference from other results reported herein that methylation of histone H3 is critical for DNA methylation.

Example 8: Generation of Heterochromatin by RIP The densely staining "constitutive heterochromatin," which is frequently found near centromeres and telomeres in eukaryotes, provides an example of how DNA sequences can direct the formation of specialized forms of chromatin. Constitutive heterochromatin is typically rich in moderately repeated sequences, such as transposons, and highly repeated sequences, such as satellite DNA, and displays a number of other identifying characteristics. It remains condensed after mitosis, replicates late in S-phase, shows low levels of genetic recombination, contains special forms of histones and, in organisms with DNA methylation, such as mammals and plants, it is hypermethylated (Hennig, Chromosoma 108:1-9, 1999). Classical studies in Drosophila demonstrated that heterochromatin is a repressive environment for most genes; rearrangements that move heterochromatic regions typically cause silencing of nearby genes in some fraction ofthe cells. Isolation of suppressors ofthe resulting variegation identified the heterochromatin protein HP1

(Su(var)3-5) (Eissenberg et al, Proc Natl Acad Sci U S A 87:9923-9927, 1990), a HMTase (Su(var)3- 9) (Rea et al, Nature 406:593-599, 2000) and an S-adenosylmethionine synthetase (Su(z)5) (Larsson et al, Genetics 143:887-896, 1996). The very much smaller heterochromatic regions of S. pombe depend on a similar set of silencing genes, including the chromo domain genes Swi6 and Clr4, which encode a HP 1 -like protein and a histone H3 MTase, respectively (Nakayama et al, Science 292: 110- 1 13, 2001). The chromo domain of HP1 has recently been shown to recognize methylated Lys 9 of histone H3 (Bannister et al, Nature 410:120-124, 2001 ; Lachner et al, Nature 410: 116-120, 2001). The finding reported herein that DNA methylation in Neurospora relies on histone methylation raises the possibility that sequences mutated by RIP, which constitute the bulk of methylated sequences of this organism and are found concentrated in centromeric DNA (Cambareri et al. , Mol Cell Biol 18:5465-5477, 1998), serve to nucleate heterochromatin. RIP detects duplicated sequences, such as transposons (Margolin et al, Genetics 149: 1787-1797, 1998; Cambareri et al. , Mol Cell Biol 18:5465-5477, 1998), during the sexual phase ofthe Neurospora life cycle and peppers them with G:C to A:T mutations (Selker, Annu. Rev. Genet. 24:579-613, 1990). Like some satellite sequences, products of RIP are bound by proteins that show limited sequence-specificity. These proteins may be responsible for recruiting heterochromatin proteins - perhaps the DIM-5 HMTase. Thus relics of RIP may underlie heterochromatin in Neurospora and serve the cell for centromere function. The observation that dim-5 strains grow irregularly and are nearly sterile in homozygous crosses, unlike other known DNA methylation mutants, implies that DIM-5 is involved in process(es) besides DNA methylation, e.g., heterochromatin formation.

Example 9: A New Paradigm: Histones as Signal Transducers for DNA Methylation The control of DNA methylation has remained enigmatic despite decades of intensive investigations in mammals, plants, and fungi. Although prokaryotic and eukaryotic DNA methyltransferases show striking structural similarities, prokaryotes offer an inappropriate paradigm for DNA methylation in eukaryotes. Bacterial DMTases require nothing more than DNA and a methyl-group donor for proper function and are sequence-specific. In contrast, eukaryotic DMTases have substantial non-catalytic domains that reflect interactions with other proteins (Colot &

Rossignol, Bioessays 21 :402-41 1, 1999) and show little sequence-specificity (Yoder et al, J Mol Biol 270:385-395, 1997).

Based on the discoveries reported herein, it is now believed that eukaryotic DMTases evolved to take their cues primarily from chromatin. A common view is that histones act as obstacles to proteins that need access to the DNA. The discovery of "chromatin remodeling" factors that can move nucleosomes (Kingston & Narlikar, Genes Dev 13:2339-2352, 1999), and of a putative chromatin remodeling factor involved in DNA methylation (Jeddeloh et al, Nat. Genet. 22:94-97, 1999) fit this view. The herein-reported discovery that DNA methylation depends on histone methylation in Neurospora suggests a alternative possibility: Proteins that require access to DNA, such as DMTases, actually depend at least to some extent on histones as cofactors. In light of this, it is further believed that chromatin remodeling factors may assist histone modification enzymes to reach their targets and/or to facilitate exchange of nucleosomes with different modification states. Histones are well suited to integrate information relevant to whether DNA in a particular region should be methylated. In addition to their proximity to DNA, histones are subject to a variety of post-translational modifications (phosphorylation, methylation, acetylation, ubiquitination, and ADP-ribosylation) that can play informational roles in the cell (Strahl & Allis, Nature 403:41-45, 2000). Acetylation, currently the best understood modification, is controlled by histone acetylases (HATs) and histone deacetylases (HDACs), which typically act as transcriptional coactivators and corepressors, respectively.

Mechanistic connections are emerging among DNA methylation, histone deacetylation, and histone methylation (Dobosy & Selker, Cell Mol Life Sci 58:721-727, 2001). The histone deacetylase inhibitor trichostatin A (TSA) causes selective loss of DNA methylation in Neurospora, suggesting that histone acetylation can influence DNA methylation (Selker, Proc Natl Acad Sci USA 95:9430- 9435, 1998). Mammalian methyl-DNA binding proteins and DMTases associate with HDACs (Dobosy & Selker, Cell Mol Life Sci 58:721-727, 2001). Moreover, in S. pombe, TSA treatment or mutation of HDAC genes causes mis-localization of Swi-6 and other defects characteristic of disruption of the Clr4 HMTase (Nakayama et al. , Science 292: 1 10-113, 2001 ; Grewal et al. , Genetics 150:563-576, 1998; Ekwall et al, Cell 91 : 1021-1032, 1997). This is perhaps because methylation of K9 of histone H3 is inhibited by acetylation of lysine 9 or 14 (Rea et al, Nature 406:593-599, 2000; Nakayama et al, Science 292:110-1 13, 2001). Phosphorylation of Ser 10 also strongly inhibits methylation of lysine 9 (Rea et al, Nature 406:593-599, 2000), providing another illustration of how histones can integrate information from multiple inputs and act as signal transducers.

Example 10: De Novo and Maintenance Methylation of DNA in Eukaryotes

A defining feature of epigenetic states is that they promote their own propagation. Thus active chromosomal regions are rarely silenced and silenced regions are rarely activated. Holliday and Pugh and Riggs recognized that the symmetry of methylated sites (5'-CpG/GpC-5') in mammalian DNA would support a simple mechanism to propagate methylation patterns; all that was required was a DMTase specific for hemimethylated sites (Bestor & Tycko, Nat Genet 12:363-367, 1996). The "maintenance methylase" model was supported by evidence that methylation states are indeed propagated and by the discovery of DMTases that prefer hemimethylated substrates. Nevertheless, the classic maintenance model does not account for some observations, such as heterogeneous methylation in cell clones, spreading of methylation and stable propagation of methylation at non-symmetrical sites, as observed in Neurospora and other eukaryotes (Singer et al, Mol. Cell. Biol. 15:5586-5597, 1995; Miao et al, J. Mol. Biol. 300:249-273, 2000; Selker et al, Science 262:1724-1728, 1993). With the demonstration herein that histone modifications can impact both de novo and maintenance DNA methylation, it is believed feasible that propagation of DNA methylation patterns in eukaryotes depends on feedback loops between modifications of chromatin proteins and DNA. Histones H3 and H4 remain tightly bound to DNA in vivo, unlike histones H2A and H2B (Kimura et al. , J Cell Biol 153 : 1341 - 1354, 2001 ), consistent with the idea that these histones are involved in the propagation of epigenetic states.

In Drosophila and S. pombe, inteφlay of heterochromatin proteins propagate epigenetic states without relying on DNA methylation (Lewis, Adv. Genet. 3:73-1 15, 1950; Allshire et al, Cell 76: 157-169, 1994; Grewal & Klar, Cell 86:95-101, 1996). If the chromo domains of Clr4 and Su(var)3-9 recognize methylated K9 of histone H3, as with HP1 (Bannister et al, Nature 410:120- 124, 2001 ; Lachner et al, Nature 410: 116-120, 2001), this might lead to preferential methylation of histones in previously methylated regions, propagating silencing.

The chromo domain is absent from the DIM-5 HMTase, and from the recently described G9a HMTase (Tachibana et al, J Biol Chem 276:25309-25317, 2001). Perhaps DNA methylation and associated factors (e.g., DMTases and methyl-DNA binding proteins) substitute for this potential self-reinforcing system. DMTases containing a chromo domain have been identified in plants (Lindroth et al, Science 292:2077-2080, 2001), suggesting that some DMTases may take cues directly from histones. A search of public databases with DIM-5 revealed a number of potential HMTases that may be involved in DNA methylation; certain of these potential HMTases are listed in Table 2.

Table 2: Examples of putative and/or known histone methyltransferases that may be involved in DNA methylation

Example 11: Structure of the Neurospora SET Domain Protein DIM-5

Histones are subject to extensive posttranslational modifications including acetylation, phosphorylation, and methylation, primarily on their N-terminal tails that protrude from the nucleosome. Evidence accumulated over the past few years suggests that such modifications constitute a "histone code" that directs a variety of processes involving chromatin (Jenuwein and Allis, Science 293: 1074-1080, 2001 ; Strahl and Allis, Nature 403:41-^15, 2000). Histone methylation represents the most recently recognized component ofthe histone code Most histone methylation occurs on lysine, though arginine methylation also occurs on histones H3 and H4 (Ma et al , Curr Biol 1 1 1981-1985, 2001 , Strahl et al , Curr Biol 11 996-1000, 2001, Wang et al , Science 293 853-857, 2001b) Lysine methylation is highly selective, with the best- characterized sites being K4 and K9 of histone H3 In general, K9 methylation is associated with transcπptionally inactive heterochromatin, while K4 methylation is associated with transcπptionally active euchromatin (Boggs et al , Nat Genet 30 73-76, 2002, Litt et al , Science 293 2453-2455, 2001 , Nakayama et al , Science 292 110- 113, 2001 , Nishioka et al , Gene Dev 16 479^189, 2002a) In addition, K9 methylation has been implicated in transcriptional silencing of euchromatic genes such as those involved in cell cycle control (Nielsen et al , Nature 412 561-565, 2001, Ogawa et al , Science 296 1 132-1136, 2002), and K4 methylation is involved in silencing of rDNA and telomere sequences in the yeast Saccharomyces cenvisiae (Bπggs et al , Genes Dev 15 3286-3295, 2001, Krogan et al , J Biol Chem 277 10753- 10755, 2002) Methylated K9 of histone H3 is specifically recognized by the chromo domain of heterochromatin protein HPl, which presumably directs the binding of additional proteins involved in the control of chromatin structure and gene expression (Bannister et al , Nature 410 120-124, 2001 , Jacobs et al , EMBO J 20 5232-5241, 2001, Lachner et al , Nature 410 1 16-120, 2001)

The discovery that some SET domain proteins are responsible for methylation of lysines in histone tails provided an important advance in our understanding ofthe workings ofthe histone code (Rea et al , Nature 406 593-599, 2000) The SET domain was originally identified in three

Drosophila genes involved in epigenetic processes, Su(var)3-9, £h(zeste), and 7rιthorax (Jenuwein et al , Cell Mol Life Sci 54 80-93, 1998) Mammalian homologs of Drosophila SU(var)3-9 were shown to specifically methylate H3 at lysine 9 (Rea et al , Nature 406 593-599, 2000) Soon thereafter, related /πstone lysine (K) /wethyl/ransferases (HKMTs) in various species (see brief description of FIG 8) were found to methylate K4, K9, K27, or K36 of H3 methylated by a protein containing no SET domain (Feng et al , Curr Biol 12 1052-1058, 2002, Lacoste et al , J Biol Chem 277 30421-30424, 2002, Ng e/ α/, Genes Dev 16 1518-1527, 2002, van Leeuwen e/ α/ , Cell 109 745-756, 2002)

The approximately 130 amino acid SET domain is found in a large number of eukaryotic proteins as well as a few bacterial proteins and is not limited to histone H3 lysine 9 methyltransferases (HKMTs) More than 60 SET domain genes have been identified in humans (Pfam database, available online at The Sanger Institute) nearly 40 are found in the genome of Arabidopsis thaliana (Baumbusch et al , Nucleic Acids Res 29 4319-4333, 2001), and about 10 each are found in Drosophila and the fungi Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Neurosopora crassa SET proteins can be grouped into families according to the sequences surrounding this distinctive domain (Baumbusch et al , Nucleic Acids Res 29 4319-4333, 2001 , Kouzaπdes, Curr Opin Genet Dev 12 198-209, 2002) The SUV39 family proteins, which methylate K9 of H3 (O'Carroll et al Mol Cell Biol 20 9423-9433, 2000, Rea et al , Nature /035844

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406:593-599, 2000; Schultz et al., Genes Dev. 16:919-932, 2002; Tamaru and Selker, Nature 414:277-283, 2001) or K9 and K27 of H3 (Tachibana e/ αt, J. Biol. Chem. 276:25309-25317, 2001) and include the most active HKMTs known to date, contain two cysteine-rich regions flanking the SET domain. These "pre-SET" and "post-SET" domains are required for histone H3 lysine 9 methyltranferase (HKMT) activity of SUV39H 1 (Rea et al. , Nature 406:593-599, 2000).

As a step to elucidate the mechanism of SET domain HKMTs, the structure of DIM-5, a K9 histone H3 methyltransferase (MTase) from TV. crassa was characterized (Tamaru and Selker, Nature 414:277-283, 2001). The discovery that this member ofthe SUV39 family is essential for DNA methylation in vivo revealed a connection between histone methylation and DNA methylation. This connection has been reinforced by the observation that a HKMT from A. thaliana is also involved in DNA methylation (Jackson et al, Nature 416:556-560, 2002).

This example provides a description ofthe elucidation ofthe crystal structure of Neurospora DIM-5, a HKMT, determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization ofthe HKMT family and the SET domain. This example is adapted from Zhang et al., Cell 1 1 1 : 1 17-127, 2002, which is incoφorated herein by reference in its entirety.

Experimental Procedures

Protein Expression and Purification -V. crassa DIM-5 protein was expressed as a GST fusion. A segment ofthe wild-type dim-5

ORF, including amino acid residues 17-318, was amplified from pGEX-5X-3/DIM-5 (Tamaru and Selker, Nature 414:277-283, 2001) and subcloned between the Bam l and £coRI sites in pGEX2T (Amersham-Pharmacia), yielding pXC379. E. coli strain BL21(DE3) Codon plus RIL (Stratagene) carrying pXC379 was grown in LB medium supplemented with 10 μM ZnS0₄ at 37°C to OD ₀₀ = 0.5, shifted to 22°C, and induced with 0.4 mM IPTG overnight at 22°C. The proteins were purified using Glutathione-Sepharose 4B (Amersham- Pharmacia), UnoQ6 (Bio-Rad), and Superdex 75 columns (Amersham-Pharmacia). The GST tag was cleaved by applying thrombin to fusion proteins bound to the Glutathione-Sepharose column, leaving five additional residues (GSHMG) in front of amino acid 17 of DIM-5. All purification buffers contained 1 mM DTT and no EDTA. The protein was stored in the Superdex 75 column buffer containing 20 mM glycine (pH 9.8), 150 mM NaCl, 1 mM DTT, and 5% glycerol. Se-containing DIM-5 (with five methionines) was expressed in a methionine auxotroph strain (B834) grown in the presence of Se- methionine, and the protein was purified similarly to the native protein. Methyl Transfer Activity Assay

The activity was assayed in a 20 μl reaction containing 50 mM glycine (pH 9.8), 2 mM DTT, 40-80 μM unlabeled AdoMet (Sigma), 0.5 μCi [methyl-³H]AdoMet (78 Ci/mmol, NEN NET155H), 0.25-0.5μg of DIM-5 protein, and 2-5 μg histones (calf thymus histones Sigma H4524, Roche 223565, or recombinant chicken erythrocyte histones, a gift from Dr. V. Ramakrishnan). The reaction was incubated at room temperature for 10—15 minutes and methylation was analyzed either by SDS-PAGE and fluorography or by precipitation with 20% TCA, filtration (Millipore GF/F filter), washing, and liquid scintillation counting. Under these conditions, DIM-5 activity was linearly related to reaction time and amount of enzyme and AdoMet and histone were saturating. For some reason, the relatively crude Sigma H4524 histone preparations generally gave 2- to 4-fold higher incoφoration than either the Roche preparations or the recombinant histones. AdoMet Binding Assay by UV Crosslinking

Twenty microliters of purified DIM-5 protein (2-5 μg) was incubated with 0.5 μCi of [methyl-³H]AdoMet (78 Ci/mmol, NEN NET155H) overnight at 4°C. Samples were added to a 96- well plate on ice and placed 8 cm from an inverted UV transilluminator (VWR, 302 nm) for 1 hour. The protein was then separated by SDS-PAGE, stained with Coomassie, and subjected to fluorography. Mutagenesis

Amino acid replacements of DIM-5 (SEQ ID NO: 3) to yield R155H, W161F, Y204F, R238H, N241Q, H242K, D282K, and Y283F were made using Quik-Change site-directed mutagenesis protocol (Stratagene) using pXC379 and primer pairs to generate CAC, TTC, TTC, CAC, CAG, AAA, AAC, and TTC codons in place of AGG, TGG, TAC, AGG, AAC, CAC, GAC, and TAT codons, respectively (see SEQ ID NO: 2). The DIM-5 mutant 3C to 3S, in which all three invariant cysteines in the post-SET region are replaced by serines, was generated by PCR using a mutagenic 3' primer. All mutants were sequenced to verify the presence ofthe intended mutation and the absence of additional mutations. The only exception is the Y204F mutant, which carries an additional Asp substitution (A24D) in the N-terminal region that was not observed in the structure. Mutant proteins, along with wild-type, were purified from 100-200 ml of induced cultures. A disposable column containing 0.5 ml of Glutathione-Sepharose 4B (Amersham-Pharmacia) was used for each mutant. The mutant proteins were separated from GST by on-column thrombin cleavage and then used for enzymatic assay (using calf thymus histones Sigma H4524 as substrate), Ado-Met binding by crosslinking analysis, and analytical gel filtration chromatography for native protein size determination. Zinc Content Analysis One sample of untreated and two samples of EDTA-treated DIM-5 protein (about 2 ml of 2 mg/ml each) was analyzed for the presence of 20 elements on a Thermo Jarrell-Ash Enviro 36 ICAP analyzer at the Chemical Analysis Laboratory ofthe University of Georgia at Athens. In order to calculate the molar ratio of Zn to protein, the precise concentration ofthe untreated DIM-5 protein was determined by amino acid analysis (averaging two independent measurements) performed at the Keck Facilities at Yale University. The extinction coefficient (29,559 M^''cm^"') derived from the amino acid analysis was used to estimate the protein concentration ofthe EDTA-treated samples. Crystallography

Purified DIM-5 protein was concentrated to about 10-15 mg/ml in 20 mM glycine (pH 9.8), 150 mM NaCl, 1 mM DTT, 5% glycerol, and 600 μM AdoHcy. Crystals were obtained using the hanging drop method, with mother liquor containing 1.1-1.2 M ammonium sulfate and 100 mM Na citrate (pH 5.4-5.6) at 16°C. Crystals belong to space group Ϋ2 2_x2χ with cell dimensions of 36.73 x 81.56 x 101.27 A. Each asymmetric unit contains one molecule. Complete data sets were collected from a native crystal near the Zn absoφtion edge (Table 3) and a SeMet-incoφorated crystal at both Se and Zn absoφtion edges. Table 3. Summary of X-Ray Diffraction Data Collection

^JThe numerical numbers are given for the whole data set/the highest resolution bin.

The data were processed using the HKL package (Otwinowski and Minor, Methods

Enzymol. 276:307-326, 1997). SOLVE (Terwilliger and Berendzen, Ada Crystallogr D55:849-861, 1999) first revealed the positions of three zinc atoms and RESOLVE (Terwilliger, Ada Crystallogr. D56:965-972, 2000) was then used to modify the electron density map. The modified map was of good quality at 2.9 A resolution to place amino acids of DIM-5 into the recognizable densities using O (Jones and Kjeldgard, Methods Enzymol. 277: 173-208, 1997). In parallel, SOLVE determined the positions of five selenium atoms: two of them (SeMet 233 and 248) were confirmed by Zn-phased map, and three of them (SeMet 75, 85, and 303) served as markers in the primary sequence during tracing. The resultant model was refined against the data collected at wavelength of 1.0332 A in the resolution range of 24.8-1.98 A, using the X-PLOR program suite (Brilnger, X-PLOR. A System for X-Ray Crystallography and NMR, 3.1 edn, New Haven, CT: Yale University, 1992). Three segments of DIM-5 were not observed in the final model: the N-terminal 8 residues (17-24) (these may not be present in the native DIM-5 protein as there is an in-frame splicing site immediately after these residues); residues 89-99 ofthe pre-SET domain (these are deleted in many ofthe SUV39 proteins) (see FIG 8); and the majority ofthe C-terminal 34 amino acids (the C terminus is also highly variable in length and sequence among SET proteins except for the three-Cys post-SET region). Among the nonglycine and nonproline residues, 86% are in most favored and 14% in additional allowed regions of a Ramachandran plot (Laskowski, J. Appl. Crystallogr. 26:283-291 , 1993).

Results and Discussion Overall Structure of DIM-5

Recombinant DIM-5 protein (residues 17 to 318 of Protein Data Bank accession number AF419248) was used for crystallographic studies (see Experimental Procedures). Electron density maps were calculated using multiwavelength anomalous diffraction data from three intrinsic zinc ions (Table 3). A model of DIM-5 was built and refined to 1.98 A resolution with a crystallographic R factor of 0.205 and R_free value of 0.258. The final model includes 1913 protein atoms (with mean B values of 26.9 A²), 3 zinc ions, and 103 water molecules, with rms deviations of 0.008 A and 1.5 A from ideality for bond lengths and angles, respectively.

The structural determination on DIM-5 allowed a structure-guided sequence alignment of SET proteins to be performed (FIG 8) that includes human SUV39 family proteins, all verified active HKMTs reported so far, and three bacterial SET proteins. The 318 residue DIM-5 protein is the smallest member ofthe SUV39 family. It contains four segments: (1) a weakly conserved aminoterminal region (light blue), (2) a pre-SET domain containing nine invariant cysteines, (3) the SET region containing signature motifs of NHXCXPN and DY, and (4) the post-SET region containing three invariant cysteines. The nine Cys pre-SET region is unique to the SUV39 family, while the post-SET region is also present in many members of SET 1 and SET2 families (Kouzarides, Curr. Opin. Genet. Dev. 12: 198-209, 2002), and even in one bacterial SET protein from Xylella fastidiosa (FIG 8). Two active human HKMTs contain neither pre- nor post-SET regions: SET7 (Wang et al, Mol. Cell 8: 1207-1217, 2001) (also called SET9 [Nishioka et al, Gene Dev. 16:479- 489, 2002]) methylates lysine 4 of histone H3 and SET8 (Fang et al, Curr. Biol. 12: 1086-1099, 2002) (also called PR-SET7 [Nishioka et al, Mol. Cell 9: 1201-1213, 2002]) methylates lysine 20 of H4.

The pre-SET residues form a 9 Cys cage enclosing a triangular zinc cluster (FIG 9A). The SET residues are folded into six β sheets surrounding the catalytic methyl transfer site, with a helical cap (αF) above the β sheets. The amino-terminal residues appear to be critical to the structural integrity ofthe molecule: the 38 residue segment extends through nearly the entire back ofthe molecule in the orientation shown (FIG 9A), providing an edge strand (βl , β2, or β3) to three separate β sheets and a 1 turn helix αA connecting to the pre-SET triangular zinc cage. The overall dimensions ofthe molecule are 60 x 50 x 30 A. The triangular zinc cluster and the cofactor binding site are approximately 38 A apart, located at opposite ends ofthe molecule along the longest dimension (FIG 9 A). A cleft can be seen running across from the cofactor binding site to the zinc cluster (FIG 9B). The Pre-SET Domain Forms a Triangular Zinc Cluster

The pre-SET domain contains nine invariant cysteine residues that are grouped into two segments of five and four cysteines separated by various numbers of amino acids (46 in DIM-5). These nine cysteines coordinate three zinc ions to form an equilateral triangular cluster (FIG 9C). Each zinc ion is coordinated by two unique cysteines (six total), and the remaining three cysteine residues (C66, C74, and C128) are each shared by two zinc atoms, thus serving as bridges to complete the tetrahedral coordination ofthe metal atoms. The distance between zinc atoms is -3.9 A, and the Zn-S distance is -2.3 A. A similar metal-thiolate cluster can be found in metallothioneins that are involved in zinc metabolism, zinc transfer, and apoptosis (reviewed in Vasak and Hasler, Curr. Opin. Chem. Biol. 4:177-183, 2000). Methallothioneins often have two metal clusters: a

(Me) Cys₉ and a (Me)₄Cysπ, where Me can be Zn²⁺, Cd²⁺, Cu²⁺, or another heavy metal. The tri-zinc cluster of DIM-5 can be superimposed perfectly upon the (Zn₂Cd)Cys₉ cluster of rat metallothionein (Robbins et al, J. Mol. Biol. 221 : 1269-1293, 1991). The SET Domain Forms the Active Site The SET domain resembles a square-sided β barrel topped by a helical cap (ocF, αG, ocH, and αi). Four β sheets— (1 | 5t 6|), (7] 16|), (4| 14T 15J 8|), and (3 9 1 1 | 10|) — form the sides of the barrel and one sheet — (2J, 12J,) — forms one end (FIG 9A). In the middle ofthe open end ofthe barrel is a crossover structure (magenta) formed by threading the βl 7-loop through an opening formed by a short loop between strands βl3 and βl4. This brings together the two most-conserved regions ofthe SET domain: the <xJ-βl3-loop (N₂₄₁HXCXPN₂₄₇) and βl7-loop (DY₂₈₃) (FIG 8). The side chains of these two highly conserved segments are involved in (1) hydrophobic structural packing (1240 of αJ and L279 and F281 of βl7), (2) intramolecular side chain-main chain interactions (after a shaφ turn at P246, the side chain of N247 interacts with the main chain carbonyl oxygen of E278 and the main chain amide nitrogen of T280), (3) AdoMet and active site formation (R238 and F239 of αJ, N241 :E278 pair, H242:D282 pair, and Y283). These binding site invariant residues are clustered together, via pair-wise interactions such as the interactions between N241 and E278 and between H242 and D282, forming an active site in a location immediately next to the AdoMet binding pocket and peptide binding cleft (see below). Enzymatic Properties of DIM-5 The DIM-5 protein is a very active HKMT in vitro. Several rather unusual properties of

DIM-5 were noticed. Under the current laboratory conditions, the enzyme is most active at ~10°C and nearly inactive at 37°C (FIG 10A). DIM-5 is extremely sensitive to salt, e.g., 100 mM NaCl inhibited its activity about 95% (FIG 10B). The enzyme also has a high pH optimum. DIM-5 showed maximal activity at -pH 9.8 (FIG 10C), although it showed strongest crosslinking to AdoMet around pH 8 (FIG 10D). Neither HKMT activity nor AdoMet binding were observed below pH 6.0. Cofactor Binding Pocket

All known HKMTs use AdoMet as the methyl donor. The most common conformation of AdoMet, or its reaction product AdoHcy, is found in the so-called consensus MTases. These MTases are built around a mixed seven-stranded β sheet, and they include more than 20 structurally characterized MTases acting on carbon, oxygen, or nitrogen atom in DNA, RNA, protein, or small molecule substrates (Cheng and Roberts, Nucleic Acids Res. 29:3784-3795, 2001). DIM-5 does not share structural similarity to any of these AdoMet-dependent proteins and appears to use a completely different means of interaction with its cofactor.

A difference electron density is observed in an open pocket on one end ofthe DIM-5 molecule opposite from the triangular zinc cluster (FIG 9A and FIG 11 ). This density was inteφreted as the cofactor product, AdoHcy, which was present during crystal growth (see Experimental Procedures). Although part ofthe AdoHcy can be fit into the density, it is difficult to fit the entire molecule, particularly because there is no recognizable density for the adenine ring of AdoHcy. This could potentially reflect flexibility ofthe cofactor bound to DIM-5. Unlike the "consensus" MTases where AdoMet/AdoHcy binds in a relatively closed pocket with hydrophobic stacking on both sides of adenine ring (Fauman et al., pp 1-38, In S-Adenosylmethionine-Dependent Methyltransferases: Structures and Functions, Cheng and Blumenthal, eds., River Edge, NJ: World Scientific, 1999), the density we observe is located in an open pocket, sitting above the antiparallel strands β5 and β6 and against the short helix αJ (FIG 9A). This environment may contribute to its flexibility or allow multiple conformations in the absence of substrate. The flexibility may also result from low pH during crystallization (pH 5.4-5.6), a condition in which no UV crosslinking of AdoMet to the protein was observed (FIG 10D). At low pH the adenine ring might not interact stably enough with DIM-5 to be crosslinked to the protein or observed in the structure.

The significance of this density is further enhanced by the highly conserved residues with which it is surrounded. Two conserved arginines (R155 of β5 and R238 of αJ) and three aromatic residues (W161 , Y204, and F239) directly contact the density (FIG 1 1). The side chains of these two arginines are locked in place by other conserved residues: the guanidino group of R155 is parallel to the plane ofthe W161 indole ring and ion pairs with D35; and the guanidino group of R238 is surrounded by three aromatic rings, F43, F239, and Y204, and its two terminal nitrogen atoms (Nε and Nη2) form hydrogen bonds to the main chain carbonyl oxygen atoms of G230 and E231, respectively (FIG 1 1 ).

Conservative substitutions were made for several ofthe residues surrounding this density: R155H, W161 F, Y204F, and R238H (see Experimental Procedures). The enzymatic activities of all the mutants were reduced ranging from a 75% reduction (W161F) to nearly inactive (R238H) (FIG 10E). The ability of these mutants to bind AdoMet, as measured by crosslinking, was also reduced but not abolished (FIG 10F). It appears that the reduced AdoMet binding alone could account for the reduction in HKMT activity for the R155H, W161 F, and Y204F mutations. The R238H mutation, however, caused a much greater reduction in HKMT activity than in AdoMet binding, suggesting that R238 may also play roles in other aspects of catalysis (see below). In SUV39H1 and SUV39H2, a histidine is in the position of R238 in DIM-5; changing this histidine to an arginine resulted in at least 20-fold increase of activity in SUV39H 1 (Rea et al, Nature 406:593-599, 2000), consistent with the greatly reduced activity in the converse R238H mutants of DIM-5. Putative Peptide Binding Cleft

The cleft along the surface emanating from the presumed cofactor binding site is the likely binding site for the substrate polypeptide (FIG 9B). One side of this cleft is formed by strand βlO (green in FIG 12A) — the outermost strand ofthe β sheet (3f 9f 1 1 J, 10J.) — and the other side is formed by the loop after strand βl 7, which is the beginning ofthe disordered carboxy-terminal residues (286-299).

Structural studies have shown that heterochromatin protein HPl binds to a methylated histone H3 peptide by inserting it as an antiparallel β strand between two 2 HPl strands, forming a hybrid three-stranded β sheet (Jacobs and Khorasanizadeh, Science 295:2080-2083, 2002; Nielsen et al, Nature 416: 103-107, 2002). Encouraged by the fact that one side ofthe DIM-5 cleft is a strand (βlO), we superimposed the HPl β strand (Drosophila HPl residues 60-62) onto DIM-5 strand βlO (residue 205-207) (FIG 12B). The superimposition placed the H3 peptide (e.g., Q5-S10 as observed in HPl) in the DIM-5 cleft (FIG 12C) and residues Y283-V284-N285 following strand βl7 on the other side ofthe peptide (FIG 12B). An induced-fit mechanism is used in HPl, in which the amino- terminal tail ofthe free HPl adopts a β strand-like conformation upon interacting with the H3 peptide (Nielsen et al, Nature 416: 103-107 ', 2002). In a similar way, binding ofthe H3 peptide may induce residues Y283-V284-N285 of DIM-5 and subsequent disordered residues to adopt a more stable β strand conformation that interacts with the peptide to form a hybrid sheet. Target Lysine Binding Site

The most interesting result ofthe docking experiment is the placement ofthe target K9 immediately next to the presumed cofactor binding site (FIG 12C) with the target nitrogen atom occupying the position of a water molecule (site 2 in FIG 1 1). It is herein proposed that water site is the likely active site of DIM-5, where the terminal amino group (NH₃) ofthe substrate lysine would form a hydrogen bond with main chain carbonyl oxygen atom of R238. Many highly conserved residues, mainly from the two signature motifs (magenta), surround this site. Side chains of N241 , H242, Y283, and Y204 form an inner circle immediately around site 2 (FIG 11). Residues E278, D282, and Y178 form an outer circle via interactions with the inner-circle residues: E278 interacts with N241, D282 interacts with H242, and Y178 interacts with Y283 via a water molecule (site 4) (FIG 1 1). Unlike protein arginine MTases or small molecule glycine N-MTase, which uses acidic residue(s) to neutralize the positive charge on the substrate amino group (Fu et al, Biochemistry 35:1 1985-1 1993, 1996; Zhang et al, EMBO J. 19:3509-3519, 2000), no acidic residue is immediately present in the proposed active site of DIM-5. Nevertheless, the combination ofthe negative dipole moment at the carboxyl end of helix αJ (R238 and F239), the negatively polarized main chain carbonyl oxygen atoms (1240 and W161), the side chain hydroxyl oxygen atoms of Y178, Y204, and Y283, and the asparagine oxygen atom of N241 might increase the nitrogen electron density enough to allow a nucleophilic attack on the AdoMet methylsulfonium group. The proton elimination step in conjunction with the methyl transfer is likely accomplished through a charge relay system involving H242 and D282, much as in protein arginine MTases (Zhang et al, EMBO J. 19:3509-3519, 2000). One observation consistent with this mechanism is the unusually high optimal pH (-10) of

DIM-5 (FIG 10C), despite the fact that AdoMet binding is much more favorable in solutions of lower pH (FIG 10D). At pH 10, the amino group of target lysine (with a typical pKa value of 10) may be partially neutralized and the conserved tyrosines Y283, Y204, and Y178 (also with typical pKa values of 10) near the active site may be deprotonated; both deprotonations would facilitate methyl transfers.

The importance ofthe proposed active site residues is supported by site-directed mutagenesis experiments. Conservative changes at three residues (N241Q, H242K, and Y283F) immediately surrounding water site 2 essentially abolished HKMT activity (FIG 10E). Y283F has the lowest residual activity, suggesting that the hydroxyl group of Y283 is critical; it is hydrogen bonded to the backbone amide nitrogen of 1240 and immediately adjacent to water site 2. Y283 is also one ofthe most conserved residues ofthe SET domain, being invariant in most ofthe SET containing proteins in the Pfam database. Mutations ofthe two residues proposed to be involved in proton elimination, H242K and D282N, abolished and reduced HKMT activity, respectively. As expected, both mutants retained AdoMet crosslinking, though at reduced levels (FIG 10F). The complete loss of AdoMet crosslinking in N241Q and Y283F mutant proteins is somewhat unexpected. For N241 Q, perhaps the longer glutamine side chain prevents the two hydrogen bonds forming between the side chain amino group of N241 and both the backbone carbonyl of W161 and the side chain of E278 (FIG 1 1). Interrupting the W161-N241-E278 interactions probably disrupts local structure, having a more deleterious effect than the replacement of side chain in the W161F mutant. It is also possible that both N241 and Y283 interact with the adenine ring of AdoMet, which is likely involved in the UV crosslinking, although not observed in the crystal.

The presumptive active site of DIM-5 is reminiscent ofthe consensus NPPY motif involved in the aminomethylation of adenine or cytosine in DNA (Blumenthal and Cheng, Nat. Struct. Biol. 8: 101-103, 2001 ; Goedecke et al, Nat. Struct. Biol. 8:121-125, 2001 ; Gong) and ofthe glutamine in peptide release factor (Heurgue-Hamard et al. , EMBO J. 21 :769-778, 2002; Nakahigashi et al, Proc. Natl. Acad. Sci. USA 99:1473-1478, 2002). Remarkably, the invariant N241 and Y283 of DIM-5 are superimposable onto the first and the last amino acids of NPPY in Taql DNA adenine MTase (FIG 12D). This suggests a potential similarity in the catalytic mechanism between histone lysine MTases and DNA amino-MTases. In the latter case, the amino group (NH2) that becomes methylated is positioned for an in-line attack on AdoMet by hydrogen bonding to the backbone carbonyl connecting the two inflexible prolines (Goedecke et al, Nat. Struct. Biol. 8: 121-125, 2001). The equivalent backbone carbonyl in DIM-5 is probably that of R238. Since this particular carbonyl needs to be relatively immobile to hinder the free rotation ofthe amino group bound at site 2, the great reduction of HKMT activity in R238H mutant could be the result of a small change or flexibility in the position ofthe backbone carbonyl oxygen atom that fails to interact properly with the target amino group.

Mono-, di-, and trimethylated lysines have been observed in histones (Duerre and Chakrabarty, J. Biol. Chem. 250:8457-8461, 1975) but very little information is available about the methylation status of individual residues. Nevertheless, it has been found that DIM-5 efficiently methylates dimethylated lysine 9 of histone H3 peptide, and DIM-5 is capable of adding 1-3 methyl groups to K9 of histone H3 peptide. It is currently believed that water sites 1 and 3 (FIG 1 1), which hydrogen bonded to site 2, may accommodate the methyl group(s) on mono- and dimethylated lysine substrates. These additional interactions may help position the nitrogen atom and enhance its reactivity. The Post-SET Domain

The C terminus, including the post-SET region, is mostly disordered in the crystal except for the segment between residues 299 and 308 (FIG 9A and 9B). This 10 residue segment, identified through M303 in selenomethionine-substituted DIM-5 protein (see Experimental Procedures), was stabilized in the interface between two crystallographic-related molecules. We hypothesize that this segment (along with the adjacent disordered residues) will adopt a different structure upon binding to substrate. The post-SET region contains three conserved cysteine residues that appear to be essential for HKMT activity in the SUV39 family. Changing all three cysteines to serines (3C-S) abolished DIM-5 activity (FIG 10E), as did a Cys to Tyr substitution at C 1279 in SETDB 1 (Schultz et al,

2002), which corresponds to C306 of DIM-5. While the exact role ofthe three post-SET cysteines cannot be determined from the current structure, one intriguing possibility is that, when coupled with the fourth cysteine from the loop formed by the signature motif N ₄₁HXCXPN ₄₇ (C244 in DIM-5), these form an additional metal binding site. Several observations are consistent with this hypothesis. Ofthe more than 50 SET protein sequences that we have examined to date, there appears to be an absolute correlation between the presence ofthe post-SET and a cysteine corresponding to C244 of DIM-5 (see FIG 8, for examples). In addition, replacing the Cys corresponding to C244 with alanine in SUV39H1 or SETDB 1 abolished HKMT activity (Rea et al, Nature 406:593-599, 2000; Schultz et al, Genes Dev. 16:919- 932, 2002). The total zinc content of DIM-5 protein is 3.51 (FIG 13 A), indicating that more than three zinc ions are present. Incubation of metal chelators, phenanthroline or EDTA, with DIM-5 protein inhibited its activity and significantly reduced AdoMet binding (FIG 13B and 13C). Interestingly, even when EDTA completely abolished DIM-5 activity, the protein still retained approximately three (2.9) zinc ions (FIG 13 A). As the triangular zinc cluster is quite stable, it is conceivable that the chelated zinc was coordinated by the three post-SET cysteines and C244 (FIG 12A), which is near the active site. Like the metal chelators, simultaneous mutation ofthe three cysteines (3C-S) also caused a complete loss of DIM-5 activity and AdoMet crosslinking (FIG 10E and 10F), consistent with the idea that the post-SET cysteines are involved in AdoMet binding. Perhaps the observed disorder ofthe post-SET is partly, or fully, responsible for the poor density of the AdoHcy in the current structure

Conclusions The crystal structure of a histone H3 lysine 9 MTase, DIM-5 from N crassa, was determined and mutational and biochemical studies were carried out to illuminate the mechanism of this enzyme The highly conserved residues ofthe pre-SET region form a triangular zinc cluster, Zn3Cys9, and residues in the SET domain are essential for the cofactor binding and methyl transfer The SET domain also has a cleft that is the likely binding site for the methylatable amino-terminal tail of histone H3 The post-SET region may also contribute to cofactor binding and catalysis by forming another zinc binding site in conjunction with a conserved cysteine near the active site These results provide insight into a common fold and the catalytic mechanism for the SUV39 family histone H3 lysine 9 MTases Finally, this work provides an example of completely unrelated, structurally distinct proteins that carry out a common function, in this case AdoMet-dependent methyl transfer

Example 12: Trimethyl-lysine 9 of Histone H3 is the Mark for DNA Methylation in Neurospora

Histone H3 methyltransferases have been implicated in various epigenetic processes In this example, it is shown that the DIM-5 histone methyltransferase, which is essential for DNA methylation in Neurospora, trimethylates H3 Lys9 Although DNA methylation and heterochromatin formation are tightly associated in organisms that show both features (Lachner et al , Curr Opin Cell Biol 14 286-298, 2002), it remains possible that they are triggered by distinct signals One possibility is that lysine 9 methylation has different consequences depending on the presence or absence of other histone modifications Another possibility, considering that an individual lysine residue can be mono-, di-, or tnmethylated (Duerre et al , J Biol Chem 250 8457-61, 1975, Zhang et al , Genes Dev 15 2343-2360, 2001), is that different methylation states of a single residue may signal different processes To explore this aspect ofthe "histone code" (Strahl et al , Nature 403 41-5, 2000), the specificity ofthe DIM-5 histone methyltransferase in vitro and in vivo was investigated

Recombinant DIM-5 was first assayed for methyltransferase activity using S-adenosyl- [methyl-³H]-Z.-methιonιne and synthetic methylated or unmodified H3 peptides Reaction products were fractionated by SDS-PAGE and assayed for incorporation of methyl groups by fluorography (FIG 14A) Consistent with the expectation that DIM-5 is a H3 lysine 9 methyltransferase, DIM-5 methylated the 1-15 unmodified peptide derived from N-terminus of H3 but not a similar peptide that was tnmethylated at lysine 9 Robust DIM-5 activity was found with a dimethyl-lysine 9 H3 peptide, which served as a poor substrate for the previously characterized SUV39H1 (Rea et al , Nature 406 593-599, 2000) and Clr4 (Nakayama et al , Science 292 110-113, 2001) H3 lysine 9 methyltransferases This result suggested that DIM-5 efficiently methylates H3 dimethyl-lysine 9 It remained formally possible, however, that the presence of dimethyl-lysine 9 altered the specificity of DIM-5, e g , leading to methylation of lysine 4 and/or lysine 14 Therefore, amino terminal sequencing was used to determine directly whether DIM-5 methylates lysine 9 of an unmodified H3 (1-20) peptide and a similar peptide with lysine 9 dimethylated Products of reactions of DIM-5, S- adenosyl-[methyl-³H]-I-methιonιne and the peptides were subjected to Edman degradation and the individual am o acid fractions were assayed for incoφoration of labeled methyl groups (FIG 14B and 14C) With both peptides, significant incoφoration occurred exclusively at position 9 This confirms that DIM-5 is a lysine 9 methyltransferase and establishes that DIM-5 efficiently trimethylates H3 lysine 9 in vitro

To further investigate the specificity of DIM-5, mass spectroscopy was used to follow the kinetics of mono-, di- and trimethyl transfer to unmodified or dimethylated lysine 9 in H3 peptides With the unmodified substrate, DIM-5 produced all three methylation states in the early phase ofthe reaction (FIG 14D and 14F) Trimethyl-lysine 9 had already became the dominant species at 30 minutes, when there was still substantial amount of unmethylated H3 peptide, and continued to increase while the relative amount of all other species decreased Dimethyl-lysine 9 was the least represented species throughout the reaction (FIG 14D) As soon as dimethyl-lysine 9 was detected (2 5 minutes), trimethyl-lysine 9 started to dominate dimethyl-lysine 9, implying that conversion from dimethyl-lysine 9 to trimethyl-lysine 9 is the fastest step catalyzed by DIM-5 Indeed, DIM-5 produced trimethyl-lysine 9 much faster from dimethyl-lysine 9 peptide than from unmodified peptide (FIG 14E and 14G) After 2 5 minutes, 60% of dimethyl-lysine 9 H3 peptide had been converted to trimethyl-lysine 9, whereas only 3% of unmodified peptide was converted Taken together, these observations suggest that DIM-5 transfers three methyl groups to lysine 9 in a processive manner Alternatively, the methylation reactions may be distributive but DIM-5 simply prefers mono-, and especially dimethylated, substrates Based on these findings, it is believed that trimethyl-lysine 9 H3 is the main product of DIM-5 in vitro An additional question is whether DIM-5 preferentially generates tnmethylated H3 lysine 9 in vivo Although the relative distribution of different methylation states of H3 lysine 9 has not been reported for any organism, antibodies generated using dimethyl-lysine 9 H3 peptides have been successfully used for chromatin immunoprecipitation (ChIP) to detect regional methylation in fission yeast (Nakayama et al , Science 292 110-3, 2001 , Noma et al , Science 293 1 150-5, 2001), mammals (Boggs et al , Nat Genet 30 73-6, 2002, Litt et al , Science 293 2453-5, 2001 ) and plants (Johnson et al , Curr Biol 12 1360, 2002) and an antibody generated against a trimethyl-lysine 9 H3 peptide was successfully used to visualize methylated H3 cytologically (Cowell et al , Chromosoma 1 11 22-36, 2002) The specificity of these antibodies was tested to explore the possibility of using them to assess Lys-9 methylation of Neurospora genomic regions with or without DNA cytosine methylation Immunoblot analysis ofthe antibodies with H3 peptides that were either unmodified, dimethylated or tnmethylated at lysine 9 demonstrated that the antibodies to dimethyl-lysine 9 and trimethyl-lysine 9 of H3 are specific for their respective epitopes (FIG 15 A) These antibodies were therefore employed to characterize the chromatin associated with methylated and unmethylated DNA regions Approximately 1.5 % of cytosines in the Neurospora genome are methylated and these appear concentrated in relics of repeat-induced point mutation (RIP) (Selker, Annu. Rev. Genet. 24:579-613, 1990). One DNA methyltransferase, encoded by the dim-2 gene, is responsible for all known DNA methylation (Kouzminova et al, EMBO Journal 20:4309-4323, 2001). Two representative methylated relics of RIP, η (Selker, Annu. Rev. Genet. 24:579-613, 1990) and punt (Margolin et al, Genetics 149: 1787-1797, 1998), and two representative unmethylated genes, pen and hH4 (Hays et al, Genetics 160:961-73, 2002) (encoding PCNA and histone H4, respectively), were chosen to analyze the methylation status of H3 lysine 9 in wild-type and dim strains. Southern hybridizations using the isoschizomers Dpnll and Sau3Al, which both recognize GATC but differ in that only Sau3Al is sensitive to cytosine methylation, show that η and punt are methylated in wild- type (WT) strain, but not in dim-5 or dim-2 mutants, whereas pen and hH4 regions are unmethylated in all the strains (FIG 15B). In addition to the antibodies generated with dimethyl (Nakayama et al, Science 292: 1 10-1 13, 2001) or trimethyl (Cowell et al, Chromosoma 1 11 :22-36, 2002) lysine 9 H3 peptides, an antibody to H3 dimethyl-lysine 4 was used which has been characterized as a marker of active euchromatin (Noma et al, Science 293:1150-5, 2001 ; Litt et al, Science 293:2453-5, 2001). Chromatin immunoprecipitation (ChIP) was performed essentially as described previously (Nakayama et al., Cell 101 :307-317, 2000) with modifications. Briefly, asexual spores (~2 x 10⁶ spores/ml) ofthe Neurospora wild-type strain N 150 (740R23-IVA) were germinated for 4.5 hours in 50 ml of Vogel's minimal liquid medium supplemented with 1.5 % sucrose and histidine at 32 °C with shaking (200 φm). Cells of fission yeast strain SPG1335 were grown to 10⁷ cells/ml in 50 ml yeast extract adenine medium as described (Noma et al, Science 293: 1150-1 155, 2001). For chromatin fixation, both cultures were mixed and paraformaldehyde was added to 2 % and incubated at 32 °C for 30 minutes with shaking (100 φm). After cell lysis, chromatin DNA was sheared to 0.5- 0.8-kb, and the soluble chromatin fraction was immunoprecipitated using 2-8 μl of antibodies to dimethyl-Lys4, dimethyl-Lys9 or trimethyl-Lys9 of histone H3. DNA was isolated from immunoprecipitated chromatin, mock control chromatin or total chromatin and subject to PCR (95 °C, 30 seconds; 56 °C, 30 seconds; 72 °C, 1 minute; 24-30 cycles). PCR reactions (25 μl) included 50 mM KC1, 10 mM Tris-HCl (pH 9.0), 0.1 % Triton X-100, 2.5 mM MgCl₂, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.2 mM dGTP, 2.5 μCi [α-³²P] dCTP and 1.25 U Taq polymerase (Promega). PCR products were fractionated in 4 % polyacrylamide gels and band intensities were quantified using a STORM 860 Phosphorimager (Molecular Dynamics).

ChIP experiments on S. pombe using the same anti-dimethyl H3 (lysine 9) detected robust enrichment of H3 dimethyl-lysine 9 in heterochromatic regions (Nakayama et al, Science 292: 1 10-3, 2001 ; Noma et al, Science 293: 1 150-5, 2001). Therefore, S. pombe chromatin was used as an internal positive control to detect H3 dimethyl-lysine 9. The S. pombe strain SPG1355 (Nakayama et al, Cell 101 :307-17, 2000) carries an endogenous ura4 gene with a small deletion (ura4DS/E) and an ectopic ura4* gene integrated into the heterochromatic cenl locus (cenl::ura4) (FIG 15C). Cultures of S. pombe strain SPG 1355 and the TV. crassa wild-type strain N 150 were mixed, the chromatin was fixed with paraformaldehyde, and immunoprecipitated with each antibody. To measure enrichment of methylated DNA relative to unmethylated DNA in the immunoprecipitated chromatin, duplex PCR was conducted with sets of primers (SEQ ID NOs: 22 and 23 for η, SEQ ID NOs: 24 and 25 for punt, SEQ ID NOs: 26 and 27 for pen, and SEQ ID NOs: 28 and 29 for hH4) to amplify a pair of methylated and unmethylated regions (η and pen or punt and hH4) from the DNA extracted from either immunoprecipitated or total chromatin. The cenl::ura4 and ura4DS/E regions of S. pombe (FIG 15C) were also amplified from the same DNA sample. PCR products were then fractionated by SDS-PAGE, quantified signals using a Phosphorimager and normalized the data based on results with total DNA. Evidence of trimethyl-lysine 9, but not dimethyl-lysine 9, was found in the regions of methylated DNA (FIG 16A and 16B). As expected based on findings in other organisms, the two active, nonmethylated genes (pen and hH4) were efficiently precipitated with the anti-dimethyl-lysine 4 H3 antibody, whereas the inactive, methylated regions (η and punt) were not. In contrast, the anti- trimethyl-lysine 9 H3 antibody preferentially precipitated both cytosine-methylated chromosomal regions. One methylated region, η, was 5.5 (± 0.9) fold enriched relative to the active gene control, pen, and the second, punt, was enriched 5.4 (± 1.6) fold relative to its control, hH4 in the chromatin precipitated with the anti-trimethyl-Iysine 9 H3 antibody. The fact that this antibody precipitated some chromatin containing the active genes could represent a low level of trimethyl-lysine 9 but may also reflect cross-reaction, e.g. with methyl-Lys27. No evidence for H3 dimethyl-lysine 9 in these regions was found using the antibodies that had revealed dimethyl-lysine 9 in other systems. Results of PCR on the S. pombe chromatin mixed in as an internal control were as expected (FIG 16C) (Noma et al, Science 293: 1150-1155, 2001). Specifically, the anti-dimethyl-lysine 4 H3 antibody preferentially precipitated ura4DS/E relative to cenl::ura4 (7.4 ± 2.6-fold), whereas the anti- dimethyl-lysine 9 H3 antibody preferentially precipitated cenl::ura4 relative to ura4DS/E (10.8 ± 4.1 -fold). These results confirmed that the dimethyl-lysine 9 antibody could immunoprecipitate chromatin with the dimethyl-lysine 9 modification and suggested that the silent regions of Neurospora and fission yeast are differentially methylated on lysine 9. Significant enrichment of cenl::ura4 relative to ura4DS/E (9.4 ± 3.8-fold) was also detected with the anti-trimethyl-lysine 9 H3 antibody (FIG 16C). This could reflect some trimethylation by Clr4 or an unidentified histone methyltransferase. These results indicate that trimethylation of lysine 9 is found in Neurospora preferentially associated with methylated DNA and that H3 dimethyl-lysine 9 is absent or extremely underrepresented in chromatin associated with methylated DNA regions, consistent with the finding that DIM-5 trimethylates lysine 9 in vitro.

To determine directly whether DIM-5 is responsible for the apparent trimethylation of lysine 9, the effect of a nonsense mutation in the SET domain of dim-5 was tested. The effect of a dim-2 null mutation was also examined to test the possibility of feedback between DNA methylation and histone methylation (FIG 17). Chromatin samples from wild-type, dim-5 or dim-2 strains were immunoprecipitated with anti-trimethyl-lysine 9, anti-dimethyl-lysine 9 or anti-dimethyl-lysine 4 antibodies and characterized by duplex PCR with all four possible combinations ofthe two methylated (η and punt) and the two unmethylated (pen and hH4) test regions. With all three strains, the anti-dimethyl-lysine 9 antibody failed to precipitate detectable chromatin associated with any of the four regions, as before. In contrast, the anti-dimethyl-lysine 4 and anti-trimethyl-lysine 9 antibodies preferentially precipitated the active (pen and hH4) and inactive, methylated (η and punt) regions, respectively, in the wild-type strain (FIG 17A and 17B). The dim-5 mutation markedly reduced the signals obtained with the trimethyl-lysine 9 antibody in the methylated regions, but did not appear to reduce the weaker signal observed with the active genes and did not completely eliminate signals with the methylated regions. Based on these findings, it is believed that DIM-5 is indeed responsible for most, if not all, ofthe H3 trimethylation at lysine 9 detected in the methylated regions but is not responsible for the lower signal observed with the non-methylated genes. The residual signal most likely reflects cross-reaction ofthe antibody with another epitope, but may reflect a low level of trimethylation by another enzyme. Unlike the mutation of dim-5, mutation of dim-2 did not reduce signals in any ofthe regions examined, consistent with the conclusion that the DIM-2 DNA methyltransferase acts downstream ofthe DIM-5 histone methyltransferase, i.e., the dim-2 gene is not required for DIM-5 activity.

Neither the dim-5 nor the dim-2 mutation affected methylation of lysine 4 detected with the anti-dimethyl-lysine 4 antibody (FIG 17). Reverse correlations have been observed for methylation of lysine 4 and lysine 9 of H3 in both fission yeast (Noma et al, Science 293:1 150-5, 2001) and mammals (Litt et al, Science 293:2453-5, 2001 ; Nishioka et al, Genes Dev 16:479-89, 2002); lysine 9 methylation is found preferentially in heterochromatin and lysine 4 methylation is found preferentially in euchromatin. Studies in vitro have demonstrated that lysine 9 methylation can inhibit a H3 lysine 4 methyltransferase (Wang et al, Mol Cell 8: 1207-17, 2001) and that H3 lysine 4 methylation can inhibit a H3 lysine 9 methyltransferase (Nishioka et al, Genes Dev 16:479-89, 2002). DIM-5 activity is also strongly inhibited by H3 lysine 4 methylation. However, the finding that the dim-5 mutation did not increase H3 lysine 4 methylation at the η or punt regions (FIG 17A and 17B) indicates that hypomethylation of H3 lysine 4 at the cytosine-methylated regions does not simply reflect competition between DIM-5 and a H3 lysine 4 methyltransferase.

This disclosure provides in certain embodiments a novel HMTase that specifically methylates the lysine 9 residue of histone H3, and nucleic acids encoding this enzyme. In other embodiments, the disclosure further provides methods of using these molecules to influence DNA methylation and/or gene expression in eukaryotes, methods of screening for compounds that interact with the provided HMTase, and more generally methods of screening for compounds that are useful in treating, ameliorating, curing, or preventing methylation-related diseases or conditions (e.g., neoplasia). It will be apparent that the precise details ofthe compositions and methods described may be varied or modified without departing from the spirit ofthe described invention. We claim all such modifications and variations that fall within the scope and spirit ofthe claims below.

Claims

CLAIMSWe claim:

1. A method for identifying a compound with potential for treating neoplasia, comprising determining histone methyltransferase (HMTase) inhibitory activity ofthe compound, wherein high HMTase inhibition activity identifies that the compound has potential for treating neoplasia.

2. The method of claim 1, wherein the HMTase inhibitory activity comprises a histone H3 methyltransferase activity.

3. The method of claim 1, comprising determining a DIM-5 inhibitory activity ofthe compound, wherein high DIM-5 inhibitory activity identifies that the compound has potential for treating neoplasia.

4. The method of claim 1, further comprising determining whether the compound inhibits tumor cell growth in a culture, wherein inhibition of tumor cell growth is further identifies that the compound has potential for treating neoplasia.

5. The method of claim 1, further comprising determining whether the compound inhibits or reverses DNA methylation in a cell, wherein inhibition or reversal of DNA methylation in the cell further identifies that the compound has potential for treating neoplasia.

6. The method of claim 1, further comprising determining whether the compound induces apoptosis of a tumor cell, wherein induction of apoptosis is further identifies that the compound has potential for treating neoplasia.

7. The method of claim 1, further comprising determining whether the compound inhibits tumor cell growth in a sample, wherein inhibition of tumor cell growth is further identifies that the compound is useful for treating neoplasia.

8. A method of selecting a compound for inhibition of neoplasia, comprising determining neoplastic cell growth inhibitory activity ofthe compound; determining HMTase inhibitory activity; and selecting a compound that exhibits neoplastic cell growth inhibitory activity and high HMTase inhibition activity as a compound to inhibit neoplasia.

9. The method of claim 8, further comprising determining whether the compound induces apoptosis in a cell; and selecting compounds that induce apoptosis.

10. A method for identifying compounds for treatment of neoplasia, comprising: determining HMTase inhibitory activity ofthe compounds; and identifying those compounds for treating neoplasia if the compounds exhibit high HMTase inhibition activity.

11. A method of reducing, preventing or reversing DNA methylation in a cell, comprising administering a hypomethylating effective amount of a HMTase inhibitory compound to the cell, thereby reducing, preventing or reversing DNA methylation in the cell.

12. The method of claim 1 1, wherein a nucleic acid in the cell is known to be or suspected of being hypermethylated.

13. The method of claim 11, wherein the cell is a protist cell, a fungal cell, a plant cell, or an animal cell.

14. The method of claim 1 1, wherein the cell is a hyper-proliferative cell.

15. The method of claim 14, wherein the hyper-proliferative cell is a mammalian tumor cell.

16. A method of treating or ameliorating a hypermethylation-related disease, condition, or disorder in a subject, comprising administering to the subject a hypomethylating effective amount of a HMTase inhibitory compound.

17. The method of claim 16, wherein the disease is a hyper-proliferative disease.

18. The method of claim 16, wherein the HMTase inhibitory compound is administered in the form of a pharmaceutical composition.

19. A method of ameliorating a tumorigenic state of a cell, comprising administering a hypomethylating effective amount of a HMTase inhibitory compound to the cell to reduce methylation of cytosine in a CpG dinucleotide in the cell, thereby ameliorating the tumorigenic state ofthe cell.

20. The method of claim 19, further comprising administering an anti-cancer agent to the cell.

21. The method of 19, wherein the HMTase inhibitory compound is provided in the form of a pharmaceutical composition.

22. The method of claim 19, wherein the cell is a cell in a subject.

23. A kit for inhibiting a DNA methyltransferase, comprising: an amount of a HMTase inhibitory compound effective to inhibit methylation of at least one DNA target.

24. The kit of claim 23, which is a kit for treating a hyper-methylation mediated disease or disorder in a subject suspected of needing such inhibition.

25. The kit of claim 23 further comprising instructions.

26. The kit of claim 24, wherein the instructions include directions for administering at least one dose ofthe therapeutic substance to the subject in need of such treatment.

27. The kit of claim 24, wherein the HMTase inhibitory compound is provided in the form of a pharmaceutical composition.

28. A purified protein comprising an amino acid sequence selected from the group consisting of

(a) SEQ ID NO: 3; (b) SEQ ID NO: 5; and

(c) conservative substitutions thereof.

29. An isolated nucleic acid molecule encoding the protein of claim 28.

30. The nucleic acid molecule of claim 29, comprising a nucleotide sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

31. A recombinant nucleic acid molecule comprising a promoter sequence operably linked to nucleic acid molecule according to claim 29.

32. A transgenic cell comprising a recombinant nucleic acid molecule according to claim 31.