# Patent

## Title
Engineered demeter 5-methylcytosine DNA glyosylase with improved yield, stability and solubility

## Abstract

Improved active DEMETER polypeptides with internal deletions are provided.

## Background
CROSS REFERENCE TO RELATED PATENT APPLICATIONS
The present application is the US National Phase entry under 35 U.S.C. §371 and claims the benefit of International Application No. PCT/US2010/031623, filed Apr. 19, 2010, which claims the benefit of U.S. Provisional Patent Application No. 61/170,978, filed Apr. 20, 2009, which are incorporated by reference for all purposes.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
The invention was made with government support under Grant Number R01 GM069415 awarded by the National Institutes of Health. The government has certain rights in the invention.


REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file -1985-1.TXT, created on Feb. 10, 2012, 61,440 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
Alleles of imprinted genes are expressed differently depending on whether they are inherited from the male or female parent. Imprinting regulates a number of genes essential for normal development in mammals and angiosperms. In mammals, imprinted genes contribute to the control of fetal growth and placental development (Constancia, M. et al., Nature, 432, 53-57 (2004)). Human diseases are linked to mutations in imprinted genes or aberrant regulation of their expression (Constancia, M. et al., Nature, 432, 53-57 (2004)). Mechanisms of distinguishing maternal and paternal alleles have been extensively characterized in mammals. Imprinted genes reside in chromosomal clusters and are regulated by differentially methylated imprinting control regions (ICRs) (Reik, W. and Walter, J., Nat Rev Genet, 2, 21-32 (2001)). Differential DNA methylation is established during oogenesis or spermatogenesis by de novo methyltransferases and maintained somatically by the CG maintenance methyltransferase Dnmt1 (Li, E. (2002). Nat Rev Genet. 3, 662-673. ICRs are subject to differential histone modifications and in some instances can act as chromatin boundaries (Delaval, K. and Feil, R., Curr Opin Genet Dev., 14, 188-195 (2004)). Other mechanisms to regulate allele-specific gene expression involve non-coding RNAs, including antisense transcripts and microRNAs (O'Neill, 2005). Polycomb group (PcG) proteins, which function in large complexes to methylate histones and modify chromatin (Cao, R. and Zang, Y., Curr Opin Genet Dev., 14, 155-164 (2004)), maintain allele-specific silencing of some imprinted genes (Delaval, K. and Feil, R., Curr Opin Genet Dev., 14, 188-195 (2004)).
The endosperm, one of the products of angiosperm double fertilization, is an important site of imprinting in plants (Gehring, M. et al., Plant Cell, 16, S203-S213 (2004)) and has functions analogous to the placenta. In flowering plants, meiosis followed by mitosis produces the female and male gametophytes. Two cells of the female gametophyte, the haploid egg and the diploid central cell, are fertilized by two haploid sperm from the male gametophyte to form the diploid embryo and triploid endosperm, respectively. The endosperm provides nutrients to the embryo during seed development and, in Arabidopsis, is almost entirely consumed by the time embryo maturation is completed.
Molecular events that take place in the female gametophyte before fertilization have an essential role in endosperm gene imprinting. The imprinting of two genes, MEA and FWA, is regulated by DEMETER (DME, also sometime abbreviated DMT), a helix-hairpin-helix DNA glycosylase (Choi, Y. et al., Cell, 110, 33-42 (2002); Kinoshita, T. et al., Science, 303, 521-523 (2004)). DME has also been referred to in the literature as Atropos (ATR). The DME plant gene product has been described to control plant phenotypes and affect DNA methylation. The DME gene product is described in, e.g., U.S. Pat. Nos. 6,476,296 and 7,109,394 as well as Choi, Y. et al., Cell, 110:33-42 (2002); Gehring, M. et al., Cell, 124:495-506 (2006).
DNA glycosylases function in the base excision repair pathway by removing damaged or mismatched bases from DNA (Scharer, O. D. and Jiricny, J., BioEssays, 23, 270-281 (2001)). Bifunctional helix-hairpin-helix DNA glycosylases have both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities. The DNA glycosylase activity removes the damaged or mispaired base by cleaving the N-glycosylic bond, creating an abasic site, whereas the lyase activity nicks the DNA. An AP endonuclease generates a 3′-hydroxyl used by a DNA repair polymerase that inserts the proper nucleotide. A DNA ligase seals the nick to complete the repair process. DNA glycosylase/lyases have not been implicated in mammalian imprinting mechanisms.
Both MEA and FWA are expressed in the central cell before fertilization and in the endosperm, from the maternal allele, after fertilization (Kinoshita, T. et al., Science, 303, 521-523 (2004); Kinoshita, T. et al., Plant Cell, 11, 1945-1952 (2004); Vielle-Calzada, J. P. et al., Genes Dev, 13, 2971-2982 (1999)). In contrast, DME is expressed in the central cell of the female gametophyte but not in the endosperm (Choi, Y. et al., Cell, 110, 33-42 (2002)). Expression of MEA and FWA in the central cell and early endosperm is dependent on DME (Choi, Y. et al., Cell, 110, 33-42 (2002); Kinoshita, T. et al., Science, 303, 521-523 (2004)).
Though maternal expression of MEA and FWA is controlled by DME, there are important distinctions regarding the regulation of expression of these genes. FWA is silent in all vegetative and reproductive tissues except for expression of the maternal allele in the female gametophyte and endosperm (Kinoshita, T. et al., Science, 303, 521-523 (2004); Soppe, W. J. J. et al., Mol Cell, 6, 791-802 (2000)). MEA is imprinted in the endosperm, but is biallelically expressed in the embryo and in other sporophytic tissues (Kinoshita, T. et al., Science, 303, 521-523 (2004)). Expression of MEA in the embryo is likely not under DME control, as DME expression is not detected in the egg cell or embryo (Choi, Y. et al., Cell, 110, 33-42 (2002)). Expression of FWA in the endosperm, and elsewhere in the plant, is associated with hypomethylation of repeats in the 5′ region of the gene (Kinoshita, T. et al., Science, 303, 521-523 (2004); Soppe, W. J. J. et al., Mol Cell, 6, 791-802 (2000)). Paternal inheritance of met1 releases FWA paternal allele silencing in the endosperm and embryo (Kinoshita, T. et al., Science, 303, 521-523 (2004)). MET1 is the homolog of Dnmt1 (Bender, J., Ann Rev Plant Biology, 55, 41-68 (2004)).
DME, MEA, and MET1 genetically interact in the female gametophyte. MEA is an E(z) homologue that functions in a PcG complex along with FIE (Kohler, C. et al., EMBO J, 22, 4804-4814 (2003)), a homologue of Eed, to repress endosperm growth. Inheritance of mutant maternal dme or mea alleles causes endosperm overproliferation, embryo arrest, and seed abortion (Choi, Y. et al., Cell, 110, 33-42 (2002); Grossniklaus, U. et al., Science, 280, 446-450 (1998); Kiyosue, T. et al., Proc Natl Acad Sci USA, 96, 4186-4191 (1999); Luo, M. et al., Proc Natl Acad Sci USA, 96, 296-301 (1999)). Seed abortion caused by dme is suppressed by maternally inherited met1 if a wild type maternal MEA allele is present (Xiao, W. et al., Developmental Cell, 5, 891-901 (2003)). Moreover, met1 can restore MEA expression in dme mutants (Xiao, W. et al., Developmental Cell, 5, 891-901 (2003)). It is known that the glycosylase activity of DME is necessary for seed viability and activation of MEA transcription (Choi, Y. et al., Proc Natl Acad Sci USA, 101, 7481-7486 (2004)). DME antagonizes MET1 by specifically removing 5′-methylcytosine from MEA in the central cell, allowing the maternal MEA allele to be expressed there before fertilization and in the endosperm after fertilization.
As mentioned above, genetic information is stored not only in the sequential arrangement of four nucleotide bases, but also in covalent modification of selected bases (see, e.g., Robertson et al., Nature Rev. Genet. 1:11-19 (2000)). One of these covalent modifications is methylation of cytosine nucleotides, particularly cytosines adjacent to guanine nucleotides in “CpG” dinucleotides. Covalent addition of methyl groups to cytosine within CpG dinucleotides is catalyzed by proteins from the DNA methyltransferase (DNMT) family (Amir et al., Nature Genet. 23:185-88 (1999); Okano et al., Cell 99:247-57 (1999)). In the human genome, CpG dinucleotides are generally under represented, and many of the CpG dinucleotides occur in distinct areas called CpG islands. A large proportion of these CpG islands can be found in promoter regions of genes. The conversion of cytosine to 5′-methylcytosine in promoter associated CpG islands has been linked to changes in chromatin structure and often results in transcriptional silencing of the associated gene. Transcriptional silencing by DNA methylation has been linked to mammalian development, imprinting and X-Chromosome inactivation, suppression of parasitic DNA and numerous cancer types (see, e.g., Li et al., Cell 69:915-26 (1992); Okano et al., Cell 99:247-57 (1999)). Detected changes in the methylation status of DNA can serve as markers in the early detection of neoplastic events (Costello et al., Nature Genet. 24:132-38 (2000)).
BRIEF SUMMARY OF THE INVENTION
The present invention provides, in part, a isolated cell comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide, the polynucleotide encoding a polypeptide comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids and wherein the polypeptide excises methylated cytosines in DNA when contacted to DNA comprising methylated cytosines.
In some embodiments, the first and second amino acid sequences are linked directly.
In some embodiments, the first and second amino acid sequences are linked indirectly via the amino acid linker In some embodiments, the linker is heterologous to the first or second amino acid sequence. In some embodiments, the linker is comprises a fragment of at least 10 amino acids of SEQ ID NO:3 but does not include the full sequence of SEQ ID NO:3. In some embodiments, the linker has 20 or fewer amino acids. In some embodiments, the linker comprises SEQ ID NO:4.
In some embodiments, the first amino acid sequence is at least 95% identical to SEQ ID NO:1. In some embodiments, the second amino acid sequence is at least 95% identical to SEQ ID NO:2. In some embodiments, the first amino acid sequence comprises SEQ ID NO:1 and the second amino acid sequence comprises SEQ ID NO:2.
In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a yeast, fungal, mammalian, plant or insect cell.
In some embodiments, the cell is a prokaryotic cell.
The present invention also provides methods of making a polypeptide that excises methylated cytosines in DNA when contacted to DNA comprising methylated cytosines. In some embodiments, the method comprises culturing the cell as described above or elsewhere herein under conditions to allow for expression of the polypeptide.
In some embodiments, the method further comprises purifying the polypeptide.
In some embodiments, the first and second amino acid sequences are linked directly.
In some embodiments, the first and second amino acid sequences are linked indirectly via the amino acid linker. In some embodiments, the linker is heterologous to the first or second amino acid sequence. In some embodiments, the linker is comprises a fragment of at least 10 amino acids of SEQ ID NO:3 but does not include the full sequence of SEQ ID NO:3. In some embodiments, the linker has 20 or fewer amino acids. In some embodiments, the linker comprises SEQ ID NO:4.
In some embodiments, the first amino acid sequence is at least 95% identical to SEQ ID NO:1. In some embodiments, the second amino acid sequence is at least 95% identical to SEQ ID NO:2. In some embodiments, the first amino acid sequence comprises SEQ ID NO:1 and the second amino acid sequence comprises SEQ ID NO:2.
In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a yeast, fungal, mammalian or insect cell.
In some embodiments, the cell is a prokaryotic cell.
The present invention also provides isolated polypeptides comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids and wherein the polypeptide excises methylated cytosines in DNA when contacted to DNA comprising methylated cytosines. In some embodiments, the polypeptides are produced by the methods described above.
In some embodiments, the first and second amino acid sequences are linked directly.
In some embodiments, the first and second amino acid sequences are linked indirectly via the amino acid linker. In some embodiments, the linker is heterologous to the first or second amino acid sequence. In some embodiments, the linker is comprises a fragment of at least 10 amino acids of SEQ ID NO:3 but does not include the full sequence of SEQ ID NO:3. In some embodiments, the linker has 20 or fewer amino acids. In some embodiments, the linker comprises SEQ ID NO:4.
In some embodiments, the first amino acid sequence is at least 95% identical to SEQ ID NO:1. In some embodiments, the second amino acid sequence is at least 95% identical to SEQ ID NO:2. In some embodiments, the first amino acid sequence comprises SEQ ID NO:1 and the second amino acid sequence comprises SEQ ID NO:2.
The present invention also provides an isolated nucleic acid comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a polypeptide, the polynucleotide encoding a polypeptide comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids and wherein the polypeptide excises methylated cytosines in DNA when contacted to DNA comprising methylated cytosines.
In some embodiments, the first and second amino acid sequences are linked directly.
In some embodiments, the first and second amino acid sequences are linked indirectly via the amino acid linker. In some embodiments, the linker is heterologous to the first or second amino acid sequence. In some embodiments, the linker is comprises a fragment of at least 10 amino acids of SEQ ID NO:3 but does not include the full sequence of SEQ ID NO:3. In some embodiments, the linker has 20 or fewer amino acids. In some embodiments, the linker comprises SEQ ID NO:4.
In some embodiments, the first amino acid sequence is at least 95% identical to SEQ ID NO:1. In some embodiments, the second amino acid sequence is at least 95% identical to SEQ ID NO:2. In some embodiments, the first amino acid sequence comprises SEQ ID NO:1 and the second amino acid sequence comprises SEQ ID NO:2.
The present invention also provides methods of detecting cytosine methylation in a DNA sample. In some embodiments, the method comprises contacting DNA with a polypeptide as described herein (e.g., comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids) such that the polypeptide excises methylated cytosines, if present, from the DNA, thereby cleaving the DNA strand having the methylated cytosine to cause a nick in the DNA; extending a primer that is complementary to a sequence in the DNA up to the nick, thereby producing an extension product; and detecting the extension product, wherein the quantity or length of the extension product indicates cytosine methylation in the DNA sample, wherein the method does not involve adding nucleotides to the extension product with a terminal transferase.
The present invention also provides methods of detecting DNA methylation in a DNA sample. In some embodiments, the method comprises contacting DNA with the polypeptide as described herein (e.g., comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids) under conditions such that the demethylase excises methylated cytosines, if present, from the DNA, thereby cleaving the DNA strand having the methylated cytosine to cause a nick in the DNA; extending a detectably-labeled primer that is complementary to a sequence in the DNA up to the nick, thereby producing an extension product; and detecting the length of the extension product, thereby detecting methylation of the DNA in the DNA sample.
The present invention also provides methods of detecting DNA methylation in a DNA sample. In some embodiments, the method comprises contacting DNA with the polypeptide as described herein (e.g., comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids) under conditions such that the polypeptide becomes covalently linked to the DNA at the site of methylated cytosines, if present, thereby cleaving the DNA strand having the methylated cytosine to cause a nick in the DNA; separating double stranded DNA covalently bound to the polypeptide from DNA not bound to the polypeptide, wherein the double stranded DNA covalently bound to the polypeptide comprises a first strand covalently linked to the polypeptide and a complementary strand not linked to the polypeptide; and amplifying DNA on the complementary strand and detecting an amplification product, thereby detecting DNA methylation in a DNA sample.
The present invention also provides methods of detecting DNA methylation in a DNA sample. In some embodiments, the method comprises contacting DNA with the polypeptide as described herein (e.g., comprising a first amino acid sequence substantially identical to SEQ ID NO:1 linked to a second amino acid sequence substantially identical to SEQ ID NO:1, wherein the first and second amino acid sequences are linked directly or by a linker having between 1-300 amino acids) under conditions such that the polypeptide excises methylated cytosines, if present, from the DNA, thereby cleaving the DNA strand having the methylated cytosine to cause a nick in the DNA; amplifying a region of DNA between two primers that are complementary to chromosomal DNA; and detecting the quantity of an amplification product, wherein the presence, absence and/or quantity of the amplification product is indicative of methylation of the DNA in the DNA sample.
DEFINITIONS
The term “a” refers to at least one of something.
“Cytosine methylation” refers to 5-methyl cytosine.
“Methylated DNA” refers to DNA comprising 5-methyl cytosine.
The term “nucleic acid” or “polynucleotide” as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids containing analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156).
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a “plant promoter” is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term “plant” includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds, embryos, endosperm, ovules, male and female gametophytes, and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of flowering plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants and plant cells of a variety of ploidy levels, including polyploid, diploid, haploid, aneuploid and hemizygous.
A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
A polynucleotide “exogenous to” an individual plant is a polynucleotide which is introduced into the plant, or a predecessor generation of the plant, by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, in planta techniques, and the like. “Exogenous,” as referred to within, is any polynucleotide, polypeptide or protein sequence, whether chimeric or not, that is initially or subsequently introduced into the genome of an individual host cell or the organism regenerated from said host cell by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation (of dicots—e.g. Salomon et al. EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983); of monocots, representative papers are those by Escudero et al., Plant J. 10:355 (1996), Ishida et al., Nature Biotechnology 14:745 (1996), May et al., Bio/Technology 13:486 (1995)), biolistic methods (Armaleo et al., Current Genetics 17:97 1990)), electroporation, in planta techniques, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T0 for the primary transgenic plant and T1 for the first generation. The term “exogenous” as used herein is also intended to encompass inserting a naturally found element into a non-naturally found location.
The phrase “host cell” refers to a cell from any organism. Exemplary host cells are derived from plants, bacteria, yeast, fungi, insects or animals, including humans. Methods for introducing polynucleotide sequences into various types of host cells are well known in the art. Host cells can be isolated from an organism rather than as part of an organism.
An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition.
“Demethylase activity” refers to the ability of a polypeptide to excise a methylated nucleotide (e.g., 5-methyl cytosine) from a DNA sequence. Demethylation activity can be assayed in vivo by expressing a candidate polypeptide in the nucleus of a cell and then assaying for a change in methylation of the cell's DNA. See, e.g., Vong, et al., Science 260:1926-1928 (1993). Changes in chromosomal methylation can be measured by comparing the ability of methylation sensitive and insensitive endonucleases to cleave DNA from a cell expressing a polypeptide suspected of having demethylase or methylase activity. Alternatively, bisulfate sequencing can be used to identify which base pairs are methylated in a DNA sequence. For a discussion of both methods, see Soppe, W. J. J. et al., Mol Cell, 6, 791-802 (2000). In vitro assays to measure demethylase activity using labeled substrates are also known to those of skill in the art. See, e.g., Vhu et al., Proc. Natl. Acad. Sci. USA 97:5135-5139 (2000). Further methods for measuring demethylase activity are provided in the Examples.
The phrase “substantially identical,” in the context of two nucleic acids or polypeptides, refers to a sequence or subsequence that has at least 40% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 40% to 100%. Exemplary embodiments include at least: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. As explained herein, the present invention provides sequences substantially identical to SEQ ID NOs:1, 2, 3, and 4, and notably polypeptides (and nucleic acids encoding such polypeptides) comprising amino acid sequences substantially identical to SEQ ID NO:1 and SEQ ID NO:2 linked directly or via a linker.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. If no range is provided, the comparison window is the entire length of the reference sequence. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
An example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:

    1) Alanine (A), Serine (S), Threonine (T);
    2) Aspartic acid (D), Glutamic acid (E);
    3) Asparagine (N), Glutamine (Q);
    4) Arginine (R), Lysine (K);
    5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
    6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins (1984)).


An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, highly stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30° C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 55° C., sometimes 60° C., and sometimes 65° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody or its functional equivalent will be most critical in specificity and affinity of binding. See Paul, Fundamental Immunology. 
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, e.g., pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990))
For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 displays a schematic drawing of the full length Arabidopsis DME polypeptide (top), the Δ677 N-terminus DME deletion (middle) and the Δ677 deletion further lacking the IDR1 region (bottom). The deleted IDR1 sequence is SEQ ID NO:3. In its place, a linker (SEQ ID NO:4) was inserted.
FIG. 2 displays a schematic drawing of (DMEΔ677ΔIDR1::lnk) , which comprises the Δ677 deletion further lacking the IDR1 region (DMEΔ677ΔIDR1::lnk) (left) (AGSSGNGSSGNG=SEQ ID NO:4). Also shown are results of an in vitro assay demonstrating that DMEΔ677ΔIDR1::lnk enzyme is active and has lyase activity on a double stranded DNA substrate with a single 5-methylcytosine residue.
FIG. 3 displays expression of DMEΔ677ΔIDR1::lnk fused to various protein fusion partners.
FIG. 4 displays results of various steps of purification of poly-His tagged (DMEΔ677ΔIDR1::lnk).
FIG. 5 displays selected results for DME full length, Δ667 and (DMEΔ677ΔIDR1::lnk).



DEMETER (DME) is a DNA glycosylase that excises 5-methylcytosine from DNA leading to active DNA demethylation. DME uniquely excises 5-methylcytosine in all sequence contexts. However, production of DME (full length and DMEΔN677) in E. coli is difficult because of its large size (1,729 amino acids) and poor solubility. As shown in FIG. 1, in this invention, we engineered DME by removing interdomain region 1 (IDR1) and replacing it with a small linker (lnk). The size of DMEΔN677ΔIDR1::lnk is only 38.9% of full-length DME, and its expression level in E. coli significantly increased. This improved both yield and solubility while preserving enzyme activity (FIG. 2). When fused with different types of tags (e.g., 6×His (SEQ ID NO:15), maltose-binding protein, small ubiquitin-like modifier, glutathione S transferase, and thioredoxin) and produced in E. coli, DMEΔN677ΔIDR1::lnk was successfully expressed and highly soluble in all cases (FIG. 3). His-tagged DMEΔN677ΔIDR1::lnk was successively purified over Nickel, Heparin, and Superdex 200 columns with improved stability and little degradation and/or truncation (FIG. 4). FIG. 5 summarizes the properties of DME, DMEΔN677, and DMEΔN677ΔIDR1::lnk.
DETAILED DESCRIPTION
I. Introduction
The present invention is based, in part, on the discovery that it is possible to delete all or part of an internal portion (referred to herein as “IDR1”) of the DEMETER amino acid sequence while maintaining protein activity. Further, the inventors have found that the internally truncated protein has substantially better expression (yield) when expressed from E. coli. It is expected that a similar improvement of expression will occur when using other prokaryotic or eukaryotic expression systems.
II. Demethylases
The present invention provides for novel DME polypeptides with internal deletions that retain demethylase activity. It was previously known that that as many as 677 amino acids can be deleted from the amino terminus of DME while retaining demethylase activity. See, e.g., U.S. patent application Ser. No. 12/006,779. The inventors have now found that a further internal portion of DME can be deleted while maintaining activity of the protein and greatly improving solubility and expression and yield when produced in cells. Accordingly, the present invention provides for polypeptide that excise methylated cytosines in DNA when contacted to DNA comprising methylated cytosines, wherein the polypeptides comprise a first amino acid sequence substantially (e.g., at least 70%, 80%, 90%, 95%) identical to SEQ ID NO:1 linked directly or linked through a linker to a second amino acid sequence substantially (e.g., at least 70%, 80%, 90%, 95%) identical to SEQ ID NO:2. The inventors have found, for example, that a heterologous linker (i.e., not a sequence or fragment thereof from SEQ ID NO:3, the IDR1 sequence) can be used to link the first and second amino acid sequences together (where the first amino acid sequence is nearer to the N-terminus of the protein than the second amino acid sequence).
A variety of linkers can be used to link the first and second amino acid sequences. In some embodiments, the first and second sequences are linked directly. In some embodiments, a linker links the first and second amino acid sequences. In some embodiments, the linker is a fragment, but not the entire sequence, of SEQ ID NO:3. For example, the linker can be a fragment of at least 2, 5, 10, 20, 50 or more amino acids of SEQ ID NO:3. In some embodiments, the fragment of SEQ ID NO:3 has fewer than 100, 50, 30, 20, or 10 amino acids.
Alternatively, a heterologous linker can be used. A wide variety of amino acid linkers are known and can be used. In some embodiments, the heterologous linker has between 2-30 amino acids, e.g., 5-25 amino acids. In some embodiments, at least a majority of the amino acids in the heterologous linker are serine, alanine or arginine. An exemplary heterologous linker is SEQ ID NO:4.
The present invention further provides numerous chimeric polypeptides with demethylase activity, the chimeric polypeptides comprising the fusion of portions of at least two different demethylases (i.e., a sequence corresponding (substantially identical) to SEQ ID NO:1 from one protein and a sequence corresponding (substantially identical) to SEQ ID NO:2 of a second enzyme) but lacking a sequence corresponding to IDR1 (e.g., lacking a sequence substantially identical to SEQ ID NO:3). Exemplary demethylases from which chimeras can be constructed include, e.g., DME (SEQ ID NO:5), ROS1 (SEQ ID NO:6), DML2 (SEQ ID NO:7), or DML3 (SEQ ID NO:8).
Optionally, the demethylases of the invention are fused with a further polypeptide sequence that can be used, e.g., to assist in purification of the demethylase and/or assist in rendering the demethylases more soluble. Examples of further polypeptides to which the demethylases can be fused include, e.g., maltose binding protein (MBP), glutathione (GST), poly-His, etc.
The present invention also provides polynucleotides encoding any of the demethylase polypeptides described herein. In some embodiments, the polynucleotides of the invention comprise an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a demethylase described herein. The promoter can be a promoter that initiates and/or controls transcription in prokaryotic and/or eukaryotic cells. The promoter can initiate and/or control transcription in plant, animal, insect or other eukaryotic cells.
One of skill will appreciate that the polypeptides of the invention can be produced by standard recombinant genetic engineering methods. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). For example, an expression cassette encoding the polypeptide can be introduced into a cell. In some embodiments, a single or multi-copy expression vector comprising the expression cassette is introduced into the cell. A wide variety of prokaryotic and eukaryotic expression systems can be used.
In some embodiments, to obtain high level expression of a polypeptide, one subclones polynucleotides encoding a polypeptide of the invention into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systems for expressing the polypeptides of the invention are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Expression of proteins from eukaryotic vectors can be also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal. Inducible expression vectors are often chosen if expression of the protein of interest is detrimental to eukaryotic cells.
Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with an polypeptides of the invention-encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of polypeptides of the invention, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra).
As desired, after the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of polypeptides of the invention, which is recovered from the culture using standard techniques identified below.
III. Detection of Methylation
As the demethylases of the invention bind to methylated DNA sequences and modify such sequences, demethylases are useful for detecting the presence or absence and/or the location (i.e., specific sequence) of cytosine methylation in DNA. Cytosine methylation is known to regulate transcription, thereby affecting biological processes in cells and organisms. Accordingly, detection of DNA methylation has numerous uses in biological research. Moreover, aberrant methylation can be a marker for aging and disease, including various cancers. See, e.g., Jones, Oncogene, 21(35):5358-5360 (2002); Esteller, Annual Review of Pharmacology and Toxicology, 45: 629-656 (2005); Li, Biochim Biophys Acta., 21704(2):87-102 (2004); Szyf, Biochem Pharmacol., 68(6):1187-97 (2004). Exemplary human genes whose methylation status is implicated in cancer and/or aging and can be detected according to the methods of the present invention include, but are not limited to, e.g., 14-3-3 Sigma, ABL1 (P1), ABO, APC, AR (Androgen Receptor), BLT1 (Leukotriene B4 Receptor), BRCA1, CALCA (Calcitonin), CASP8 (CASPASE 8), Caveolin 1, CD44, CFTR, COX2, CSPG2 (Versican), CX26 (Connexin 26), Cyclin A1, DBCCR1, ECAD (E-cadherin), Endothelin Receptor B, EPHA3, EPO (Erythropoietin), ER (Estrogen Receptor), FHIT, GPC3 (Glypican 3), GST-pi, H19, H-Cadherin (CDH13), HIC1, hMLH1, HOXA5, IGF2 (Insulin-Like Growth Factor II), IGFBP7, IRF7, LKB1, LRP-2 (Megalin), MDGI (Mammary-derived growth inhibitor), MDR1, MDR3 (PGY3), MGMT (O6 methyl guanine methyl transferase), MT1a (metallothionein 1), MUC2, MYOD1, N33, NEP (Neutral Endopeptidase 24.1)/CALLA, NF-L (light-neurofilament-encoding gene), NIS (sodium-iodide symporter gene), p14/ARF, p15 (CDKN2B), p16 (CDKN2A), p27KIP1, p57 KIP2, PAX6, PgR (Progesterone Receptor), RAR-Beta2, RASSF1, RB1 (Retinoblastoma), TERT, TESTIN, TGFBRI, THBS1 (Thrombospondin-1), TIMP3, TLS3 (T-Plastin), Urokinase (uPA), VHL (Von-Hippell Lindau), WT1, ZO2 (Zona Occludens 2). Accordingly, the present invention provides for detecting methylation of these or other mammalian (e.g., human) gene sequences, including, e.g., promoters thereof), by any methylation detection method provided herein. The DNA samples can be obtained from any mammal, including humans, and can be any biological sample that contains DNA, include, but not limited to, tissue biopsies (e.g., solid tumors or tissues suspect of having cancer or pre-cancerous tissue), blood samples, stool samples, etc.
The DNA methylation detection methods of the present invention can be used to detect DNA methylation, including for use in determining a diagnosis or prognosis, or, e.g., for monitoring progress of a disease or drug therapy.
Demethylases useful for the DNA methylation detection methods described herein include any demethylase that causes a nick when excising a methylated cytosine.
A. Methods Involving Primer Extension
Numerous methods of DNA methylation detection are provided herein. In some embodiments, the methods take advantage of the DNA “nicking” activity of the polypeptides of the present invention. “Nicking” refers to an activity of demethylases in which at least one strand of the DNA double helix is cleaved at or adjacent to a methylated nucleotide (e.g., a methylated cytosine) on the phosphodiester backbone of the DNA. See, e.g., Gehring, M. et al., Cell, 124:495-506 (2006).
In some embodiments, the methods comprise nicking DNA with a demethylase of the invention and using primer extension from a DNA sequence adjacent to a DNA region of interest to detect the nick and thus the methylated nucleotide. In these embodiments, the primer extension continues until it is stopped by the nick. Determination of the length of the extension product, with knowledge of the chromosomal sequence and the sequence to which the primer hybridizes on the chromosomal sequence, allows one to determine where the methylation occurs in the chromosome. For example, if the extension product is 100 nucleotides long, then the methylated nucleotide is approximately 100 nucleotides from the site to which the primer hybridizes. Determination of the quantity of the primer extension product is indicative of the amount of methylated DNA. For example, if only 50% of the chromosomal copies are methylated (or if 50% of the cells in a sample from which DNA is obtain have methylated copies) then there will be a long extension product at the limits of primer extension and a shorter extension product which results from the nick stopping the extension, each in approximately equal amounts. In some embodiments, multiple different length extension products of different quantity are detected, thereby detecting a complicated heterogeneous methylation pattern of a sample, thereby detecting methylation at multiple sites on the DNA.
Primer extension can be performed by any method that allow for polymerase-based extension of a primer hybridized to genomic DNA. In some embodiments, simple primer extension involves addition of a primer and DNA polymerase to genomic DNA under conditions to allow for primer hybridization and primer extension by the polymerase. Of course, such a reaction includes the necessary nucleotides, buffers, and other reagents known in the art for primer extension.
In some embodiments, primer extension occurs during a nucleic acid amplification reaction. A non-limiting example of nucleic acid amplification is the polymerase chain reaction (PCR). Additional examples of amplification reactions include the ligase chain reaction (LCR), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, et al., J. Clin. Microbiol. 33:1856-1859 (1995)), nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2 (1991), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999)); Hatch et al., Genet. Anal. 15(2):35-40 (1999)) and branched DNA signal amplification (bDNA) (see, e.g., Iqbal et al., Mol. Cell. Probes 13(4):315-320 (1999)). Other amplification methods known to those of skill in the art include CPR (Cycling Probe Reaction), SSR (Self-Sustained Sequence Replication), SDA (Strand Displacement Amplification), QBR (Q-Beta Replicase), Re-AMP (formerly RAMP), RCR (Repair Chain Reaction), TAS (Transcription Based Amplification System), and HCS. In embodiments involving amplification, one or more primers are extended by amplification following nicking of the genomic DNA template with a demethylase of the invention, and the amplification product quantity or length is determined. Any number of methods are known for detecting amplification products, including, e.g., real-time amplification techniques, including those involving probes that specifically detect amplification products in real time, such as TaqMan® probes, Molecular Beacons and the like.
In some embodiments, the primer is detectably labeled (e.g., at its 5′ end or otherwise located to not interfere with 3′ extension of the primer) and following primer extension, the length and/or quantity of the labeled extension product is detected by detecting the label.
In some embodiments, the primer extension products are detected by adding a nucleotide sequence to the 3′ end of the extension product. A non-limiting example of this process includes “tailing” with a single nucleotide (e.g., one of A, C, T, or G) using terminal tranferase and subsequent amplification using a primer that hybridizes to the tailed homopolymeric sequence. An example of such a method is described in Choi, Y. et al., Cell, 110:33-42 (2002). In some embodiments, this method is performed using demethylases comprising less than the full length DME protein sequences (or substantially identical sequences thereof) as disclosed herein or using chimeric demethylases (or substantially identical sequences thereof) as described herein. In some embodiments, the primer extension methods of the invention do not involving adding nucleotides to the extension product with a terminal transferase.
B. Methods Involving Covalent Bonding of Demethylase to DNA
The present invention also provides methods of detecting DNA methylation by taking advantage of the ability of the polypeptides of the invention to bind to methylated DNA. The demethylases of the invention temporarily bind to methylated DNA in the process of introducing a nick by cleavage of the phosphodiester backbone of DNA. This temporary binding of the DNA can be converted to a covalent bond if the demethylase and DNA are contacted together in the presence of a reducing agent. The presence of the reducing agent results ins a chemical reduction reaction resulting in covalent linkage of the demethylase to the DNA at the site of the methylated nucleotide. Exemplary non-limited reducing agents include NaBH4.
Once the demethylase is covalently bound to the DNA, the DNA/demethylase complex can be separated from other DNA (and, e.g., protein, carbohydrates and other cellular constituents) in the sample using any agent that has affinity for the demethylase. For example, a reagent that specifically binds to the demethylase can be bound to a solid surface, the demethylase/DNA mixture can be contacted to the agent under conditions in which the agent binds to the demethylase and then washed, thereby removing any DNA not bound to the demethylase. Exemplary agents that bind the demethylases of the invention include, but are not limited to antibodies that bind the demethylases. In alternate embodiments, the demethylase can be engineered to include an epitope or other tag that is recognized by an affinity agent. For example, a poly-His sequence can be genetically engineered to either end of the demethylase. Nickel can then be used as an affinity agent to bind the poly-His demethylase bound to DNA. In another non-limiting embodiment, the demethylase is biotinylated and the affinity agent is streptavidin or another molecule with affinity for biotin. In yet another embodiment, an epitope tag is genetically engineered into the demethylase and an antibody that binds to the epitope is used to bind to the demethylase.
Once the demethylase/DNA complex is separated from non-bound DNA, the double stranded DNA strand bound to the demethylase is detected directly. Alternatively, the DNA bound to the demethylase is double stranded and the doubles-stranded DNA is denatured after the demethylase/DNA complex is separated from non-bound DNA, and the resulting intact strand of DNA is detected. Detection can include any nucleic acid detection method known in the art, e.g., nucleic acid amplification techniques such as those relying on PCR.
IV. Kits
For use in diagnostic, prognostic, research applications and other uses described herein, kits are also provided by the invention. The kits of the invention may comprise any or all of the reagents to perform the methods described herein. Such kits may include any or all of the following: at least one demethylase polypeptide of the invention, or nucleic acid encoding a demethylase, primers for primer extension (optionally detectably labeled), a DNA polymerase (optionally a thermostable polymerase capable of carrying out PCR under standard conditions), a terminal transferase, hybridization probes (optionally labeled) for detecting extension products, etc.
In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
V. Therapeutic Use of Demethylases
The present invention also provides for use of the demethylase polypeptides of the invention to reduce methylation in cells, including cells in vivo as well as ex vivo (e.g., cells extracted from an individual, treated with a demethylase and then returned to an individual). Thus, demethylases of the invention can be used for the purpose of modulating the activity of target genes through chromatin architecture in animal cells as well as plant cells. For example, in some embodiments, a demethylase of the invention is used to catalytically remove 5-MeC from target gene DNA in several ways: e.g., (1) by fusing the demethylase to a sequence specific DNA binding protein, or (2) by fusing the demethylase to a subunit of the target repressor complex such as MeCP2 or Sin3. When combined with cell, tissue, or developmentally specific promoters, a demethylase of the invention can be used to modulate specific sets of target genes.
In addition, reactive oxygen species, partially reduced species that are produced as intermediates of aerobic respiration, are powerful oxidizing agents that escape the mitochondria and attach via cellular components. Ionizing radiation and other agents that generate free radicals also produce reactive oxygen species that can attack the genome and cause lesions that are thought to have a key role in causing cancer and ageing. For example, 7,8-dihydro-8-oxoguanine (oxoG) is a very deleterious adduct generated by oxidation of the guanine base in DNA. The oxoG protein can pair with either cytosine or adenine during DNA replication. Thus, oxoG residues in DNA give rise to G/C to T/A transversion mutations. These transversions are common somatic mutations found in human cancers. Demethylases of the invention, such as those described herein, represent a defense against oxoG by catalysing the expulsion of the oxoG. Thus, in some embodiments, enhancing demethylase activity is a method to reduce the incidence of mutations in animal cells. Also, a demethylase of the invention can be used to catalytically remove oxoG from a target gene by fusing a demethylase of the invention to a sequence specific DNA binding protein. When combined with a cell, tissue, or developmentally specific promoters a demethylase of the invention can be used to modulate repair of target genes.
As described above, the polypeptides of the invention can be targeted to chromosomal regions of interest by linking the polypeptides of the invention, including fragments with demethylase activity, to a DNA-binding domain that binds a target sequence. For example, it is known that an enzyme that methylates DNA (Dam methylase) can be targeted to specific sites in the genome (B. V. Steensel and S. Henikoff, Nature Biotechnology 18:424-428 (2000)). Specifically, the methylase was tethered to the DNA-binding domain of GAL4. When recombinant GAL4-methylase protein was expressed in transgenic Drosophila, targeted methylation occurred in a region of a few kilobases surrounding the GAL4 DNA binding sequence. In a analogous fashion, a demethylase of the invention can be tethered (e.g., as a translational fusion or chemically linked) to proteins that interact at specific sites in the genome). As a result, specific targeted regions of the genome are hypomethylated by a demethylase of the invention. As discussed above, typically hypomethylation promotes transcription of genes (S. E. Jacobsen, Current Biology 9, 617 (1999). The invention provides compositions and methods for demethylation of a desired area of the chromosome by targeting a demethylase of the invention to those regions. Thus, these embodiments provide additional ways to activate transcription of a desired gene in a targeted chromosomal region.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Summary
DEMETER (DME) is a DNA glycosylase that excises 5-methylcytosine from DNA leading to active DNA demethylation. DME uniquely excises 5-methylcytosine in all sequence contexts. However, production of DME (full length and DMEΔN677) in E. coli is difficult because of its large size (1,729 amino acids) and poor solubility. As shown in FIG. 1, in this invention, we engineered DME by removing interdomain region 1 (IDR1) and replacing it with a small linker (Ink). The size of DMEΔN677ΔIDR1::lnk is only 38.9% of full-length DME, and its expression level in E. coli significantly increased. This improved both yield and solubility while preserving enzyme activity. When fused with different types of tags (e.g., 6×His (SEQ ID NO:15), maltose-binding protein, small ubiquitin-like modifier, glutathione S transferase, and thioredoxin) and produced in E. coli, DMEΔN677ΔIDR1::lnk was successfully expressed and highly soluble in all cases. His-tagged DMEΔN677ΔIDR1::lnk was successively purified over Nickel, Heparin, and Superdex 200 columns with improved stability and little degradation and/or truncation.
Introduction
DME is a DNA glycosylase that excises 5-methylcytosine from DNA (Gehring et al., 2006). DME is primarily expressed in the central cell of the female gametophyte in Arabidopsis and catalyzes female-specific DNA demethylation at target loci such as MEDEA, FWA, and FIS2 (Choi et al., 2002, Kinoshita et al., 2004, Jullien et al., 2006). Asymmetric DNA methylation between the paternal and maternal alleles is epigenetically inherited to the zygotic endosperm, where the less methylated maternal allele is expressed while the hypermethylated paternal allele silenced. Such monoallelic expression according to the parent-of-origin, that is called gene imprinting, is crucial for endosperm development and seed viability.
DME encodes a 1,729 amino acid peptide (Choi et al., 2002). A centrally located DNA glycosylase domain (1167-1368) contains a helix-hairpin-helix (HhH) motif. As an enzyme initiating the base excision repair (BER), the HhH motif in DME catalyzes excision of 5-methylcytosine, and the substitution of catalytic residues (K1286Q and D1304N) abolishes the enzyme activity (Gehring et al., 2006). In addition, DME has four cysteine residues adjacent to the DNA glycosylase domain that hold a [4Fe-4S] cluster in place. This cluster, found in many DNA glycosylases, is thought to play a role in DNA binding and/or structural stability. We found that site-directed mutagenesis on the residues comprising the [4Fe-4S] cluster disrupted 5-methylcytosine excision activity, proposing its necessity in DME function (U.S. patent application Ser. No. 12/006,779).
There are three other DME-like DNA glycosylases in Arabidopsis- ROS 1, DML2, and DML3 (Choi et al., 2002, Penterman et al., 2007). Besides the above mentioned DNA glycosylase domain, two additional conserved domains (Domain A and Domain B) are present in this family (FIG. 1). These domains flank the central DNA glycosylase domain. Even though their function is still elusive with no homology to other proteins, these domains are inevitable for DME activity, suggesting their essential role in excision of 5-methylcytosine. These conserved domains are connected with interdomain regions IDR1 and IDR2, the former is variable in size (395 amino acids for DME and 73 for DML3) while the latter comprises approximately 50 amino acids in all DME and DME-like proteins (FIG. 1). Unlike highly conserved glycosylase domain and Domains A and B, these IDR1 and IDR2 show little homology between the DME family members.
In the previous study, 537 amino acids at the N-terminus of DME were removed and fused with a maltose binding protein (MBP) for expression in E. coli (Gehring et al., 2006). This fusion protein MBP-DMEΔN537 specifically excised 5-methylcytosine from DNA in all sequence contexts (Gehring et al., 2006), suggesting that N-terminal 537 amino acids are dispensable for base excision activity. DMEΔN537 was soluble and active only when fused with MBP. However, the size of MBP-DMEΔN537 (1,587 amino acids; Mw=177.1 kDa) is still too big to be stably expressed at a high level in E. coli. 
In this patent application, we report that engineered DME which has both a further N-terminal truncation and an internal IDR1 deletion displays improved yield, stability and solubility, while retaining essential 5-methylcytosine glycosylase activity.
Results
IDR1 of DMEΔN677 was removed and replaced with a short linker peptide (lnk) as shown in FIG. 1. At the same time, two unique restriction enzyme sites PstI and KpnI were incorporated at both 5′- and 3′ ends of ink for further manipulation at this region. This DMEΔN677ΔIDR1::lnk fragment consists of 673 amino acids with all three conserved domains preserved (FIG. 1). The size of DMEΔN677ΔIDR1::lnk is only 38.9% of full-length DME owing to the removal of both nonconserved N-terminal region and IDR1.
In order to test 5-methylcytosine excision activity, DMEΔN677ΔIDR1::lnk was fused with MBP and expressed in E. coli. The resulting MBP-DMEΔN677ΔIDR1::lnk was purified over an amylose resin and reacted with methylated DNA substrate as described by Gehring et al. (2006). As shown in FIG. 2, MBP-DMEΔN677ΔIDR1::lnk was able to excise 5-methylcytosine to the same extent with MBP-DMEΔN677. This implies that IDR1 is unnecessary for DME activity and that a connection of Domain A to a glycosylase domain via a short linker peptide does not affect the overall folding and assembly of the protein.
We next examined the expression of DMEΔN677ΔIDR1::lnk fused with different types of fusion proteins—6×His (SEQ ID NO:15), glutathione S-transferase (GST), small ubiquitin-like modifier (SUMO), MBP, and thioredoxin (Trx). DMEΔN677ΔIDR1::lnk was expressed at a higher level in E. coli than DMEΔN677 with no IDR1 deletion. In addition, unlike DMEΔN677 that was soluble only when fused with MBP, DMEΔN677ΔIDR1::lnk was soluble in all cases (FIG. 3). This suggests that a deletion of IDR1 improves both yield and solubility of the protein when expressed in E. coli. 
Purification of MBP-DMEΔN677 barely reached >90% of purity due possibly to largely unstructured regions present in IDR1, which are often responsible for the instability of the protein. We fused DMEΔN677ΔIDR1::lnk with 6×His and expressed in E. coli. As shown in FIG. 4, 6×His (SEO ID NO:15) tagged DMEΔN677ΔIDR1::lnk was successively purified over HisTrap, Heparin, and Superdex 200 columns with improved purity at each step. Much less amount of small molecular weight fragments was observed in the final stage of purification compared to DMEΔN677 (FIG. 4). Such small fragments might represent early-truncated expression or degradation of the protein. However, a deletion of IDR1 appears to stabilize the protein with minimal truncation or degradation problems. FIG. 5 summarizes the properties of DME, DMEΔN677, and DMEΔN677ΔIDR1::lnk.
Materials and Methods
Construction of c2x-DMEΔN677ΔIDR1::lnk
The DMEΔN677 sequence was cloned into the pMAL-c2x vector (NEB) at XbaI and SalI sites (c2x-DMEΔN677). Primers JH151 (5′-TGA AGA GCC ATT ACC ACT TGA TCC TGC AGG AGG GAA TCG AGC AGC TAG-3′ (SEQ ID NO:9)) and JH152 (5′-AGT GGT AAT GGC TCT TCA GGA AAC GGT ACC AGC CAG TGG GAT AGT CTC-3′(SEQ ID NO:10)) were extended on the c2x-DMEΔN677 to create a linker sequence. The underlined sequences are complementary to each other. Following the treatment of DpnI to remove template DNA, the product was annealed and then PCR-amplified with primers JH-RNseqF (5′-CAG ATG TCC GCT TTC TGG TAT G-3′ (SEQ ID NO:11)) and JH153 (5′-CAT ACA GTG TTC GTT GAT CGA G-3′ (SEQ ID NO:12)) creating DMEΔN677ΔIDR1::lnk in which IDR1 is replaced with a short linker peptide (lnk, AGSSGNGSSGNG (SEQ ID NO:4)). The PCR product was digested with XbaI and XhoI and reinserted into the c2x-DMEΔN677 at the corresponding sites.
Construction of DMEΔN677ΔIDR1::lnk fusion with 6×His (SEQ ID NO:15), GST, SUMO, MBP, and Trx
The c2x-DMEΔN677ΔIDR1::lnk plasmid was PCR-amplified with primers JH154 (5′-TTA AGG ATC CTA CAA AGG AGA TGG TGC AC-3′ (SEQ ID NO:13)) and JH155 (5′-TTA AGT CGA CTT AGG TTT TGT TGT TCT TCA ATT TGC-3′ (SEQ ID NO:14)). The product was digested with BamHI and Sall and then cloned into the following pET-19b (Novagen) derivatives at the corresponding sites: pBG100 (6×His), pBG101 (GST), pBG102 (SUMO), pLM302 (MBP), and pLM304 (Trx) (provided by Brandt Eichman at Vanderbilt University).
Production and Purification of DMEΔN677ΔIDR1:lnk
The c2x-DMEΔN677ΔIDR1::lnk plasmid was transformed into Rosetta2 cells (Novagen) for expression. The other constructs were transformed into Rosetta2 (DE3) cells (Novagen). Expression of DMEΔN677ΔIDR1::lnk was induced with IPTG at 18° C. for 5 hrs. MBP-DMEΔN677ΔIDR1::lnk was purified over an amylose resin (NEB). The other proteins were first purified using HisTrap FF (GE Healthcare) utilizing 6×His (SEQ ID NO:15) in the fusion peptide. The subsequent purifications over Heparin HP (GE Healthcare) and Superdex 200 (GE Healthcare) columns were done according to the manufacturer's protocol.
5-Methylcytsine Excision Assay
The in vitro 5-methylcytosine excision assay was done as described by Gehring et al. (2006).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. An isolated polypeptide consisting of:
a first amino acid sequence, wherein the first amino acid sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 1,
a second amino acid sequence, wherein the second amino acid sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, and
optionally a linker, wherein the linker consists of 1-300 amino acids,
wherein the first and second amino acid sequences are linked directly or by the linker and wherein the polypeptide excises methylated cytosines in DNA when contacted to DNA comprising methylated cytosines.

2. The polypeptide of claim 1, wherein the first and second amino acid sequences are linked directly.

3. The polypeptide of claim 1, wherein the first and second amino acid sequences are linked by the linker.

4. The polypeptide of claim 3, wherein the linker is heterologous to the first or second amino acid sequence.

5. The polypeptide of claim 3, wherein the linker consists of 10-300 amino acids of the amino acid sequence of SEQ ID NO: 3.

6. The polypeptide of claim 3, wherein the linker consists of 20 or fewer amino acids.

7. The polypeptide of claim 3, wherein the linker consists of the amino acid sequence of SEQ ID NO: 4.

8. The polypeptide of claim 1, wherein the first amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

9. The polypeptide of claim 1, wherein the second amino acid sequence is at least 95% identical to the amino acid sequence of SEQ ID NO: 2.

10. The polypeptide of claim 1, wherein the first amino acid sequence consists of the amino acid sequence of SEQ ID NO: 1 and the second amino acid sequence consists of the amino acid sequence of SEQ ID NO: 2.

11. A method of making the polypeptide of claim 1, the method comprising, culturing an isolated cell under conditions to allow for expression of the polypeptide of claim 1, wherein the isolated cell comprises an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding the polypeptide of claim 1 thereby making the polypeptide of claim 1.

12. The method of claim 11, further comprising purifying the polypeptide.

13. The method of claim 11, wherein the cell is a eukaryotic cell.

14. The method of claim 13, wherein the cell is a yeast, fungal, mammalian or insect cell.

15. The method of claim 11, wherein the cell is a prokaryotic cell.