The Human Dnmt2 Has Residual DNA-(Cytosine-C5) Methyltransferase Activity

The human Dnmt2 protein is one member of a protein family conserved from Schizosaccharomyces pombe and Drosophila melanogaster to Mus musculus and Homo sapiens. It contains all of the amino acid motifs characteristic for DNA-(Cytosine-C5) methyltransferases, and its structure is very similar to prokaryotic DNA methyltransferases. Nevertheless, so far all attempts to detect catalytic activity of this protein have failed. We show here by two independent assay systems that the purified Dnmt2 protein has weak DNA methyltransferase activity. Methylation was observed at CG sites in a loose ttnCGga(g/a) consensus sequence, suggesting that Dnmt2 has a more specialized role than other mammalian DNA methyltransferases. The human Dnmt2 protein is one member of a protein family conserved from Schizosaccharomyces pombe and Drosophila melanogaster to Mus musculus and Homo sapiens. It contains all of the amino acid motifs characteristic for DNA-(Cytosine-C5) methyltransferases, and its structure is very similar to prokaryotic DNA methyltransferases. Nevertheless, so far all attempts to detect catalytic activity of this protein have failed. We show here by two independent assay systems that the purified Dnmt2 protein has weak DNA methyltransferase activity. Methylation was observed at CG sites in a loose ttnCGga(g/a) consensus sequence, suggesting that Dnmt2 has a more specialized role than other mammalian DNA methyltransferases. In mammals, DNA methylation is the only known physiological modification of DNA. It primarily occurs at CG sites, which are methylated to 70–80% and encode epigenetic information on the DNA. DNA methylation is implicated in the regulation of gene expression, control of development, X chromosome inactivation, parental imprinting, and the protection of the genome against parasitic genetic elements such as transposons, retrotransposons, and viruses (for review see Refs. 1Jeltsch A. Chembiochem. 2002; 3: 274-293Crossref PubMed Google Scholar, 2Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1560) Google Scholar, 3Li E. Nat. Rev. Genet. 2002; 3: 662-673Crossref PubMed Scopus (1584) Google Scholar, 4Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar). Aberrant methylation is among the most important causes of the inactivation of tumor suppressor genes in cancer (5Jones P.A. Baylin S.B. Nat. Rev. Genet. 2002; 3: 415-428Crossref PubMed Google Scholar). DNA- (Cytosine-C5) methyltransferases (MTases) 1The abbreviations used are: Mtase, AdoMet, S-adenosylmethionine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HPLC, high pressure liquid chromatography; dNK, deoxynucleoside kinase. are characterized by a set of highly conserved amino acid motifs (6Kumar S. Cheng X. Klimasauskas S. Mi S. Posfai J. Roberts R.J. Wilson G.G. Nucleic Acids Res. 1994; 22: 1-10Crossref PubMed Scopus (394) Google Scholar) that easily allows the identification of putative enzymes based on genomic sequences. In mammals, four candidate enzymes have been identified (1Jeltsch A. Chembiochem. 2002; 3: 274-293Crossref PubMed Google Scholar, 7Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1611) Google Scholar). Dnmt1 is known to have a high preference for hemimethylated CG sites and has an important role in maintenance of methylation (8Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar, 9Fatemi M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (200) Google Scholar). Dnmt3a and Dnmt3b do not recognize preexisting patterns of methylation and are de novo MTases (10Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 11Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar, 12Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (193) Google Scholar). However, it is also clear that in vivo the functions of maintenance and de novo methylation overlap, because accurate maintenance of the methylation at certain sequences also requires the presence of Dnmt3a and 3b and de novo methylation also relies on support by Dnmt1 (13Liang G. Chan M.F. Tomigahara Y. Tsai Y.C. Gonzales F.A. Li E. Laird P.W. Jones P.A. Mol. Cell. Biol. 2002; 22: 480-491Crossref PubMed Scopus (453) Google Scholar, 14Kim G.D. Ni J. Kelesoglu N. Roberts R.J. Pradhan S. EMBO J. 2002; 21: 4183-4195Crossref PubMed Scopus (301) Google Scholar, 15Rhee I. Bachman K.E. Park B.H. Jair K.W. Yen R.W. Schuebel K.E. Cui H. Feinberg A.P. Lengauer C. Kinzler K.W. Baylin S.B. Vogelstein B. Nature. 2002; 416: 552-556Crossref PubMed Scopus (1040) Google Scholar, 16Fatemi M. Hermann A. Gowher H. Jeltsch A. Eur. J. Biochem. 2002; 269: 4981-4984Crossref PubMed Scopus (199) Google Scholar). Dnmt1, Dnmt3A, and Dnmt3B are large proteins comprising an N-terminal part implicated in protein targeting and regulation and a C-terminal part that contains all 10 motifs characteristic for DNA-(Cytosine-C5) MTases. All three MTases are essential in mammals. Knock-out mice die during embryogenesis or shortly after birth (8Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar, 11Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar). The fourth putative DNA MTase in the genome of mammals, Dnmt2, has been discovered in 1998 (17Yoder J.A. Bestor T.H. Hum. Mol. Genet. 1998; 7: 279-284Crossref PubMed Scopus (226) Google Scholar, 18Van den Wyngaert I. Sprengel J. Kass S.U. Luyten W.H. FEBS Lett. 1998; 426: 283-289Crossref PubMed Scopus (42) Google Scholar). Expression of Dnmt2 has been found in many human and mouse tissues albeit at low levels (10Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 17Yoder J.A. Bestor T.H. Hum. Mol. Genet. 1998; 7: 279-284Crossref PubMed Scopus (226) Google Scholar, 18Van den Wyngaert I. Sprengel J. Kass S.U. Luyten W.H. FEBS Lett. 1998; 426: 283-289Crossref PubMed Scopus (42) Google Scholar). Lacking a large N-terminal part, the human protein comprises 391 amino acid residues, which show clear homology to prokaryotic DNA-(Cytosine-C5) MTases. Members of the Dnmt2 family are found in many species including man and mice, which also contain other DNA MTases, but also in D. melanogaster and S. pombe where these enzymes are the only obvious DNA MTase candidate genes. The strict conservation of all of the motifs characteristic for prokaryotic Cytosine-C5 MTases in Dnmt2 including the catalytically important amino acid residues suggests a function as DNA MTase. This hypothesis was supported by the finding that D. melanogaster DNA contains methylated cytosines that most probably is attributed to the activity of Dnmt2 (19Gowher H. Leismann O. Jeltsch A. EMBO J. 2000; 19: 6918-6923Crossref PubMed Scopus (168) Google Scholar, 20Lyko F. Ramsahoye B.H. Jaenisch R. Nature. 2000; 408: 538-540Crossref PubMed Scopus (362) Google Scholar). The structure of Dnmt2 was solved in complex with AdoMet, the cofactor for DNA methylation (21Dong A. Yoder J.A. Zhang X. Zhou L. Bestor T.H. Cheng X. Nucleic Acids Res. 2001; 29: 439-448Crossref PubMed Scopus (195) Google Scholar). It shows strong similarities to other DNA MTases, supporting the assumption that the protein is a DNA MTase. Interaction with DNA could be detected in gel shift experiments. In addition, it has been reported that Dnmt2 covalently binds to DNA (21Dong A. Yoder J.A. Zhang X. Zhou L. Bestor T.H. Cheng X. Nucleic Acids Res. 2001; 29: 439-448Crossref PubMed Scopus (195) Google Scholar). This observation also connects the protein to DNA MTases, which form a covalent enzyme-DNA intermediate during catalysis (for review see Refs. 1Jeltsch A. Chembiochem. 2002; 3: 274-293Crossref PubMed Google Scholar, 22Bestor T.H. Verdine G.L. Curr. Opin. Cell Biol. 1994; 6: 380-389Crossref PubMed Scopus (240) Google Scholar, 23Cheng X. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 293-318Crossref PubMed Scopus (287) Google Scholar). Nevertheless, despite considerable efforts no catalytic activity of Dnmt2 could be detected so far (10Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 17Yoder J.A. Bestor T.H. Hum. Mol. Genet. 1998; 7: 279-284Crossref PubMed Scopus (226) Google Scholar, 18Van den Wyngaert I. Sprengel J. Kass S.U. Luyten W.H. FEBS Lett. 1998; 426: 283-289Crossref PubMed Scopus (42) Google Scholar, 21Dong A. Yoder J.A. Zhang X. Zhou L. Bestor T.H. Cheng X. Nucleic Acids Res. 2001; 29: 439-448Crossref PubMed Scopus (195) Google Scholar). In addition, Dnmt2-deficient embryonic stem cells are viable and do not show any obvious difference in the DNA methylation pattern (10Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar) and Dnmt2 knock-out animals are viable and fertile with minor defects. 2E. Li, personal communication. Cloning, Site-directed Mutagenesis, and Protein Purification— Dnmt2 cDNA was obtained from the Deutsches Ressourcenzentrum für Genomforschung GmbH (Berlin, Germany) (clone number IMAGp998C184250Q2) amplified by PCR and cloned into pET28a+ (Novagen). A Dnmt2 variant in which the highly conserved active site residues Cys-79 and Glu-119 were exchanged by Ala was prepared by site-directed mutagenesis following standard procedures (24Jeltsch A. Lanio T. Methods Mol. Biol. 2002; 182: 85-94PubMed Google Scholar). The sequence of all of the expression constructs was verified by DNA sequencing. Dnmt2 and the Dnmt2 mutant were expressed in BL21 Rosetta(DE3) pLysS cells (Novagen) and purified to homogeneity by nickel-nitrilotriacetic acid affinity chromatography following a procedure described for Dnmt3a with the exception that no protease inhibitors were added (12Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (193) Google Scholar). A MALDI-TOF mass spectrometry analysis revealed masses of the M + H+ ions of 46631.0 and 46537.1 Da for Dnmt2 (Fig. 1B) and the Dnmt2 mutant (data not shown), respectively. These values are within experimental accuracy identical to the corresponding theoretical masses (46629.7 and 46538.6 Da) calculated after removal of the initial Met residue. A mock Dnmt2 preparation was carried out using BL21 Rosetta(DE3) pLysS cells containing the pET28a + vector without the Dnmt2 insert. Mass Spectrometry—Molecular masses were determined by MALDI-TOF mass spectrometry using a Vision 2000 mass spectrometer (Finnegan MAT, Bremen, Germany). The protein samples were de-salted using Centricon-30 concentrators (Amicon, Beverly, MA). 1 μl of protein solution was mixed with 1 μl of matrix solution (9 mg of 2,5-dihydroxybenzoic acid and 1 mg of 2-hydroxy-5-methoxybenzoic acid/ml, 0.1% (v/v) TFA, 30% (v/v) acetonitrile) and allowed to air-dry. Ions were generated by irradiation with a pulsed nitrogen laser (emission wavelength: 337 nm; laser power density: ∼106 watts cm–2), and positive ions were accelerated and detected in the reflector mode. Spectra were calibrated using bovine serum albumin (Sigma) as external standard. DNA and Oligodeoxynucleotides—Purified oligodeoxynucleotides were purchased from MWG-Biotech (Ebersberg, Germany). DNA methylation experiments were carried out with a 558-mer PCR fragment derived from pAT153 that contains 34 CG sites. The fragment was produced by PCR and purified over PCR spin columns (Qiagen), and its concentration was determined from A 260 nm. DNA Methylation Reactions—For the DNA methylation reactions, 1–2 μg of DNA was incubated with 10 μm Dnmt2 in 40 μl of methylation buffer (20 mm HEPES, pH 7.5, 1 mm EDTA) containing 1 mm AdoMet (Sigma) for at the methylation reactions, DNA was purified by or by over PCR spin columns a the DNA was incubated at the with the of a Dnmt2 mock positive the DNA was using of for 1 at of of the was carried out as described (19Gowher H. Leismann O. Jeltsch A. EMBO J. 2000; 19: 6918-6923Crossref PubMed Scopus (168) Google Scholar, A. F. M. M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Jeltsch A. Lett. 2001; PubMed Google Scholar). The DNA was to using μg of μg of and of The S. was by G. für Germany). It is also as from μg of the DNA was to with buffer A mm pH were at a of at using a of A and mm pH 30% buffer buffer 40 buffer and buffer B. a containing was corresponding to or were or were for during the The were in in and and in μl of of the in the and analysis were carried out as described (19Gowher H. Leismann O. Jeltsch A. EMBO J. 2000; 19: 6918-6923Crossref PubMed Scopus (168) Google Scholar, H. Jeltsch A. Lett. 2001; PubMed Google Scholar). μl of the samples were incubated with deoxynucleoside by H. für Germany) in buffer mm pH mm containing μl of for at μl of the were to Germany) at The was in The was in and in the with The was and the was using an was carried out as described C. E. A. G. Nucleic Acids Res. 2001; 29: PubMed Scopus Google Scholar, C. J. C. 2002; PubMed Scopus Google Scholar). methylated or DNA (20 was incubated for at and were added to of and and the was incubated for at The DNA was purified over PCR spin columns and incubated with for at The solution was by the of pH and with The of the DNA was amplified by PCR using and cloned using for of the DNA incubated with Dnmt2 and of the control DNA were We have cloned human Dnmt2 into a expression vector and expressed and purified the protein to homogeneity (Fig. A MALDI-TOF mass spectrometry analysis the mass of the protein within the experimental mass of M + 46631.0 theoretical Da) (Fig. were detected at This shows that the purified protein not covalently or DNA. The protein was incubated with DNA generated by PCR in the of cofactor as as in the presence of AdoMet or and on However, no covalent complex with DNA could be detected (data not This suggests that the covalent complex reported (21Dong A. Yoder J.A. Zhang X. Zhou L. Bestor T.H. Cheng X. Nucleic Acids Res. 2001; 29: 439-448Crossref PubMed Scopus (195) Google Scholar) only with DNA sequences. catalytic activity of Dnmt2 was using or a PCR fragment derived from pAT153 and In addition, protection were using to methylation of However, in with (10Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 17Yoder J.A. Bestor T.H. Hum. Mol. Genet. 1998; 7: 279-284Crossref PubMed Scopus (226) Google Scholar, 18Van den Wyngaert I. Sprengel J. Kass S.U. Luyten W.H. FEBS Lett. 1998; 426: 283-289Crossref PubMed Scopus (42) Google Scholar, 21Dong A. Yoder J.A. Zhang X. Zhou L. Bestor T.H. Cheng X. Nucleic Acids Res. 2001; 29: 439-448Crossref PubMed Scopus (195) Google no catalytic activity could be have used purified Dnmt2 at high in these this that the activity of Dnmt2 is than that of Dnmt1 M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (200) Google Scholar) or Dnmt3a or Dnmt3b (10Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 12Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (193) Google Scholar). However, are for this because the of is and many DNA MTases cofactor after cofactor with the in high of the purified are which is low are to be In addition, the that be from protection experiments are because methylation only be detected the sequence of the with that of the MTase. have also the activity of Dnmt2 using an assay that is very and (19Gowher H. Leismann O. Jeltsch A. EMBO J. 2000; 19: 6918-6923Crossref PubMed Scopus (168) Google Scholar, H. Jeltsch A. Lett. 2001; PubMed Google Scholar). DNA was methylated in and to and the were by The were with deoxynucleoside using and the of was by with methylated from the after (Fig. The of the from the was by at 260 nm. was with DNA after with Dnmt2 and AdoMet, that the of methylation was the from the the of the was and the were using and to (Fig. The of the on the is in using DNA. However, also in the DNA incubated with Dnmt2, a clear was detected (Fig. The in the in to and which in the of the of the of the DNA. A is not because the has a preference for M. B. J. A. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). was detected in DNA not incubated with a DNA MTase (data not of 1 μg of DNA was incubated with DNA MTase Dnmt2 or Dnmt2 mock preparation to and to were and with dNK, and by The of the and the as in A were determined in independent experiments (data not out that the methylation be to a of the Dnmt2 Dnmt2 was expressed in cells that do not contain a DNA-(Cytosine-C5) MTase. In addition, a control was carried out using the cells containing the vector without Dnmt2 insert. were and as the expression and the was to affinity chromatography the as the Dnmt2 The DNA was incubated with the of the mock as used in the Dnmt2 methylation experiments and to the assay procedure as the DNA incubated with However, no was (Fig. that the methylation activity is attributed to the Dnmt2 the of the in an to the of the from the in the was observed and as from the of the with the DNA. the of in the have added of to the samples and the of and the of these the of in the DNA could be a of of as with as observed in human DNA from a Dnmt2 variant was prepared in which the highly conserved active site residues Cys-79 and Glu-119 were exchanged by The variant was and its was by MALDI-TOF mass spectrometry mass of M + 46537.1 theoretical 46538.6 of the DNA with the active site a of was detected (Fig. A analysis revealed that the of obtained after with the Dnmt2 variant was than the obtained with This that the active site residues of Dnmt2 are important for the that Dnmt2 is an active DNA MTase. in the Dnmt2 variant the two most important catalytic residues are one have a in activity. However, obtained similar with the Dnmt3a MTase where also activity was after the removal of these residues S. Li F. Gowher H. Jeltsch A. J. Mol. Biol. PubMed Scopus Google Scholar). This that in to the catalytic DNA MTases also have other to the methylation such as of the and the cofactor and to the of the catalytic activity of Dnmt2 with an independent DNA was incubated with Dnmt2 in and to M. F. S. A. 1992; PubMed Scopus Google Scholar, J. M. Nucleic Acids Res. 1994; 22: PubMed Scopus Google Scholar). This procedure all of the residues to to after by methylated cytosines are to the DNA was and were a of DNA incubated with the Dnmt2 mock preparation were In the residues were detected in to in the control DNA However, because of in the PCR of the not all of these cytosines to methylated cytosines in the DNA. of the cytosines detected in the control DNA and in the DNA were observed at corresponding to a in the DNA. these cytosines have been by which be because be used to DNA C. J. C. 2002; PubMed Scopus Google Scholar). In addition, three cytosines in the control DNA at corresponding to in the DNA. the control was not be the of during or after of the to However, the of cytosines observed at the of or in the DNA is highly in the control DNA only three cytosines were observed at corresponding to a in the DNA. In the cytosines were found at such sites. This of cytosines found at the of found at the of for DNA but for the control be to and is a strong that Dnmt2 is an active DNA MTase. observed residues in the the of be to be on the of the to has been from the of the of the methylation in a We have the sequences the where methylation has been observed in the in the control three cytosines were observed that from or of the to three or four cytosines of the DNA also could be the of this one a The most of the of the of and from a was at with to the where a is highly This shows that Dnmt2, similar to all of the other mammalian DNA methyltransferases identified so methylation of CG sites. all of the other sequence are one an of in as the following be and a preference for is no preference for any is and at A is most and A are a consensus sequence of ttnCGga(g/a) be derived for Dnmt2 from It is to that Dnmt2 recognize its CG site in a sequence However, the number of methylated sequences so far a on the of Dnmt2, and the consensus sequence be and as a of sequence for DNA methylation by Dnmt2 have a of for many species including man and in human genes have been found B. C. J. M. 2001; PubMed Scopus Google Scholar, Li P.W. R.J. G.G. M. 2001; PubMed Scopus Google Scholar). However, in many the functions of the genes identified are of the proteins in the S. genome not show clear homology to any protein of known of all for the functions of proteins be derived on the of amino acid sequence homology G. A. C. R. Genet. Full Text PDF PubMed Scopus Google Scholar, B. Genet. Full Text PDF PubMed Scopus Google Scholar). of a human protein to the is Dnmt2, which shows clear amino acid sequence to DNA-(Cytosine-C5) MTases and also has a structure the structure of enzymes from this Nevertheless, catalytic activity of Dnmt2 could not been detected by the function of Dnmt2 as DNA MTases has been a assay that is very and show here that Dnmt2 is an active DNA MTase. low in activity the to detect activity in and in part the weak observed after of Dnmt2 in many of DNA by Dnmt2 and analysis of the could a loose consensus sequence for Dnmt2 that Dnmt2 also DNA at CG residues similar to all of the other mammalian DNA MTases identified so the other MTases for the function of Dnmt2, which also could the weak knock-out The consensus sequence out that the sites as by the that this sequence has been reported for a mutant of the S. pombe Dnmt2 E. J. J. Mol. Biol. PubMed Scopus Google Scholar) and by the observation that methylation has been detected in M. S. A. 2001; PubMed Scopus Google and However, is the sequence for the E. which have the methylation activity observed in the protein preparation of the The methylation identified in human DNA could be attributed to other Dnmt3a or which also DNA at sites (12Gowher H. Jeltsch A. J. Mol. Biol. 2001; 309: 1201-1208Crossref PubMed Scopus (193) Google Scholar, A. I. J. T. T. H. S. Nucleic Acids Res. 2001; 29: PubMed Scopus Google Scholar, Ramsahoye B.H. M. Li E. 2002; PubMed Scopus Google Scholar). The consensus sequence derived from the in methylation with the site of which have important in regulation of gene expression in and with many other proteins in regulation (for review see Nat. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). it has been that CG methylation DNA by A. H. Mol. Cell. Biol. PubMed Scopus Google Scholar, Y. J. A. M. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google the consensus sequence suggests that Dnmt2 could be in the regulation of DNA and by an Dnmt2 has a more specialized role in vivo than the other known mammalian DNA MTases. it be that Dnmt2 is the only mammalian DNA MTase that not have a large N-terminal which is in the targeting and regulation of the MTases. It is to that Dnmt1, which is highly active in is not active after removal of the N-terminal part M. Hermann A. Pradhan S. Jeltsch A. J. Mol. Biol. 2001; 309: 1189-1199Crossref PubMed Scopus (200) Google Scholar). the catalytic of Dnmt1 on by the N-terminal It is that an protein for Dnmt2 is in certain tissues where it could the role of the N-terminal part in Dnmt1 and the activity of However, it be that the catalytic of Dnmt3a and Dnmt3b are active in the of the N-terminal of the proteins H. Jeltsch A. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google that not all of the mammalian MTases an N-terminal part for activity. We Li, and Cheng for of to by B. is with