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Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells

Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells 2536–2540 Nucleic Acids Research, 1998, Vol. 26, No. 11  1998 Oxford University Press Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells Masaki Okano, Shaoping Xie and En Li* Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, 149, 13th Street, Charlestown, MA 02129, USA Received March 11, 1998; Revised and Accepted April 15, 1998 DDBJ/EMBL/GenBank accession nos AF045888, AF045889 ABSTRACT and tissue specific DNA methylation patterns are established during development are poorly understood. The major obstacle We have shown previously that de novo methylation has been the lack of information about the enzymes that catalyze activities persist in mouse embryonic stem (ES) cells de novo methylation and demethylation (11). The enzyme homozygous for a null mutation of Dnmt1 that encodes encoded by Dnmt1 functions primarily as a maintenance the major DNA cytosine methyltransferase. In this methyltransferase which transfers methyl groups to cytosine in study, we have cloned a putative mammalian DNA hemi-methylated CpG sites after DNA replication (12). Although methyltransferase gene, termed Dnmt2, that is homo- this enzyme can also methylate unmethylated DNA in vitro, no logous to pmt1 of fission yeast. Different from pmt1 in evidence has been established so far for its role as a de novo which the catalytic Pro-Pro-Cys (PPC) motif is ‘mu- methyltransferase in vivo. Recently, we showed that ES cells tated’ to Pro-Ser-Cys, Dnmt2 contains all the con- homozygous for a null mutation of Dnmt1 contained residual served methyltransferase motifs, thus likely encoding levels of methyl cytosine and retained the ability to methylate a functional cytosine methyltransferase. However, provirus DNA de novo (2). This result provides the first genetic baculovirus-expressed Dnmt2 protein failed to methy- evidence for the existence of an independently encoded de novo late DNA in vitro. To investigate whether Dnmt2 DNA methyltransferase in mammalian cells. functions as a DNA methyltransferase in vivo, we In this study, we report the cloning of a mammalian gene Dnmt2 inactivated the Dnmt2 gene by targeted deletion of the that shares homology with the pmt1 gene of fission yeast (13), and putative catalytic PPC motif in ES cells. We showed encodes a protein which contains all the conserved methyltrans- that endogenous virus was fully methylated in ferase motifs. We provide genetic evidence that Dnmt2 is not Dnmt2-deficient mutant ES cells. Furthermore, newly essential for maintenance methylation nor for de novo methyla- integrated retrovirus DNA was methylated de novo in tion of viral DNA in ES cells. infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA MATERIALS AND METHODS in ES cells. Cloning of the mammalian Dnmt2 gene INTRODUCTION A search of the dbEST database was performed with the DNA methylation at the C-5 position of cytosine in CpG TBLASTN program (14) using bacterial cytosine methyltrans- dinucleotides is the major form of DNA modification in ferases as queries. Two human EST clones (GenBank accession vertebrate animals. DNA methylation has been shown to be nos N31314 and R95731) were found to match the M.HgiGI essential for mammalian development as inactivation of Dnmt1, sequences. The clones were obtained from American Type a major maintenance DNA cytosine methyltransferase, results in Culture Collection (ATCC, MD) and sequenced by the MGH genome-wide demethylation and embryonic lethality (1,2). The sequencing core facility. The deduced amino acid sequences of function of DNA methylation has been implicated in a diverse the clones share a significant homology with the yeast pmt1 (13). range of biological processes. Molecular and genetic studies have The insert DNA of these clones were cut out by EcoRI/NotI demonstrated that DNA methylation plays critical roles in digestion and used as probes for screening cDNA libraries. regulation of parent-origin-specific expression of imprinted Nine positive clones were obtained by screening a mouse ES genes (3–5) and X chromosome inactivation (6,7). Recently, cell cDNA library (Clontech, CA) and sequenced. Two of them growing evidence also suggests that DNA methylation is contained uninterrupted ORFs corresponding to the entire ORF involved in carcinogenesis (8–10). of pmt1, but lacked a stop codon upstream of the first ATG. The While the function of DNA methylation has been studied human cDNA clones were obtained by screening a human heart extensively, mechanisms by which DNA methylation is regulated cDNA library (Clontech, CA). Of 14 positive clones, one showed *To whom correspondence should be addressed. Tel: +1 617 726 4345; Fax: +1 617 726 5806; Email: [email protected] 2537 Nucleic Acids Research, 1998, Vol. 26, No. 11 2537 Nucleic Acids Research, 1994, Vol. 22, No. 1 a continuous ORF with a stop codon upstream of the putative initiation codon. RNA preparation and northern analysis Total RNA was prepared from ES cells, ovary and testis using the GTC-CsCl centrifugation method (15), fractionated on a formaldehyde denatured 1% agarose gel by electrophoresis, and transferred to nylon membranes. A poly A+ RNA blot of mouse tissues was obtained from Clontech, CA. All blots were hybridized with a random-primed 540 bp EcoRI–PstI cDNA fragment of the mouse Dnmt2 in a standard hybridization solution containing 50% formamide at 42C, washed with 0.2× SSC, 0.1% SDS at 65C, and exposed to X-ray film. Construction of the gene targeting vector A 14 kb SmaI–XhoI genomic DNA fragment of the Dnmt2 gene was isolated from a 129/Sv genomic DNA library and subcloned into the pBluescript vector. A 1 kb StuI–SnaBI genomic DNA fragment containing exons encoding the putative catalytic domain (the PPC motif) was removed and replaced by the IRES-βgeo cassette with a splicing accept site (16). The resulting gene targeting vector contains a 9.2 kb fragment upstream and a 3.7 kb fragment downstream of the IRES-βgeo cassette (Fig. 3). Generation of Dnmt2-deficient mutant ES cell lines Transfection of J1 ES cells with linearized targeting vector DNA and subsequent G418 selection and cloning of drug-resistant colonies were carried out as described previously (1). Genomic Figure 1. The comparison of the deduced amino acid sequences between the DNA from G418-resistant clones was digested with BamHI and mammalian Dnmt2 and the yeast pmt1. The identical amino acids are analyzed by Southern blot hybridization using the probe pXhR5 shadowed. The conserved DNA methyltransferase motifs (I–X) are marked (Fig. 3). To generate ES cell lines homozygous for the mutation, with roman numerals. The stop codons are indicated with asterisks (*) and gaps with dots (...). cells of a heterozygous clone were subject to selection in medium containing a high concentration of G418 (0.5 mg/ml of pure form) (17). cDNA libraries using the EST clones as probes, and a full length Analysis of de novo and maintenance methylation of cDNA was constructed with overlapping cDNA fragments after provirus DNA DNA sequencing. The deduced amino acid sequences of the human and mouse cDNA showed 81% identity and revealed that Infection of wild-type and Dnmt2-deficient ES cells with the both genes contained all the conserved cytosine methyltransfer- sup MoMuLV -1 virus, DNA preparation from infected ES cells, ase motifs (Fig. 1) (18). The same gene was independently cloned analysis of de novo and maintenance methylation of endogenous by Yoder and Bestor, and was named Dnmt2 (19). A BLAST or newly integrated viral DNA by Southern analysis of HpaII/ search of GenBank with human and mouse cDNA sequences MspI digested DNA were carried out as described previously (2). identified pmt1 of fission yeast Schizosaccharomyces pombe as the most closely related sequences, sharing 42% identity at the RESULTS AND DISCUSSION amino acid level. The yeast pmt1 contains all other conserved methyltransferase motifs except that the catalytic Pro-Pro-Cys Cloning of the mammalian homologs of the yeast pmt1 motif was ‘mutated’ to Pro-Ser-Cys (13). gene To determine whether Dnmt2 has methyltransferase activity, One of the approaches that we took in search of a de novo DNA the mouse cDNA was expressed in Escherichia coli or in insect methyltransferase in mammalian cells was to screen the dbEST cells using the baculovirus expression system. Methyltransferase database using the amino acid sequences of different prokaryotic activity assay was carried out using either poly(dI-dC) or λ phage methyltransferases as query sequences (14). When sequences of DNA as substrates under a standard assay condition which could the bacterial restriction methyltransferase M.HgiGI were used as detect residual levels of enzyme activity in protein extracts query sequences, two EST clones of the same gene (GenBank prepared from the Dnmt1 null mutant ES cells (2). Despite the accession nos N31314 and R95731) were found to give presence of large amounts of Dnmt2 protein in both bacterial and significant matches. Sequencing analysis of the EST clones insect cell extracts, no methyltransferase activities were detected revealed that they contained three of the highly conserved so far (data not shown). At the moment, it is not clear why the methyltransferase motifs. Multiple cDNA clones of the gene recombinant proteins have no detectable activities. The following were isolated subsequently by screening human and mouse two possibilities are considered: (i) the recombinant Dnmt2 2538 Nucleic Acids Research, 1998, Vol. 26, No. 11 Dnmt2-deficient ES cells are viable To investigate the role of Dnmt2 in development, we generated a m1 putative null allele of Dnmt2, termed Dnmt2 , by deletion of the exons encoding the putative catalytic PPC motif through homologous recombination in ES cells (Fig. 3A). Of 85 G418-resistant colonies analyzed by Southern blot hybridization, six were positive for homologous recombination (Fig. 3B). To Figure 2. Dnmt2 expression in organs and ES cells. A blot with 2 μg of poly A+ RNA from mouse tissues (Clontech, CA) is shown on the left, and a blot generate ES cell lines homozygous for the mutation, cells of a with 20 μg of total RNA from ES cells, ovary and testis is shown on the right. heterozygous ES cell line were cultured in medium containing a He, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Mu, skeletal muscle; Ki, high concentration of G418 (0.5 mg/ml) for 14 days. Of 29 kidney; Te, testis; ES, ES cells; and Ov, ovary. Note that three Dnmt2 transcripts colonies analyzed, two were homozygous for the mutant allele of sizes 1.6, 2.6 and 4.0 kb were detected in mouse tissues, and the 1.6 kb transcript was the most abundant one in the organs examined. (Fig. 3C). The Dnmt2 homozygous ES cells appeared to be normal in growth and morphology after consecutive passaging for more than 20 generations (data not shown), suggesting that protein may lack the natural conformation or modification, or is Dnmt2 function is not essential. very unstable in vitro; (ii) Dnmt2 may require cofactors to catalyze methylation reaction. Further studies are necessary to De novo and maintenance methylation of provirus investigate these possibilities. m1 m1 DNA in Dnmt2 /Dnmt2 ES cells Since Dnmt2 transcripts were detected in ES cells, we speculated Dnmt2 expression in mouse organs and ES cells that Dnmt2 might be required for de novo methylation. We Dnmt2 expression in mouse ES cell lines and various organs were showed previously that ES cells homozygous for a Dnmt1 null analyzed by northern hybridization using a full length cDNA mutation were able to methylate provirus DNA de novo. We fragment as probes. We showed that three Dnmt2 transcripts of carried out a similar analysis of de novo methylation of integrated 1.6, 2.6 and 4.0 kb were detected in mouse tissues, and the 1.6 kb provirus DNA in infected Dnmt2 mutant ES cells. transcript was the most abundant one in most tissues examined First, we examined methylation status of endogenous virus in m1 m1 (Fig. 2). Dnmt2 appeared to express ubiquitously but at very low Dnmt2 /Dnmt2 ES. DNA isolated from wild-type and m1 m1 levels in mouse tissues, with relatively high levels in the heart, Dnmt2 /Dnmt2 ES cells was digested with the methylation- lung, kidney and testis (Fig. 2). Dnmt2 expression was also sensitive restriction enzyme HpaII or its isoschizomer MspI that detected in mouse ES cells (Fig. 2), suggesting that Dnmt2 might cuts CCGG sequences regardless of whether CpG sites are be responsible for the residual methyltransferase activity detected methylated or not, and was then subject to Southern blot in Dnmt1 null ES cells. hybridization with a MoMuLV cDNA probe that hybridizes with Figure 3. Targeted disruption of the Dnmt2 gene. (A) The wild-type Dnmt2 genomic locus (top), the targeting vector (middle), and the targeted allele (bottom). The location of the exons (solid bars), PC motif, ENV motif and the IRES-βgeo cassette are shown. The 1.3 kb XhoI–EcoRV genomic fragment was used as a probe for Southern analysis, and the 10.6 and 9.0 kb BamHI fragments from wild-type and targeted alleles, respectively, are indicated as dashed lines. Sm, SmaI; B, BamHI; Xh, XhoI; St, StuI; Sn, SnaBI; Rv, EcoRV; SA, splicing acceptor; and pA, poly (A) signal. (B) Southern blot hybridization of genomic DNA from wild-type and targeted ES cell clones. DNA was digested with BamHI, blotted and hybridized to the probe shown in (A). (C) Southern analysis of genomic DNA from a heterozygous and two homozygous mutant ES cell clones. 2539 Nucleic Acids Research, 1998, Vol. 26, No. 11 2539 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 4. Methylation of endogenous provirus DNA in the Dnmt2 null mutant ES cells. Genomic DNA was isolated from ES cells, digested with HpaII (H) or MspI (M), blotted and hybridized to the MoMuLV cDNA probe (1). +/– and m1 m1 m1 –/– are Dnmt2 /+ and Dnmt2 /Dnmt2 cells while n/n and c/c are n n c c Dnmt1 /Dnmt1 and Dnmt1 /Dnmt1 ES cells, respectively (2). endogenous provirus DNA (1). We showed that endogenous virus m1 m1 DNA in Dnmt2 /Dnmt2 ES cells was methylated to the same levels as in wild-type cells (Fig. 4). This result indicates that Dnmt2 is not required for the maintenance methylation of genomic DNA. m1 m1 To examine whether Dnmt2 /Dnmt2 ES cells were able to methylate foreign DNA such as newly integrated provirus DNA, we sup Figure 5. De novo methylation of provirus DNA in Dnmt2 mutant ES cells. infected Dnmt2 mutant ES cells with the MoMuLV -1 retrovirus sup (A) Schematic diagrams of the MoMuLV -1 provirus genome (top), the 3′ and analyzed the methylation status of newly integrated provirus LTR region (middle), the size marker, the location of the πAN7 probe and the DNA 2–4 days after infection. DNA was digested with KpnI and five HpaII/MspI sites (bottom) (2). (B) Genomic DNA was isolated from infected 3T3 cells (lanes 1–3), infected wild-type (lanes 4–6 and 9–11), HpaII, or with KpnI and MspI as controls, and analyzed by Southern uninfected wild-type (lanes 7 and 8), infected heterozygous mutant (lanes 12 blot hybridization using the πAN7 probe that would recognize a and 13) and infected homozygous mutant (lanes 14 and 15) at day 0 (lane 9), 1.45 kb KpnI fragment of infected viral DNA but not the day 2 (lanes 10, 12 and 14 ) and day 4 (lanes 4–6, 11, 13 and 15) post-infection. endogenous proviruses (Fig. 5A). We found that the newly DNA was digested with MspI/KpnI (lanes 1, 4 and 7), HpaII/KpnI (lanes 2, 5 m1 m1 integrated virus DNA was methylated in Dnmt2 /Dnmt2 ES and 8–15), or KpnI alone (lanes 3 and 6), blotted and hybridized to the πAN7 sup probe. Mov, MoMuLV -1 virus infected; M, MspI; H, HpaII; K, KpnI. cells as efficiently as in wild-type cells as shown by the presence of an HpaII-resistant 1.45 kb fragment (Fig. 5B), indicating that Dnmt2 is not an essential component of the de novo methyltransferases. The lack of detectable methyltransferase activities in vitro and in vivo raises interesting possibilities that Dnmt2 might encode a sequence-specific DNA methyltransferase which methylates contains all the conserved methyltransferase motifs except that only a small number of target sequences in the genome, or it may motif VI has an EET sequence rather than the ENV sequence that methylate cytosine in non-CpG sequences such as CpNpG. It is is conserved in almost all the known DNA cytosine methyltrans- also possible that Dnmt2 is simply not a functional cytosine DNA ferases. The methyltransferase activity of masc1 encoded proteins methyltransferase, despite having all the conserved DNA methyl- has not been reported. Sequence analysis indicates that masc1 is transferase motifs. Dnmt2 may be involved in cellular processes distantly related to Dnmt1 and Dnmt2 (data not shown). It remains to be seen whether a mammalian homologue of masc1 exists, and other than DNA methylation, such as DNA repair by binding to whether it functions as a de novo DNA methyltransferase. mismatched nucleotides as the bacterial cytosine methyltransfer- ases (21–23), DNA recombination and carcinogenesis. Since Dnmt2 is not essential for de novo methylation in ES ACKNOWLEDGEMENTS cells, additional DNA methyltransferases that catalyze de novo methylation are predicted to be present in mammalian cells. It is We thank Dr Austin Smith for the plasmid GT1.8Iresβgeo(Sal), formally possible that both Dnmt1 and Dnmt2 are de novo Lian Yu for excellent technical assistance, and members of our methyltransferases and can functionally compensate each other. laboratory for discussion. This work was supported by grants Recently, a gene known as masc1 was cloned through homology- from Bristol-Myers/Squibb and NIH (GM52106 to E.L.). M.O. based screening using a PCR amplification method, and genetic was a fellow of the Japanese Society for the Promotion of analysis has revealed that masc1 is involved in de novo Science. methylation in Ascobolus (24). The protein encoded by masc1 2540 Nucleic Acids Research, 1998, Vol. 26, No. 11 13 Wilkinson, C. R., Bartlett, R., Nurse, P. and Bird, A. P. (1995) Nucleic REFERENCES Acids Res., 23, 203–210. 14 Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. 1 Li, E., Bestor, T. H. and Jaenisch, R. (1992) Cell, 69, 915–926. (1990) J. Mol. Biol., 215, 403–410. 2 Lei, H., Oh, S. P., Okano, M., Juttermann, R., Goss, K. A., Jaenisch, R. 15 Chomzynski, P. and Sacchi, N. (1991) Anal. Biochem., 162, 156–159. and Li, E. (1996) Development, 122, 3195–3205. 16 Mountford, P., Zevnik, B., Duwel, A., Nichols, J., Li, M., Dani, C., 3 Li, E., Beard, C. and Jaenisch, R. (1993) Nature, 366, 362–365. Robertson, M., Chambers, I. and Smith, A. (1994) Proc. Natl. Acad. Sci. 4 Bartolomei, M. S. (1997) In Reik, W. and Sorani, A. (eds), Genomic USA, 91, 4303–4307. Imprinting: Frontiers in Molecular Biology. IRL Press, Oxford, pp. 53–69. 17 Mortensen, R. M., Conner, D. A., Chao, S., Geisterfer-Lowrance, A. A. 5 Neumann, B. and Barlow, D. P. (1996) Curr. Opin. Genet. Dev., 6, and Seidman, J. G. (1992) Mol. Cell. Biol., 12, 2391–2395. 159–163. 18 Kumar, S., Cheng, X., Klimasauskas, S., Mi, S., Posfai, J., Roberts, R. and 6 Monk, M. (1995) Dev. Genet., 17, 188–197. Wilson, G. G. (1994) Nucleic Acids Res., 22, 1–10. 7 Brockdoff, N. (1997) In Reik, W. and Sorani, A. (eds), Genomic 19 Yoder, J. A. and Bestor, T. H. (1998) Hum. Mol. Genet., 7, 279–284. Imprinting: Frontiers in Molecular Biology. IRL Press, Oxford, pp. 20 Pinarbasi, E., Elliott, J. and Hornby, D. P. (1996) J. Mol. Biol., 257, 191–210. 804–813. 8 Laird, P. W. and Jaenisch, R. (1996) Annu. Rev. Genet., 30, 441–464. 21 Klimasauskas, S. and Roberts, R. J. (1995). Nucleic Acids Res., 23, 9 Baylin, S. B., Herman, J. G., Graff, J. R., Vertino, P. M. and Issa, J. P. 1388–1395. (1998) Adv. Cancer Res., 72, 141–196. 22 Yang, A. S., Shen, J. C., Zingg, J. M., Mi, S. and Jones, P. A. (1995) 10 Jones, P. A. and Gonzalgo, M. L. (1997) Proc. Natl. Acad. Sci. USA, 94, Nucleic Acids Res., 23, 1380–1387. 2103–2105. 23 Renbaum, P. and Razin, A. (1995) Gene, 157, 177–179. 11 Li, E. (1997) In Reik, W. and Sorani, A. (eds), Genomic Imprinting: 24 Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., Rossignol, Frontiers in Molecular Biology. IRL Press, Oxford, pp. 1–20. J. L., Noyer-Weidner, M., Vollmayr, P., Trautner, T. A. and Walter, J. 12 Bestor, T. H. (1992) EMBO J., 11, 2611–2617. (1997) Cell, 91, 281–290. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells

Okano, Masaki; Xie, Shaoping; Li, En
Nucleic Acids Research , Volume 26 (11) – Jun 1, 1998
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Oxford University Press
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© 1998 Oxford University Press
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0305-1048
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10.1093/nar/26.11.2536
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2536–2540 Nucleic Acids Research, 1998, Vol. 26, No. 11  1998 Oxford University Press Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells Masaki Okano, Shaoping Xie and En Li* Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, 149, 13th Street, Charlestown, MA 02129, USA Received March 11, 1998; Revised and Accepted April 15, 1998 DDBJ/EMBL/GenBank accession nos AF045888, AF045889 ABSTRACT and tissue specific DNA methylation patterns are established during development are poorly understood. The major obstacle We have shown previously that de novo methylation has been the lack of information about the enzymes that catalyze activities persist in mouse embryonic stem (ES) cells de novo methylation and demethylation (11). The enzyme homozygous for a null mutation of Dnmt1 that encodes encoded by Dnmt1 functions primarily as a maintenance the major DNA cytosine methyltransferase. In this methyltransferase which transfers methyl groups to cytosine in study, we have cloned a putative mammalian DNA hemi-methylated CpG sites after DNA replication (12). Although methyltransferase gene, termed Dnmt2, that is homo- this enzyme can also methylate unmethylated DNA in vitro, no logous to pmt1 of fission yeast. Different from pmt1 in evidence has been established so far for its role as a de novo which the catalytic Pro-Pro-Cys (PPC) motif is ‘mu- methyltransferase in vivo. Recently, we showed that ES cells tated’ to Pro-Ser-Cys, Dnmt2 contains all the con- homozygous for a null mutation of Dnmt1 contained residual served methyltransferase motifs, thus likely encoding levels of methyl cytosine and retained the ability to methylate a functional cytosine methyltransferase. However, provirus DNA de novo (2). This result provides the first genetic baculovirus-expressed Dnmt2 protein failed to methy- evidence for the existence of an independently encoded de novo late DNA in vitro. To investigate whether Dnmt2 DNA methyltransferase in mammalian cells. functions as a DNA methyltransferase in vivo, we In this study, we report the cloning of a mammalian gene Dnmt2 inactivated the Dnmt2 gene by targeted deletion of the that shares homology with the pmt1 gene of fission yeast (13), and putative catalytic PPC motif in ES cells. We showed encodes a protein which contains all the conserved methyltrans- that endogenous virus was fully methylated in ferase motifs. We provide genetic evidence that Dnmt2 is not Dnmt2-deficient mutant ES cells. Furthermore, newly essential for maintenance methylation nor for de novo methyla- integrated retrovirus DNA was methylated de novo in tion of viral DNA in ES cells. infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA MATERIALS AND METHODS in ES cells. Cloning of the mammalian Dnmt2 gene INTRODUCTION A search of the dbEST database was performed with the DNA methylation at the C-5 position of cytosine in CpG TBLASTN program (14) using bacterial cytosine methyltrans- dinucleotides is the major form of DNA modification in ferases as queries. Two human EST clones (GenBank accession vertebrate animals. DNA methylation has been shown to be nos N31314 and R95731) were found to match the M.HgiGI essential for mammalian development as inactivation of Dnmt1, sequences. The clones were obtained from American Type a major maintenance DNA cytosine methyltransferase, results in Culture Collection (ATCC, MD) and sequenced by the MGH genome-wide demethylation and embryonic lethality (1,2). The sequencing core facility. The deduced amino acid sequences of function of DNA methylation has been implicated in a diverse the clones share a significant homology with the yeast pmt1 (13). range of biological processes. Molecular and genetic studies have The insert DNA of these clones were cut out by EcoRI/NotI demonstrated that DNA methylation plays critical roles in digestion and used as probes for screening cDNA libraries. regulation of parent-origin-specific expression of imprinted Nine positive clones were obtained by screening a mouse ES genes (3–5) and X chromosome inactivation (6,7). Recently, cell cDNA library (Clontech, CA) and sequenced. Two of them growing evidence also suggests that DNA methylation is contained uninterrupted ORFs corresponding to the entire ORF involved in carcinogenesis (8–10). of pmt1, but lacked a stop codon upstream of the first ATG. The While the function of DNA methylation has been studied human cDNA clones were obtained by screening a human heart extensively, mechanisms by which DNA methylation is regulated cDNA library (Clontech, CA). Of 14 positive clones, one showed *To whom correspondence should be addressed. Tel: +1 617 726 4345; Fax: +1 617 726 5806; Email: [email protected] 2537 Nucleic Acids Research, 1998, Vol. 26, No. 11 2537 Nucleic Acids Research, 1994, Vol. 22, No. 1 a continuous ORF with a stop codon upstream of the putative initiation codon. RNA preparation and northern analysis Total RNA was prepared from ES cells, ovary and testis using the GTC-CsCl centrifugation method (15), fractionated on a formaldehyde denatured 1% agarose gel by electrophoresis, and transferred to nylon membranes. A poly A+ RNA blot of mouse tissues was obtained from Clontech, CA. All blots were hybridized with a random-primed 540 bp EcoRI–PstI cDNA fragment of the mouse Dnmt2 in a standard hybridization solution containing 50% formamide at 42C, washed with 0.2× SSC, 0.1% SDS at 65C, and exposed to X-ray film. Construction of the gene targeting vector A 14 kb SmaI–XhoI genomic DNA fragment of the Dnmt2 gene was isolated from a 129/Sv genomic DNA library and subcloned into the pBluescript vector. A 1 kb StuI–SnaBI genomic DNA fragment containing exons encoding the putative catalytic domain (the PPC motif) was removed and replaced by the IRES-βgeo cassette with a splicing accept site (16). The resulting gene targeting vector contains a 9.2 kb fragment upstream and a 3.7 kb fragment downstream of the IRES-βgeo cassette (Fig. 3). Generation of Dnmt2-deficient mutant ES cell lines Transfection of J1 ES cells with linearized targeting vector DNA and subsequent G418 selection and cloning of drug-resistant colonies were carried out as described previously (1). Genomic Figure 1. The comparison of the deduced amino acid sequences between the DNA from G418-resistant clones was digested with BamHI and mammalian Dnmt2 and the yeast pmt1. The identical amino acids are analyzed by Southern blot hybridization using the probe pXhR5 shadowed. The conserved DNA methyltransferase motifs (I–X) are marked (Fig. 3). To generate ES cell lines homozygous for the mutation, with roman numerals. The stop codons are indicated with asterisks (*) and gaps with dots (...). cells of a heterozygous clone were subject to selection in medium containing a high concentration of G418 (0.5 mg/ml of pure form) (17). cDNA libraries using the EST clones as probes, and a full length Analysis of de novo and maintenance methylation of cDNA was constructed with overlapping cDNA fragments after provirus DNA DNA sequencing. The deduced amino acid sequences of the human and mouse cDNA showed 81% identity and revealed that Infection of wild-type and Dnmt2-deficient ES cells with the both genes contained all the conserved cytosine methyltransfer- sup MoMuLV -1 virus, DNA preparation from infected ES cells, ase motifs (Fig. 1) (18). The same gene was independently cloned analysis of de novo and maintenance methylation of endogenous by Yoder and Bestor, and was named Dnmt2 (19). A BLAST or newly integrated viral DNA by Southern analysis of HpaII/ search of GenBank with human and mouse cDNA sequences MspI digested DNA were carried out as described previously (2). identified pmt1 of fission yeast Schizosaccharomyces pombe as the most closely related sequences, sharing 42% identity at the RESULTS AND DISCUSSION amino acid level. The yeast pmt1 contains all other conserved methyltransferase motifs except that the catalytic Pro-Pro-Cys Cloning of the mammalian homologs of the yeast pmt1 motif was ‘mutated’ to Pro-Ser-Cys (13). gene To determine whether Dnmt2 has methyltransferase activity, One of the approaches that we took in search of a de novo DNA the mouse cDNA was expressed in Escherichia coli or in insect methyltransferase in mammalian cells was to screen the dbEST cells using the baculovirus expression system. Methyltransferase database using the amino acid sequences of different prokaryotic activity assay was carried out using either poly(dI-dC) or λ phage methyltransferases as query sequences (14). When sequences of DNA as substrates under a standard assay condition which could the bacterial restriction methyltransferase M.HgiGI were used as detect residual levels of enzyme activity in protein extracts query sequences, two EST clones of the same gene (GenBank prepared from the Dnmt1 null mutant ES cells (2). Despite the accession nos N31314 and R95731) were found to give presence of large amounts of Dnmt2 protein in both bacterial and significant matches. Sequencing analysis of the EST clones insect cell extracts, no methyltransferase activities were detected revealed that they contained three of the highly conserved so far (data not shown). At the moment, it is not clear why the methyltransferase motifs. Multiple cDNA clones of the gene recombinant proteins have no detectable activities. The following were isolated subsequently by screening human and mouse two possibilities are considered: (i) the recombinant Dnmt2 2538 Nucleic Acids Research, 1998, Vol. 26, No. 11 Dnmt2-deficient ES cells are viable To investigate the role of Dnmt2 in development, we generated a m1 putative null allele of Dnmt2, termed Dnmt2 , by deletion of the exons encoding the putative catalytic PPC motif through homologous recombination in ES cells (Fig. 3A). Of 85 G418-resistant colonies analyzed by Southern blot hybridization, six were positive for homologous recombination (Fig. 3B). To Figure 2. Dnmt2 expression in organs and ES cells. A blot with 2 μg of poly A+ RNA from mouse tissues (Clontech, CA) is shown on the left, and a blot generate ES cell lines homozygous for the mutation, cells of a with 20 μg of total RNA from ES cells, ovary and testis is shown on the right. heterozygous ES cell line were cultured in medium containing a He, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Mu, skeletal muscle; Ki, high concentration of G418 (0.5 mg/ml) for 14 days. Of 29 kidney; Te, testis; ES, ES cells; and Ov, ovary. Note that three Dnmt2 transcripts colonies analyzed, two were homozygous for the mutant allele of sizes 1.6, 2.6 and 4.0 kb were detected in mouse tissues, and the 1.6 kb transcript was the most abundant one in the organs examined. (Fig. 3C). The Dnmt2 homozygous ES cells appeared to be normal in growth and morphology after consecutive passaging for more than 20 generations (data not shown), suggesting that protein may lack the natural conformation or modification, or is Dnmt2 function is not essential. very unstable in vitro; (ii) Dnmt2 may require cofactors to catalyze methylation reaction. Further studies are necessary to De novo and maintenance methylation of provirus investigate these possibilities. m1 m1 DNA in Dnmt2 /Dnmt2 ES cells Since Dnmt2 transcripts were detected in ES cells, we speculated Dnmt2 expression in mouse organs and ES cells that Dnmt2 might be required for de novo methylation. We Dnmt2 expression in mouse ES cell lines and various organs were showed previously that ES cells homozygous for a Dnmt1 null analyzed by northern hybridization using a full length cDNA mutation were able to methylate provirus DNA de novo. We fragment as probes. We showed that three Dnmt2 transcripts of carried out a similar analysis of de novo methylation of integrated 1.6, 2.6 and 4.0 kb were detected in mouse tissues, and the 1.6 kb provirus DNA in infected Dnmt2 mutant ES cells. transcript was the most abundant one in most tissues examined First, we examined methylation status of endogenous virus in m1 m1 (Fig. 2). Dnmt2 appeared to express ubiquitously but at very low Dnmt2 /Dnmt2 ES. DNA isolated from wild-type and m1 m1 levels in mouse tissues, with relatively high levels in the heart, Dnmt2 /Dnmt2 ES cells was digested with the methylation- lung, kidney and testis (Fig. 2). Dnmt2 expression was also sensitive restriction enzyme HpaII or its isoschizomer MspI that detected in mouse ES cells (Fig. 2), suggesting that Dnmt2 might cuts CCGG sequences regardless of whether CpG sites are be responsible for the residual methyltransferase activity detected methylated or not, and was then subject to Southern blot in Dnmt1 null ES cells. hybridization with a MoMuLV cDNA probe that hybridizes with Figure 3. Targeted disruption of the Dnmt2 gene. (A) The wild-type Dnmt2 genomic locus (top), the targeting vector (middle), and the targeted allele (bottom). The location of the exons (solid bars), PC motif, ENV motif and the IRES-βgeo cassette are shown. The 1.3 kb XhoI–EcoRV genomic fragment was used as a probe for Southern analysis, and the 10.6 and 9.0 kb BamHI fragments from wild-type and targeted alleles, respectively, are indicated as dashed lines. Sm, SmaI; B, BamHI; Xh, XhoI; St, StuI; Sn, SnaBI; Rv, EcoRV; SA, splicing acceptor; and pA, poly (A) signal. (B) Southern blot hybridization of genomic DNA from wild-type and targeted ES cell clones. DNA was digested with BamHI, blotted and hybridized to the probe shown in (A). (C) Southern analysis of genomic DNA from a heterozygous and two homozygous mutant ES cell clones. 2539 Nucleic Acids Research, 1998, Vol. 26, No. 11 2539 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 4. Methylation of endogenous provirus DNA in the Dnmt2 null mutant ES cells. Genomic DNA was isolated from ES cells, digested with HpaII (H) or MspI (M), blotted and hybridized to the MoMuLV cDNA probe (1). +/– and m1 m1 m1 –/– are Dnmt2 /+ and Dnmt2 /Dnmt2 cells while n/n and c/c are n n c c Dnmt1 /Dnmt1 and Dnmt1 /Dnmt1 ES cells, respectively (2). endogenous provirus DNA (1). We showed that endogenous virus m1 m1 DNA in Dnmt2 /Dnmt2 ES cells was methylated to the same levels as in wild-type cells (Fig. 4). This result indicates that Dnmt2 is not required for the maintenance methylation of genomic DNA. m1 m1 To examine whether Dnmt2 /Dnmt2 ES cells were able to methylate foreign DNA such as newly integrated provirus DNA, we sup Figure 5. De novo methylation of provirus DNA in Dnmt2 mutant ES cells. infected Dnmt2 mutant ES cells with the MoMuLV -1 retrovirus sup (A) Schematic diagrams of the MoMuLV -1 provirus genome (top), the 3′ and analyzed the methylation status of newly integrated provirus LTR region (middle), the size marker, the location of the πAN7 probe and the DNA 2–4 days after infection. DNA was digested with KpnI and five HpaII/MspI sites (bottom) (2). (B) Genomic DNA was isolated from infected 3T3 cells (lanes 1–3), infected wild-type (lanes 4–6 and 9–11), HpaII, or with KpnI and MspI as controls, and analyzed by Southern uninfected wild-type (lanes 7 and 8), infected heterozygous mutant (lanes 12 blot hybridization using the πAN7 probe that would recognize a and 13) and infected homozygous mutant (lanes 14 and 15) at day 0 (lane 9), 1.45 kb KpnI fragment of infected viral DNA but not the day 2 (lanes 10, 12 and 14 ) and day 4 (lanes 4–6, 11, 13 and 15) post-infection. endogenous proviruses (Fig. 5A). We found that the newly DNA was digested with MspI/KpnI (lanes 1, 4 and 7), HpaII/KpnI (lanes 2, 5 m1 m1 integrated virus DNA was methylated in Dnmt2 /Dnmt2 ES and 8–15), or KpnI alone (lanes 3 and 6), blotted and hybridized to the πAN7 sup probe. Mov, MoMuLV -1 virus infected; M, MspI; H, HpaII; K, KpnI. cells as efficiently as in wild-type cells as shown by the presence of an HpaII-resistant 1.45 kb fragment (Fig. 5B), indicating that Dnmt2 is not an essential component of the de novo methyltransferases. The lack of detectable methyltransferase activities in vitro and in vivo raises interesting possibilities that Dnmt2 might encode a sequence-specific DNA methyltransferase which methylates contains all the conserved methyltransferase motifs except that only a small number of target sequences in the genome, or it may motif VI has an EET sequence rather than the ENV sequence that methylate cytosine in non-CpG sequences such as CpNpG. It is is conserved in almost all the known DNA cytosine methyltrans- also possible that Dnmt2 is simply not a functional cytosine DNA ferases. The methyltransferase activity of masc1 encoded proteins methyltransferase, despite having all the conserved DNA methyl- has not been reported. Sequence analysis indicates that masc1 is transferase motifs. Dnmt2 may be involved in cellular processes distantly related to Dnmt1 and Dnmt2 (data not shown). It remains to be seen whether a mammalian homologue of masc1 exists, and other than DNA methylation, such as DNA repair by binding to whether it functions as a de novo DNA methyltransferase. mismatched nucleotides as the bacterial cytosine methyltransfer- ases (21–23), DNA recombination and carcinogenesis. Since Dnmt2 is not essential for de novo methylation in ES ACKNOWLEDGEMENTS cells, additional DNA methyltransferases that catalyze de novo methylation are predicted to be present in mammalian cells. It is We thank Dr Austin Smith for the plasmid GT1.8Iresβgeo(Sal), formally possible that both Dnmt1 and Dnmt2 are de novo Lian Yu for excellent technical assistance, and members of our methyltransferases and can functionally compensate each other. laboratory for discussion. This work was supported by grants Recently, a gene known as masc1 was cloned through homology- from Bristol-Myers/Squibb and NIH (GM52106 to E.L.). M.O. based screening using a PCR amplification method, and genetic was a fellow of the Japanese Society for the Promotion of analysis has revealed that masc1 is involved in de novo Science. methylation in Ascobolus (24). The protein encoded by masc1 2540 Nucleic Acids Research, 1998, Vol. 26, No. 11 13 Wilkinson, C. R., Bartlett, R., Nurse, P. and Bird, A. P. (1995) Nucleic REFERENCES Acids Res., 23, 203–210. 14 Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. 1 Li, E., Bestor, T. H. and Jaenisch, R. (1992) Cell, 69, 915–926. (1990) J. Mol. Biol., 215, 403–410. 2 Lei, H., Oh, S. P., Okano, M., Juttermann, R., Goss, K. A., Jaenisch, R. 15 Chomzynski, P. and Sacchi, N. (1991) Anal. Biochem., 162, 156–159. and Li, E. 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Journal

Nucleic Acids Research – Oxford University Press

Published: Jun 1, 1998

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