It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

Reference

Adams
R. L. F.
,
Burdon
R. H.
,
McKinnon
K.
,
Rinaldi
A.
(
1983
)
Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase.
FEBS lett
163
,
194
198
Ariel
M.
,
Robinson
E.
,
McCarrey
J. R.
,
Cedar
H.
(
1995
)
Gamete-specific methylation correlates with imprinting of the murine Xist gene.
Nature Genet
9
,
312
315
Beard
C.
,
Li
E.
,
Jaenisch
J.
(
1995
)
Loss of methylation activates Xist in somatic but not in embryonic cells.
Genes Dev
9
,
2325
2334
Bestor
T. H.
,
Ingram
V. M.
(
1983
)
Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA.
Proc. Natl. Acad. Sci. USA
80
,
559
5563
Bestor
T. H.
,
Laudano
A. P.
,
Mattaliano
R.
,
Ingram
V. M.
(
1988
)
Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases.
J. Mol. Biol
203
,
971
983
Bestor
T. H.
(
1992
)
Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain.
EMBO J
11
,
2611
2617
Brandeis
C. V.
,
Kafri
T.
,
Ariel
M.
,
Chaillet
J. R.
,
McCarrey
J. R.
,
Razin
A.
,
Cedar
H.
(
1993
)
The ontogeny of allele-specific methylation associated with imprinted genes in the mouse.
EMBO J
12
,
3669
3677
Cedar
H.
,
Solage
A.
,
Glaser
G.
,
Razin
A.
(
1979
)
Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.
Nucl. Acids Res
6
,
2125
2132
Chaillet
J. R.
,
Vogt
T. F.
,
Beier
D. R.
,
Leder
P.
(
1991
)
Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis.
Cell
66
,
77
83
Chapman
V.
,
Forrester
L.
,
Sanford
J.
,
Hastie
N.
,
Rossant
J.
(
1984
)
Cell lineage-specific undermethylation of mouse repetitive DNA.
Nature
307
,
284
286
Chen
L.
,
MacMillan
A. M.
,
Chang
W.
,
Ezaz-Nikpay
K.
,
Lane
W. S.
,
Verdine
G. L.
(
1991
)
Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase.
Biochemistry
30
,
11018
11025
DeChiara
T. M.
,
Robertson
E. J.
,
Efstradis
A.
(
1991
)
Parental imprinting of the mouse insulin-like growth factor II gene.
Cell
64
,
859
859
Eden
S.
,
Cedar
H.
(
1994
)
Role of DNA methylation in the regulation of transcription.
Curr. Opinion Genet. & Dev
4
,
255
259
Friedman
S.
,
Ansari
N.
(
1992
)
Binding of the EcoRII methyltransferase to 5-fluorocytosine-containing DNA. Isolation of a bound peptide.
Nucl. Acids Res
20
,
3241
3248
Gruenbaum
Y.
,
Cedar
H.
,
Razin
A.
(
1982
)
Substrate and sequence specificity of a eukaryotic DNA methylase.
Nature
295
,
620
622
Jähner
D.
,
Stuhlmann
H.
,
Stewart
C. L.
,
Harbers
K.
,
Löhler
J.
,
Simon
I.
,
Jaenisch
R.
(
1982
)
De novo methylation and expression of retroviral genomes during mouse embryogenesis.
Nature
298
,
623
628
Jeppesen
P.
,
Turner
B. M.
(
1993
)
The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression.
Cell
74
,
281
289
Kafri
T.
,
Ariel
M.
,
Brandeis
M.
,
Shemer
R.
,
Urven
L.
,
McCarrey
J.
,
Cedar
H.
,
Razin
A.
(
1992
)
Developmental pattern of gene specific DNA methylation in the mouse embryo and germ line.
Genes Dev
6
,
705
715
Kumar
S.
,
Cheng
X.
,
Klimasauskas
S.
,
Mi
S.
,
Posfai
J.
,
Roberts
R.
,
Wilson
G. G.
(
1994
)
The DNA (cytosine-5) methyltransferases.
Nucl. Acids Res
22
,
1
10
Lau
M. M. H.
,
Stewart
C. E. H.
,
Liu
Z.
,
Bhatt
H.
,
Rotwein
P.
,
Stewart
C. L.
(
1995
)
Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality.
Genes Dev
8
,
2953
2963
Lauster
R.
,
Trautner
T. A.
,
Noyer-Widner
M.
(
1989
)
Cytosine-specific type II DNA methyltransferases. A conserved enzyme core with variable target-recognizing domains.
J. Mol. Biol
206
,
305
312
Leighton
P. L.
,
Ingram
R. S.
,
Eggenschwiler
J.
,
Efstratiadis
A.
,
Tilghman
S. M.
(
1995
)
Disruption of imprinting caused by deletion of the H19 gene region in mice.
Nature
375
,
34
39
Leonhardt
H.
,
Page
A. W.
,
Weier
H.-U.
,
Bestor
T. H.
(
1992
)
A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei.
Cell
71
,
865
873
Li
E.
,
Bestor
T. H.
,
Jaenisch
R.
(
1992
)
Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.
Cell
69
,
915
926
Li
E.
,
Beard
C.
,
Jaenisch
R.
(
1993
)
Role for DNA methylation in genomic imprinting.
Nature
366
,
362
365
Monk
M.
,
Boubelik
M.
,
Lehnert
S.
(
1987
)
Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development.
Development
99
,
371
382
Nan
X.
,
Tate
P.
,
Li
E.
,
Bird
A.
(
1996
)
DNA methylation specifies chromosomal localization of MeCP2.
Mol. Cell. Biol
16
,
414
421
Posfai
J.
,
Bhagwat
A. S.
,
Posfai
G.
,
Roberts
R. J.
(
1989
)
Predictive motifs derived from cytosine methyltransferases.
Nucl. Acids Res
17
,
2421
2435
Reik
W.
,
Weiher
H.
,
Jaenisch
R.
(
1987
)
Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: Selective cloning of proviral and flanking host sequences.
Proc. Natl. Acad. Sci. USA
82
,
1141
1145
Stewart
C.
,
Stuhlmann
H.
,
Jähner
D.
,
Jaenisch
R.
(
1982
)
De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.
Proc. Natl. Acad. Sci. USA
79
,
4098
4102
Stöger
R.
,
Kubicka
P.
,
Liu
C.-G.
,
Kafri
T.
,
Razin
A.
,
Cedar
H.
,
Barlow
D. P.
(
1993
)
Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal.
Cell
73
,
61
71
Tazi
J.
,
Bird
A.
(
1990
)
Alternative chromatin structure at CpG islands.
Cell
60
,
909
920
Tremblay
K. D.
,
Saam
J. R.
,
Ingram
R. S.
,
Tilghman
S. M.
,
Bartolomei
M. S.
(
1995
)
A paternal-specific methylation imprinting marks the alleles of the mouse H19 gene.
Nature Genet
9
,
407
413
Tucker
K. L.
,
Beard
C.
,
Dausman
J.
,
Jackson-Grusby
L.
,
Laird
P.W.
,
Lei
H.
,
Li
E.
,
Jaenisch
R.
(
1996
)
Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes.
Genes Dev
10
,
1008
1020
Wang
Z.
,
Fung
M. R.
,
Barlow
D. P.
,
Wagner
E. F.
(
1995
)
Regulation of embryonic growth and lysosomal targeting by the imprinted Igf-2/Mpr gene.
Nature
372
,
464
467
Wu
J. C.
,
Santi
D. V.
(
1987
)
Kinetic and catalytic mechanism of HhaI methyltransferase.
J. Biol. Chem
262
,
4778
4786
Wyszynski
M. W.
,
Gabbara
S.
,
Kubareva
E. A.
,
Oretskaya
T. S.
,
Gromova
E. S.
,
Shabaraova
E. A.
,
Bhagwat
A. S.
(
1993
)
The cytosine conserved among DNA methylases is required for transfer, but not for specific DNA binding.
Nucl. Acids. Res
21
,
295
301
Zuccotti
M.
,
Monk
M.
(
1995
)
Methylation of the mouse Xist gene in sperm and eggs correlates with imprinted Xist expression and paternal X-inactivation.
Nature Genet
9
,
316
320
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