Cell, Vol. 99, 247–257, October 29, 1999, Copyright 1999 by Cell Press

DNA Methyltransferases Dnmt3a and Dnmt3b
Are Essential for De Novo Methylationand Mammalian Development
Masaki Okano,* Daphne W. Bell,† Daniel A. Haber,†and En Li*‡* Cardiovascular Research Center† Cancer Center
Massachusetts General Hospital
Department of Medicine
Harvard Medical School
Charlestown, Massachusetts 02129

Summary
The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly
during early development and gametogenesis in mice.
Here we demonstrate that two recently identified DNA
methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting
blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted
methylation patterns. Dnmt3a and Dnmt3b also exhibit
nonoverlapping functions in development, with Dnmt3b
specifically required for methylation of centromericminor satellite repeats. Mutations of human DNMT3B
are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3bfunction as de novo methyltransferases that play important roles in normal development and disease.

Introduction
Covalent modification of DNA by methylation of cytosine
residues is a heritable and reversible epigenetic process, which is involved in regulation of a diverse rangeof biological processes in vertebrate animals, plants,
and fungi (reviewed by Colot and Rossignol, 1999). In
mammals, DNA methylation is essential for normal embryonic development (reviewed by Li, 1997), and it plays
important roles in the regulation of gene expression, X
chromosome inactivation, genomic imprinting, chromatin modification, and silencing of endogenous retroviruses (for reviews, see Jähner and Jaenisch, 1984;
Brockdorff, 1997; Surani, 1998; Ng and Bird, 1999). Altered DNA methylation patterns have been implicatedin tumorigenesis (reviewed by Ehrlich, 1999; Jones and
Laird, 1999).
DNA methylation changes in a highly orchestrated
way during mouse development, involving genomewide as well as gene-specific demethylation and denovo methylation (reviewed by Razin and Cedar, 1993).
The fertilized egg first undergoes a wave of demethyl-‡ To whom correspondence should be addressed (e-mail: en@

cvrc.mgh.harvard.edu).

ation during preimplantation development, which erasespart of the inherited parental methylation pattern. The
DNA of blastocysts is thus relatively undermethylated.
After implantation, the embryo undergoes a wave ofde novo methylation that establishes a new embryonicmethylation pattern (Monk et al., 1987; Sanford et al.,
1987; Howlett and Reik, 1991; Kafri et al., 1992). The
biological function of this dynamic reprogramming of
DNA methylation in early development is unknown. Demethylation and de novo methylation also occur during
gametogenesis and have been shown to play a criticalrole in the establishment of parental-specific methylation marks in imprinted genes (Chaillet et al., 1991;
Stöger et al., 1993; Tremblay et al., 1995).
Riggs (1975) and Holliday and Pugh (1975) first proposed that heritable DNA methylation provides a mechanism for the developmental regulation of gene expression. They predicted the existence of a maintenancemethyltransferase that methylates unmethylated sites
with great difficulty but easily methylates hemi-methylated sites. The existence of such maintenance methyltransferase activity was subsequently demonstrated byexperiments showing the clonal inheritance of methylation patterns in mammalian cells (Wigler et al., 1981;
Stein et al., 1982) and by the cloning of the first mammalian DNA methyltransferase, now known as Dnmt1 (Bestor et al., 1988). Purified Dnmt1 protein methylates DNA
containing hemimethylated CpG dinucleotides more efficiently than unmethylated DNA in vitro (Bestor, 1992).
The Dnmt1 protein has been localized to DNA replication
foci, suggesting that maintenance methylation might becoupled to DNA replication (Leonhardt et al., 1992). Further, inactivation of the mouse Dnmt1 gene by genetargeting results in extensive demethylation of all sequences examined (Li et al., 1992; Lei et al., 1996). Together, these findings strongly suggest that Dnmt1 functions as a major maintenance methyltransferase in vivo,
ensuring “replication” of DNA methylation patterns after
each round of cell division.
De novo methylation activity is detected predominantly in early embryos and embryonic carcinoma (EC)cells (Jähner et al., 1982; Palmiter et al., 1982; Stewart etal., 1982). Although Dnmt1 can methylate unmethylated
DNA in vitro, evidence for its involvement in de novo
methylation during development remains sketchy. In
contrast, genetic studies suggest that enzymes otherthan Dnmt1 might be responsible for de novo methylation in vivo, since ES cells completely lacking Dnmt1
are still capable of methylating retroviral DNA de novo
(Lei et al., 1996). The search for the mammalian de novo
methyltransferase has yielded several new methyltransferases, including members of the Dnmt2 and the Dnmt3
families. Although the Dnmt2 protein contains all theconserved motifs shared by known prokaryotic and eukaryotic DNA cytosine methyltransferases, inactivationof Dnmt2 in mouse ES cells by gene targeting perturbs
neither de novo methylation nor maintenance methylation of proviral DNA, making it unlikely that Dnmt2 isthe enzyme responsible for global de novo methylation(Okano et al., 1998a).

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Figure 1. Targeted Disruption of Dnmt3a and Dnmt3b(A and B) Gene targeting at the Dnmt3a locus (A) and Dnmt3b locus (B). The top lines represent the wild-type genomic DNA structures, whichcontain exons (shaded boxes) encoding the highly conserved motifs IV (PC) and VI (ENV). The middle lines represent various targeting vectors
in which motifs IV and VI are deleted to create null mutations. The bottom lines represent the mutant loci resulting from homologous
recombination between targeting vectors and wild-type loci. H, HindIII; Ap, ApaI; Nr, NruI; Xh, XhoI; B, BamHI; R, EcoRV; A, AatII; Xb, XbaI;S, ScaI; Hyg, PGK-hygromycin cassette; Pur, PGK-puromycin cassette.
(C–E) Establishment of the mutant ES cell lines. J1, a wild-type line; 36, a Dnmt1 null mutant line (Lei et al., 1996); 154a, a Dnmt3a heterozygousline; 6aa, a Dnmt3a homozygous mutant line (bgeo/hyg); 77aa, a Dnmt3a homozygous mutant line (bgeo/bgeo); 207b, a Dnmt3b heterozygousline; 8bb and 11bb, independent Dnmt3b homozygous lines (bgeo/hyg); 7aabb and 8aabb, independent lines homozygous for both Dnmt3a(bgeo/bgeo) and Dnmt3b (hyg/puro) mutations (derived from 77aa). (C) Southern analysis of the genotype of mutant lines. Genomic DNA wasdigested with HindIII (Dnmt3a), BamHI (Dnmt3b), or EcoRV (Dnmt1). wt, wild-type alleles; KO, mutant alleles. (D) Northern analysis of total
RNA (20 mg per lane) from wild-type and mutant ES cells. The same blot was hybridized repeatedly with cDNA probes for Dnmt3a, Dnmt3b,
or Oct3/4. It should be noted that smaller and faint transcripts derived from the PGK-hygromycin targeted allele of Dnmt3a were detectedfrom line 6aa, which was not used for generation of the double mutant ES cells and mice. (E) Immunoblot analysis with anti-Dnmt1 antibodies.
All cell lines except Dnmt1 null ES cells (c/c) expressed Dnmt1 protein.

The murine Dnmt3 family consists of two genes,
Dnmt3a and Dnmt3b, which are highly expressed inundifferentiated ES cells but downregulated after differentiation and expressed at low levels in adult somatic
tissues (Okano et al., 1998b). We have previously shownthat recombinant Dnmt3a and Dnmt3b proteins methylate CpG dinucleotides in various native and synthetic
DNA substrates and show no preference for hemimethylated DNA (Okano et al., 1998b). These results raised
the possibility that Dnmt3a and Dnmt3b encode thelong-sought de novo methyltransferases, known to behighly active in ES cells and early embryos. To test thishypothesis, we inactivated both genes by gene targetingin ES cells and mice. We show here that both Dnmt3a
and Dnmt3b are required for genome-wide de novo
methylation and are essential for mammalian development.

Results
Inactivation of Both Dnmt3a and Dnmt3b Disrupts
De Novo Methylation in ES Cells
Since both Dnmt3a and Dnmt3b encode functional DNA
methyltransferases with similar biochemical properties
and both genes are expressed at high levels in ES cells(Okano et al., 1998b), we speculated that Dnmt3a andDnmt3b might have overlapping functions. To test thishypothesis, we inactivated Dnmt3a, Dnmt3b, or bothgenes (four alleles) in ES cells by gene targeting. Several
gene targeting vectors were constructed by deletionof part of the catalytic domain containing the highly
conserved PC and ENV motifs (motifs IV and VI) (reviewed by Kumar et al., 1994) and by insertion of different selection markers, including the IRES-bgeo, PGKhygromycin, and PGK-puromycin cassettes (Figures 1A

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Figure 2. Disruption of De Novo Methylationof Proviral DNA in ES Cells Homozygous for
Both Dnmt3a and Dnmt3b Mutations
Genomic DNA was isolated from ES cells 1,
4, or 8 days after infection with MoMuLVsup1 retrovirus (d1, d4, and d8), or before infection (no), and digested with KpnI and HpaII.
The methylation status of newly integrated
MoMuLVsup-1 proviral DNA was analyzed bySouthern hybridization using the supF probe,
pAN7. The 1.48 kb Kpn I (K) fragment in the
39 LTR region contains five HpaII/MspI sites(lollipops) as shown in the lower panel. KpnI(K) digestion yields a 1.48 kb band, which
represents complete methylation, while double digestion with KpnI and MspI (K/M) yields
a 1.0 kb band, which represents unmethylated status. Cell lines: 3T3, NIH3T3; 1/1, wild
type ES cells; c/c, Dnmt1 null mutant ES cells;
3a2/2 3b2/2, Dnmt3a and Dnmt3b double homozygous lines, 7aabb and 8aabb; 3a2/2, Dnmt3a homozygous lines, 6aa and 77aa; and 3b2/2, Dnmt3b homozygous lines, 8bb and 11bb.
Note that de novo methylation of proviral DNA was completely blocked in cells lacking both Dnmt3a and Dnmt3b.

and 1B; and see Experimental Procedures). Since missense mutations of the PC motifs result in complete
inactivation of DNA methyltransferase activities (Kumar
et al., 1994), we predicted that our deletion mutations
would be functionally null.
ES cell lines homozygous for single gene disruption(Dnmt3a2/2 or Dnmt3b2/2) or for mutations of both genes([Dnmt3a2/2, Dnmt3b2/2]) were successfully isolated(Figure 1C, see Experimental Procedures). Northern
analysis using cDNA probes corresponding to the catalytic domain did not detect any stable transcripts from
Dnmt3a and Dnmt3b targeted loci, indicating that thecatalytic domain was not expressed from the mutant
genes (Figure 1D). All homozygous ES cell lines retained
undifferentiated morphology and expressed Oct3/4, an
undifferentiated ES cell marker (Figure 1D). We also
showed that Dnmt1 protein was expressed at approximately the same levels in all Dnmt3 mutant cell lines asin wild-type cells (Figure 1E).
Proviral DNA derived from new retroviral infection is
actively methylated de novo in EC and ES cells (Stewart
et al., 1982; Lei et al., 1996). To examine whether ES
cells lacking Dnmt3a, Dnmt3b, or both enzymes havede novo methylation activity, we infected these ES celllines with a recombinant retrovirus, MoMuLVsup-1 (Figure
2, lower panel), and analyzed the methylation status ofproviral DNA using the CpG methylation-sensitive enzyme HpaII. We showed that the unmethylated 1.0 kbband was shifted to the fully methylated 1.48 kb bandin wild-type ES cells over a period of 8 days, indicativeof active de novo methylation, but remained unmethylated in NIH3T3 cells, known to have low de novo methyltransferase activity (Figure 2). As shown previously, theproviral DNA was partially methylated in Dnmt1 null mutant ES cells (c/c), demonstrated by the presence ofmultiple bands ranging from 1.0 kb to 1.48 kb (Figure2) (Lei et al., 1996). Although Dnmt3a2/2 or Dnmt3b2/2
single mutant ES cell lines methylate proviral DNA normally, the [Dnmt3a2/2, Dnmt3b2/2] double mutant ES
cells completely lacked de novo methylation activity(Figure 2). These results demonstrate that Dnmt3a andDnmt3b are essential for de novo methylation in ES cells,

and that these two gene products are redundant in thisfunction.
Dnmt3a and Dnmt3b Are Essential
for Embryonic Development
To determine whether Dnmt3a and Dnmt3b are also
required for de novo methylation during embryonic development, we generated Dnmt3-deficient mice frommultiple ES cell lines heterozygous for either Dnmt3a or
Dnmt3b mutations. We first determined the expression
patterns of Dnmt3a and Dnmt3b during early development, taking advantage of the fact that both Dnmt3a
and Dnmt3b mutant genes were tagged by insertionof the IRES-bgeo cassette (Figures 1A and 1B). The
expression pattern of Dnmt3 genes could thus be determined by analyzing the lacZ expression pattern fromthe Dnmt3-IRES-bgeo fusion gene (Mountford et al.,1994). We showed by X-gal staining of heterozygous
embryos that Dnmt3a was moderately expressed in embryonic ectoderm and weakly in mesodermal cells of
E7.5 embryos (Figures 3A and 3B). At E8.5 and E9.5,
Dnmt3a expression became ubiquitous with increased
expression in the somites and the ventral part of theembryo (Figures 3C and 3D). Dnmt3b, on the other hand,
was highly expressed in the embryonic ectoderm, neural
ectoderm, and chorionic ectoderm at E7.5, although
weak expression was also detected in mesodermal andendodermal cells (Figures 3E and 3F). At later stages,
Dnmt3b expression was detected predominantly in theforebrain and eyes but weakly throughout the embryo(Figures 3G and 3H). It is noteworthy that in early embryos Dnmt3a and Dnmt3b show overlapping expression in the embryonic ectoderm, with Dnmt3b expressedat higher levels than Dnmt3a, suggestive of possible
overlapping functions.
Both Dnmt3a1/2 and Dnmt3b1/2 heterozygous mice
were grossly normal and fertile. Dnmt3a2/2 andDnmt3b2/2 mice were then derived by intercrosses ofheterozygous mice. Dnmt3a2/2 mice developed to term
and appeared to be normal at birth. However, most
homozygous mutant mice became runted and died atabout 4 weeks of age (Figure 4A). In contrast, no viable

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Figure 3. The Expression Patterns of Dnmt3a and Dnmt3b during Early Embryonic Development
Whole-mount X-gal staining of Dnmt3a1/2 embryos (A–D) or Dnmt3b1/2 embryos (E–H), which carry the IRES-bgeo marker driven by theendogenous Dnmt3a or Dnmt3b promoter, respectively. Embryos were dissected out at E7.5 (A, B, E, and F), E8.5 (C and G), and E9.5 (D and
H). Sagittal sections of E7.5 embryos after X-gal staining (B and F) reveal overlapping expression of Dnmt3a and Dnmt3b in the embryonicectoderm and distinct expression of Dnmt3b in the chorion. The expression of Dnmt3a appears to be ubiquitous at E8.5 and E9.5, whereas
Dnmt3b is expressed at high levels in the anterior head region and eyes. The unstained embryos on the right in (D) and (H) are wild-type
littermates. A, anterior; P, posterior; al, allantois; am, amnion; ch, chorion; ee, embryonic ectoderm; m, mesoderm; and ne, neuroectoderm.

Dnmt3b2/2 mice were recovered at birth. Dissection ofembryos at different stages of development revealed
that Dnmt3b2/2 embryos had multiple developmental
defects including growth impairment and rostral neuraltube defects with variable severity at later stages ofdevelopment (Figures 4B and 4C), though most of themappeared to develop normally before E9.5. The detailed
tissue and cellular defects in Dnmt3a2/2 and Dnmt3b2/2
mice are under investigation.
To test whether Dnmt3a and Dnmt3b might have overlapping function during early embryonic development,
we analyzed the phenotypes of offspring derived fromintercrosses of [Dnmt3a1/2, Dnmt3b1/2] compound heterozygous mice. After dissection and genotype analysis
of 198 embryos, we found that all [Dnmt3a2/2,
Dnmt3b2/2] double homozygous embryos (n 5 5, expecting 1/16 of total offspring) showed smaller size andabnormal morphology at E8.5 and E9.5 and died before
E11.5 (Figures 4E and 4H). The [Dnmt3a2/2, Dnmt3b2/2]
embryos lacked somites and did not undergo embryonicturning, indicating that their growth and morphogenesis
were arrested shortly after gastrulation (Figure 4E).
These embryonic defects are very similar to those of
Dnmt1 null mutant embryos (Figure 4F) (Lei et al, 1996)
but evidently more severe than those of Dnmt3a2/2 or
Dnmt3b2/2 single mutant mice. Our results thus demonstrate that Dnmt3a and Dnmt3b have overlapping functions during early embryogenesis.
De Novo Methylation Is Impaired in [Dnmt3a2/2,
Dnmt3b2/2] Embryos
To investigate whether the Dnmt3 mutant embryos
are defective in de novo methylation, we examined

global genomic methylation patterns in E9.5 Dnmt3a2/2,
Dnmt3b2/2, and [Dnmt3a2/2, Dnmt3b2/2] mutant embryos. The endogenous C-type retroviruses and intracisternal A particle (IAP) repeats at copy numbers of about
100 and 1000 per haploid genome, respecitvely, are
known to be interspersed in the mouse genome andmethylated at high levels in embryos and adult tissues(Sanford et al., 1987; Howlett and Reik, 1991; Li et al.,1992). We found that the endogenous C-type retroviral
DNA was methylated at normal levels in Dnmt3a2/2 embryos, but slightly undermethylated in Dnmt3b2/2 embryos, and highly undermethylated in [Dnmt3a2/2,
Dnmt3b2/2] embryos (Figure 5A). Nevertheless, a significant amount of methylation was retained in [Dnmt3a2/2,
Dnmt3b2/2] embryos when compared to Dnmt1 null mutant embryos, which lose almost all methylation (Lei etal., 1996) (Figure 5A). Similar results were obtained whenthe methylation status of the IAP repeats was examined(Figure 5B). In contrast to hypomethylation of these repetitive sequences, methylation of the 59 upstream region of the imprinted gene H19 was unaffected in[Dnmt3a2/2, Dnmt3b2/2] embryos (Figure 5C). This result
is consistent with the observation that this region of
H19 is subject to neither demethylation nor de novo
methylation during early embryogenesis (Tremblay etal., 1995).
To further determine whether the loss of methylationin repetitive sequences in [Dnmt3a2/2, Dnmt3b2/2] embryos was caused by impaired de novo methylation,
we compared methylation levels between [Dnmt3a2/2,
Dnmt3b2/2] embryos and blastocysts. We found thatmethylation levels of C-type retroviral DNA and the IAP

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Figure 4. Developmental Defects of Dnmt3a and Dnmt3b Single and Double Mutant Mice(A) A Dnmt3a2/2 mutant mouse and a wild-type littermate at 18 days after birth. (B) An E11.5 Dnmt3b2/2 mutant embryo and its normal
littermate. The enlarged head region of the mutant embryo shows rostral neural defects (C). (D) Side view of an E9.5 wild-type embryo. (E)Dorsal view of an E9.5 [Dnmt3a2/2, Dnmt3b2/2] embryo at the same magnification as (D). The [Dnmt3a2/2, Dnmt3b2/2] embryo was growth
retarded and failed to turn and form somites and was morphologically indistinguishable from the Dnmt1 null embryos (c/c) as shown in (F)(Lei et al. 1996). (G) Genotyping of one litter of E9.5 embryos by Southern hybridization. Genomic DNA was digested with BamHI and hybridized
to the Dnmt3a probe and the Dnmt3b 39 probe (Figures 1A and 1B) simultaneously. (H) Analysis of embryos derived from intercrosses of[Dnmt3a1/2, Dnmt3b1/2] compound heterozygous mice. Fourteen litters for E8.5–9.5 and eight litters for E11.5–15.5 were examined. Numbers
in parenthesis represent the number of abnormal embryos. The mutant phenotypes include growth defects, neural defects, and others withvariable penetrance. Res, resorbtion. (*) Of 16 resorbed embryos, 11 were collected from three litters, suggesting natural resorption due toabnormal pregnancy of the mother. All five double homozygous mutant embryos were growth arrested at the late gastrulation stage as shownin (E).

repeats in [Dnmt3a2/2, Dnmt3b2/2] embryos were essentially identical to those in blastocysts (Figures 5D
and 5E). This result suggests that global methylationlevels did not increase, and that de novo methylation
did not occur in [Dnmt3a2/2, Dnmt3b2/2] embryos after
implantation. In contrast, the methylation in C-type retroviral DNA and IAP repeats in E9.5 Dnmt1 null mutant
embryos was significantly lower than that in blastocysts(Figures 5D and 5E), indicating that inactivation of Dnmt1
resulted in demethylation of sequences which were
methylated at the time of implantation. As summarized
in Figure 5F, our results indicate that inactivation of both
Dnmt3a and Dnmt3b blocks de novo methylation in early
embryos but does not affect maintenance methylationof bulk genomic DNA. On the other hand, inactivationof Dnmt1 results in global demethylation, consistent
with the fact that Dnmt1 is required for maintenancemethylation.
Methylation of Repetitive Sequences and Imprinted
Genes in Dnmt3-Deficient ES Cells
To determine whether inactivation of Dnm3a andDnmt3b had any effect on global methylation of genomic

DNA in ES cells, we examined the methylation status ofa number of repetitive sequences and unique genes.
We found that the dispersed C-type retroviruses and
IAP repeats were slightly demethylated in [Dnmt3a2/2,
Dnmt3b2/2] double homozygous lines, but in neither
Dnmt3a2/2 or Dnmt3b2/2 single mutant lines nor two
independent [Dnmt3a2/2, Dnmt3b1/2] lines isolated fromthe same experiment as the [Dnmt3a2/2, Dnmt3b2/2]
mutant lines (Figures 6A and 6B). The major satellite
DNA is composed of tandem repeats at copy numbersof 700,000 that are located in the centromeric region(Hastie, 1989). We showed that the major satellite
DNA was also slightly demethylated in [Dnmt3a2/2,
Dnmt3b2/2] mutant cells, but not in other Dnmt3 mutant
lines (Figure 6C). It should be noted that the repetitiveelements in [Dnmt3a2/2, Dnmt3b2/2] cells were not demethylated as extensively as in cells homozygous forthe Dnmt1 mutations (Figures 6A–6C), indicating that
Dnmt3a and Dnmt3b are involved, but do not play amajor role, in keeping normal levels of methylation ofrepetitive sequences in ES cells.
We also examined the DNA methylation status ofseveral imprinted genes and Xist, which had been

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Figure 6. DNA Methylation in Dnmt3a and Dnmt3b Mutant ES Cells

Figure 5. Disruption of De Novo Methylation but Not Maintenance
Methylation in [Dnmt3a2/2, Dnmt3b2/2] Double Mutant Embryos(A) Methylation of C-type retroviral DNA in E9.5 embryos. DNA wasdigested with HpaII or MspI (M) and probed with pMO. The genotypeof embryos is indicated at the top as described in Figure 2.
(B) Methylation of IAP repeats in E9.5 embryos. DNA was digested
with HpaII or MspI (M) and probed with the IAP probe.
(C) Methylation of the upstream region of H19. DNA was digested
with SacI and HhaI and hybridized to the 59 upstream probe.
(D and E) Comparison of methylation levels of C-type retrovirus (D)or IAP (E) in E9.5 [Dnmt3a2/2, Dnmt3b2/2] embryos, E9.5 Dnmt1 nullembryos (c/c), and blastocysts. DNA from E9.5 embryos was loaded
at different quantities (1 or 1/4) for better comparison. DNA wasdigested with HpaII or MspI (M) and probed with pMO (D) or IAP(E). The methylation level in [Dnmt3a2/2, Dnmt3b2/2] embryos iscomparable to that in blastocysts. In contrast, methylation levels in
c/c embryos were lower than those in blastocysts. BL, blastocysts.
(F) Schematic diagram of methylation changes in wild-type (1/1),[Dnmt3a2/2, Dnmt3b2/2] double mutant (3a2/2,3b2/2), and Dnmt1
null mutant embryos (c/c) during early development. During preimplantation development, the bulk DNA undergoes demethylation(dashed line), resulting in hypomethylation of genomic DNA at theblastocyst stage. After implantation of blastocysts, de novo methylation takes place, resulting in a rapid increase in methylation (yellowline). In [Dnmt3a2/2, Dnmt3b2/2] embryos, de novo methylation is
impaired, but preexisting methylation patterns remain unaffected(red line), whereas in Dnmt1 null mutants embryos, maintenancemethylation is disrupted, resulting in rapid loss of methylation in all
sequences (green line). This suggests that Dnmt3a and Dnmt3b
encode de novo methyltransferases (green), while Dnmt1 encodesa maintenance methyltransferase (red).

(A–C) Genomic DNA from various ES cell lines as described in Figure
2 was digested with HpaII (A and B) or MaeII (C) and hybridized toprobes for endogenous C-type retrovirus repeat (A), IAP repeats(B), and major satellite repeats (C). DNA digested with MspI (M) is
a control for complete lack of methylation. (n/n), a Dnmt1 hypomorphic homozygous line, 52 (Li et al., 1992).
(D) Methylation of region 2 of Igf2r. Genomic DNA was digested
with PvuII (P), PvuII and MspI (P/M), or PvuII and HpaII (not indicated)and hybridized to the region 2 probe.
(E) Methylation of the 59 upstream region of H19. Genomic DNA wasdigested with SacI and HhaI or SacI alone (Sc). The right panel
shows H19 methylation in two additional [Dnmt3a2/2, Dnmt3b2/2]
lines.
(F) Methylation of the DMR2 region of Igf2. Genomic DNA was digested with BamHI (B), BamHI and MspI (B/M), or BamHI and HpaII.
(G) Methylation of the 59 region of the Xist gene. Genomic DNA
isolated from undifferentiated ES cells (d0) or cells that were differentiated in vitro for 4, 8, and 16 days (d4, d6, d8, and d16) was
digested with EcoRV and HhaI.

extensively characterized in wild-type and Dnmt1 mutant ES cells (Tucker et al., 1996). It has been shownthat an intronic CpG island, known as “region 2”, inthe Igf2 receptor (Igf2r) gene is methylated when it isinherited from the mother, but unmethylated when it isinherited from the father (Stöger et al., 1993). We foundthat the region 2 sequence (presumably, of the maternalallele) in [Dnmt3a2/2, Dnmt3b2/2] ES cells was resistant
to digestion by a methylation-sensitive restriction enzyme (Figure 6D). Similarly, the 59 upstream region of
H19 gene was methylated in three of the four
[Dnmt3a2/2, Dnmt3b2/2] ES cell lines (Figure 6E). The
partial demethylation in one of the lines is probably fortuitous, representing clonal variation, as methylation ofMammalian De Novo Methyltransferases
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were demethylated to approximately the same level asin Dnmt3b2/2 cells, suggesting that disruption of
Dnmt3a did not contribute to the demethylation of these
repeats. Similar demethylation of the minor satellite repeats was also detected in E9.5 Dnmt3b2/2 and[Dnmt3a2/2, Dnmt3b2/2] embryos, but not in Dnmt3a2/2
embryos (Figure 7B). These results suggest that the minor satellite repeats, in contrast to other repetitive sequences, are specific targets of Dnmt3b. It should be
noted that the minor satellite repeats in Dnmt1 null mutant ES cells or embryos were demethylated to levels
lower than those in Dnmt3b2/2 ES cells or embryos (Figures 7A and 7B), suggesting that Dnmt3b might contribute to methylation of a subset of minor satellite repeats.

Figure 7. Methylation of Minor Satellite Repeats by Dnmt3b
Genomic DNA from ES cells (A) or embryos (B) was digested with
HpaII and hybridized to the pMR150 probe. Note that minor satellite
repeats were hypomethylated to the same extent in Dnmt3b2/2 and[Dnmt3a2/2, Dnmt3b2/2] mutants.

H19 in the other three lines was stable over 20 passages(data not shown). However, to our surprise, a differentially methylated region in the Igf2 gene, known as DMR2
(Feil et al, 1994), was almost completely demethylated in[Dnmt3a2/2, Dnmt3b2/2] cells, but not in the Dnmt3a2/2,
Dnmt3b2/2, or [Dnmt3a2/2, Dnmt3b1/2] cells (Figure 6F).
The methylation level of this region in [Dnmt3a2/2,
Dnmt3b2/2] cells was comparable to that in Dnmt1 nullmutant cells (Figure 6F). We also analyzed methylationof the Xist promoter region in ES cells before and after
differentiation. This region is moderately methylated inwild-type ES cells, and the methylation level increases(by de novo methylation) upon differentiation of ES cells(Figure 6G). Similar to the Igf2 DMR2 region, the 59 regionof Xist was also highly demethylated in undifferentiated[Dnmt3a2/2, Dnmt3b2/2] cells and did not undergo denovo methylation upon differentiation in vitro. These observations suggest that some genomic sequences are
subject to demethylation of unknown mechanisms, and“maintenance” of methylation patterns of these sequences in ES cells requires persistent de novo methylation by Dnmt3 proteins.
Dnmt3b Is Required for Methylation of Centromeric
Minor Satellite Repeats
Despite their overlapping function in de novo methylation of various sequences in vivo, distinct functional
properties of Dnmt3a and Dnmt3b are suggested by
the observation that Dnmt3a2/2 and Dnmt3b2/2 micedisplayed different development defects. This raised thepossibility that Dnmt3a and Dnmt3b might methylate
different sets of sequences in the genome. Minor satellite DNA consists of tandem repeats at copy numbers of
50,000–100,000, located in centromeric regions (Hastie,1989). We showed that the minor satellite repeats were
substantially demethylated in Dnmt3b2/2, but not in
Dnmt3a2/2 ES cells (Figure 7A). Furthermore, the minor
satellite repeats in [Dnmt3a2/2, Dnmt3b2/2] ES cells

Mutations of Human DNMT3B in ICF Syndrome
Recently, the human DNMT3B gene has been mapped
to 20q11.2 (Robertson et al., 1999; Xie et al., 1999), a
locus associated with ICF syndrome (immunodeficiency, centromeric instability, facial anomalies) (Wijmenga et al., 1998). This rare autosomal recessive disorder is characterized by variable immunological defects, centromeric heterochromatin instability, and facial
anomalies (Smeets et al., 1994). DNA methylation studies have shown that the classical satellites II and III,
which are major components of constitutive heterochromatin, are hypomethylated in this syndrome (Jeanpierre
et al. 1993), similar to the hypomethylation of centromeric minor satellite repeats in Dnmt3b2/2 mutant mice
(Figure 7). This observation, together with the genetic
linkage data, raised the possibility that ICF syndrome
may be associated with dysfunction of DNMT3B.
To determine whether DNMT3B is indeed disrupted inthis syndrome, we analyzed immortalized lymphoblasts
derived from an affected individual and her parents (Carpenter et al., 1988). Nucleotide sequencing of the
DNMT3B transcript from the proband revealed two mutations, consistent with a compound heterozygote. A
heterozygous guanidine to adenosine mutation, resulting in a substitution of alanine to threonine at codon
603, was present in the proband and in her mother (Figure 8B). This missense mutation occurs in a region between motifs I and IV, within the catalytic domain of
DNMT3B (Xie et al., 1999). A second heterozygouschange in the DNMT3B transcript from the proband wasobserved at codon 744, consisting of a 9 bp insertion
encoding three amino acids: serine, threonine, and proline (Figure 8C). This insertion occurred upstream of theexon 23 splice acceptor site and was derived from theadjacent intronic sequence (Xie et al., 1999). The insertion is within the conserved region of the catalytic domain, which is likely to be disrupted by insertion of aproline residue. Analysis of genomic sequence revealed
a guanidine to adenosine mutation within intron 22, located 11 nucleotides 59 to the normal splice acceptor
site. This mutation results in the generation of a novel
splice acceptor site (AG9TA), which is presumably responsible for the insertion of the adjacent 9 nucleotides
into the altered transcript (Figure 8D). Neither the intronic mutation nor the altered DNMT3B transcript waspresent in either parent, suggesting that it may be theconsequence of a de novo germline event in the proband. Analysis of immortalized lymphoblasts from 50

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together with the chromosomal linkage and characteristic hypomethylation of centromeric repeats, suggestthat mutations of DNMT3B are responsible for ICF syndrome.
Discussion

Figure 8. Mutations of DNMT3B in Patient with ICF Syndrome(A) Methylation of centromeric satellites II repeats in a proband with
ICF syndrome and her parents. DNA was digested with BstBI andprobed using a labeled oligonucleotide specific to the satellites II
repeats (Ji et al., 1997).
(B) G to A substitution at nucleotide position 1807 of DNMT3B,
resulting in an alanine to threonine missense mutation in codon 603,
detected both in the proband and her mother.
(C) Insertion within the DNMT3B transcript of the proband of 9 bp,
encoding serine, threonine, and proline, between codons 743 and744. The insertion is derived from the intronic sequence flanking
the 59 splice site of exon 23. Analysis of uncloned RT–PCR products
shows equal representation of the wild-type and mutant transcripts
in the unsynchronized sequence. In the upper panel, analysis of theplus strand shows the sequence of exon 20 (left; uppercase letters),
which is normally spliced to that of exon 23 (right; uppercase letters).
The arrowhead denotes the normal splice site between exons 20
and 23. Inserted intronic sequence is shown in lowercase letters.
In the lower panel, analysis of the minus strand (converted to sensesequence) shows the sequence of exon 23 (right; uppercase letters)
and exon 20 (left; uppercase letters) and confirms the 9 bp insertionof intronic sequence (lowercase letters).
(D) Intronic G to A substitution, 11 nucleotides 59 of the exon 23
splice acceptor site. The mutation leads to a novel splice acceptor
site (AG9TA), resulting in insertion of the adjacent nine intronic nucleotides into the DNMT3B transcript. The novel splice acceptor site
is denoted by arrowhead, and the first two nucleotide of the insertion
are in lowercase letters.

healthy individuals (i.e., 100 alleles) failed to identify
either the missense mutation or the altered transcript,
indicating that both mutations are unlikely to represent
polymorphic variants in the population. These results,

Dnmt3a and Dnmt3b Encode Functional
De Novo Methyltransferases
De novo methylation and maintenance methylation are
two distinct processes that are required for the establishment and mitotic inheritance of tissue-specific methylation patterns. An ongoing debate since the cloningof Dnmt1 has been whether de novo methylation andmaintenance methylation are carried out by Dnmt1 alone
or by two or more distinct enzymes. Biochemical andgenetic studies have demonstrated that Dnmt1 is the
major maintenance methyltransferase. In this study, weshow that two recently identified DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo
methylation in ES cells and early embryos. Inactivation ofboth Dnmt3a and Dnmt3b disrupts de novo methylationof proviral DNA in ES cells (Figure 2) and genome-wide
de novo methylation occurring during early development, but it has no discernible effect on the maintenanceof preexisting methylation patterns (see Figure 5F). Ourfindings thus provide genetic evidence that Dnmt3a andDnmt3b encode DNA methyltransferases which are primarily responsible for de novo methylation during development.
While it remains possible that Dnmt1 might be involved in de novo methylation of some unique sequences in vivo, Dnmt1 is unlikely to be the predominant
de novo methyltransferase in development as was previously claimed (Yoder et al., 1997). Our observations
that Dnmt1 fails to methylate retroviral DNA and various
repetitive sequences de novo in ES cells and embryos
lacking both Dnmt3a and Dnmt3b suggest that Dnmt1
alone probably cannot initiate de novo methylation in
vivo. It is unknown why Dnmt3a and Dnmt3b can carryout de novo methylation efficiently in vivo, while theyshow lower methyltransferase activity than Dnmt1 invitro (Lei et al., 1996; Okano et al., 1998b). It is possiblethat efficient de novo methylation by Dnmt3a andDnmt3b requires the interaction of these proteins withother proteins or with chromatin. Further studies will be
required to elucidate the mechanisms by which Dnmt3a
and Dnmt3b catalyze de novo methylation in vitro andin vivo.
De Novo Methylation Is an Essential
Developmental Process
Genome-wide de novo methylation occurring during
early development plays a major role in the establishment of embryonic methylation patterns (Razin and Cedar, 1993). In this study, we show that inactivation ofboth Dnmt3a and Dnmt3b causes early embryonic lethality, demonstrating that de novo methylation is an
essential process for mammalian development. As illustrated in Figure 5F, Dnmt3a and Dnmt3b carry out denovo methylation in early postimplantation embryos, resulting in a rapid increase in the methylation of bulk
DNA while Dnmt1 is required for the maintenance ofMammalian De Novo Methyltransferases
255

all methylation in the genome. Both establishment andmaintenance of a hypermethylated genome are essential for embryonic development beyond gastrulation. De
novo methylation may play an important role in organizing or compartmentalizing the genome during tissue
differentiation, such that tissue-specific gene expression patterns can be established. DNA methylation has
been implicated in chromatin remodeling mediated bymethyl CpG-binding proteins (Ng and Bird, 1999), andits disruption may result in global genomic deregulation,
abrogating embryonic development. De novo methylation may also play important roles in regulation of X
chromosome inactivation and genomic imprinting in
mammals. Dnmt3a and Dnmt3b are likely to be responsible for de novo methylation of Xist (on the active X) andthe CpG islands on the inactive X during X inactivationand of imprinted genes during gametogenesis. Further
studies will be required to test these hypotheses.
Distinct Functions of Dnmt3a and Dnmt3b
in Development and Disease
The persistent expression of Dnmt3a and Dnmt3b after
gastrulation suggests that de novo methylation is notstrictly confined to early embryos as previously thought.
The distinct expression patterns of Dnmt3a and Dnmt3b
suggest that de novo methylation may also play an important role during late development. Mice lacking either
Dnmt3a or Dnmt3b display different defects and die at
different stages of development (Figures 4A–4C), indicating that they have distinct functions during development. Dnmt3b may play more important roles during
early development and methylate a broader spectrumof target sequences, whereas Dnmt3a may methylate a
set of genes or sequences that are critical during latedevelopment or after birth.
One important observation is that Dnmt3b, but not
Dnmt3a, specifically methylates the centromeric minor
satellite repeats in ES cells and developing embryos(Figure 7). Although the function of methylation of centromeric repeats is unknown, our finding that DNMT3B
is mutated in human ICF syndrome, a disease characterized by hypomethylation of centrometric satellites II and
III and centromeric heterochromatin instability, suggests that methylation of centromeric repeats may play
an important role in maintaining chromosome stability.
It remains to be determined how these mutations might
affect enzymatic activity of DNMT3B, but the fact thatboth mutations reside in the catalytic domain suggeststhat they may cause at least partial loss of function. We
presume that complete loss-of-function mutations in
DNMT3B would be lethal in humans, as it is in mice.
The Dnmt3b-deficient mice may provide a useful animalmodel for studying cellular and developmental defects
in ICF syndrome. Further studies will be required toelucidate specific roles of Dnmt3a and Dnmt3b in normal
development and disease.
Experimental ProceduresConstruction of Targeting Vectors
To construct Dnmt3a targeting vectors, a 13.5 kb ApaI-XhoI Dnmt3a
genomic fragment was cloned into pBluescript II SK vector. A 0.3
kb ApaI-NruI fragment containing exons encoding the PC and ENV
motifs was then replaced either by the IRES-bgeo cassette with the

splicing accept site (Mountford et al., 1994) or a PGK-hygromycin
cassette in the same transcriptional orientation as Dnmt3a (Figure1A). To construct Dnmt3b targeting vectors, a 2.4 kb AatII-XbaI
fragment containing the PC and ENV motifs was deleted from a 14
kb EcoRV-ScaI Dnmt3b genomic fragment in the pBluescript II SK
vector and replaced by the IRES-bgeo cassette, the PGK-hygromycin cassette, or the PGK-puromycin cassette (Figure 1B). The
IRES-bgeo and PGK-hygromycin cassettes were placed in the sametranscriptional orientation as Dnmt3b, while the PGK-puromycin
cassette was in the opposite orientation. All targeting vectors werelinearized with NotI prior to transfection into ES cells.
Generation of Mutant ES Cell Lines and Mice
Targeting vectors for Dnmt3a or Dnmt3b with IRES-bgeo cassettes
were electroporated into J1 ES cells, which were subsequently selected in G418-containing medium as described previously (Li etal. 1992). Genomic DNA isolated from G418-resistant colonies wasdigested with HindIII (for Dnmt3a) or BamHI (for Dnmt3b) and analyzed by Southern hybridization using a 0.3 kb SphI-ApaI fragment
as a probe for Dnmt3a, or a 0.5 kb BamHI-HindIII fragment as a 59
probe and 0.7 kb ScaI-BamHI fragment as a 39 probe for Dnmt3b(see Figures 1A and 1B). Targeting frequency was about 80% forboth Dnmt3a and Dnmt3b. Chimeric mice and F1 heterozygotes
were generated from multiple Dnmt3a or Dnmt3b mutant ES celllines as described (Li et al. 1992). Mutant mice were maintained on
a 129SvJae 3 C57BL/6 hybrid background and analyzed.
To generate ES cell lines homozygous for Dnmt3a or Dnmt3b
mutations, the second wild-type allele was disrupted with the hygromycin targeting vectors. Targeting frequency was 9/96 and 11/154
for Dnmt3a and Dnmt3b, respectively. Dnmt3a homozygous lines
were also generated by selection of heterozygous (bgeo/1) cells in
medium containing high concentration of G418 (6 mg/ml active form)
for 14 days. Of 96 resistant colonies, eight were found to be homozygous for Dnmt3a mutation (bgeo/bgeo). To generate ES cell lines
lacking both Dnmt3a and Dnmt3b, the Dnmt3a homozygous (bgeo/bgeo) ES cells were subject to two sequential rounds of gene targeting using Dnmt3b targeting vectors containing hygromycin andpuromycin. The targeting frequency was 19/154 and 20/96 for hygromycin and puromycin vectors, respectively.
Infection of ES Cells with Retrovirus
Infection of wild-type and Dnmt3a or Dnmt3b mutant ES cells with
the MoMuLVsup-1 virus, DNA preparation, and analysis of de novo
methylation of newly integrated provirus were carried out as described (Lei et al., 1996), except that single infection for 6 hr insteadof multiple rounds of infection was done.
DNA Preparation and Methylation Analysis
Blastocysts were collected from superovulated C57BL/6 females
after mating with C57BL/6 male mice using the standard protocol(Hogan et al. 1994). DNA from about 100 blastocysts was digested
and loaded on one lane (Figures 5D and 5E). DNA from embryos,
yolk sac, and cell lines was isolated, digested with methylationsensitive or -insensitive enzymes, and analyzed by Southern hybridization as previously described (Lei et al., 1996). Probes for methylation analysis include the following: pAN7 for supF of MoMuLVsup-1(Lei et al., 1996); pMO for endogeous C-type retroviruses (Li et al.,1992); pSAT for major satellite repeats (gift from A. Bird); pMR150
for minor satellite repeats (Chapman et al., 1984); IAP (Walsh et al.,1998); the Igf2r region 2 probe (Stöger et al., 1993); the H19 upstream
region (Tremblay et al., 1995); DMR2 or “probe 6” for Igf2 (Feilet al., 1994); 59 region of Xist cDNA (gift from T. Sado); and an
oligonucleotide probe (59-TCGAGTCCATTCGATGAT-39) specific tohuman classic satellite II (Ji et al., 1997).
RNA Preparation and Northern Analysis
Total RNA prepared from ES cells was analyzed using standard
Northern hybridization protocols. Dnmt3a- and Dnmt3b-specificprobes were a 1.05 kb (nt 2615–nt 3661, AF068625) fragment anda 1.0 kb EST clone (AA116694), respectively. Both probes overlap
with the C-terminal catalytic domain. The Oct3/4 probe was a 720
bp cDNA fragment obtained by RT–PCR.

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Immunoblot Analysis
ES cells were lysed in SDS loading buffer and quickly heated at
708C for 15 min. The cell lysates were fractionated by SDS–PAGE(8%) and blotted onto nitrocellulose membrane (Millipore). Immunoblot was carried out using anti-Dnmt1 antibody, pATH56, as described (Li et al., 1992).
Whole-Mount X-Gal Staining and Histology
Male mice heterozygous for either Dnmt3a or Dnmt3b mutation
were crossed with wild-type or heterozygous female. Embryos at
E7.5–E9.5 were dissected out of decidua, fixed, stained with X-gal,
and analyzed by histology as described (Hogan et al., 1994). Embryos were sectioned at 7 mm and counterstained with nuclear fast
red.
Analysis of Mutations in the Human DNMT3B Gene
For mutational analysis of patients with ICF syndrome, EBV immortalized lymphoblasts were obtained from Coriell Cell Repositoriesof the NIGMS Human Genetic Mutant Cell Repository. GM08714,
proband; GM08728, mother; and GM08729, father. Total cellular
RNA was extracted using the GTC-CsCl2 method. Uncloned RT–PCR
products spanning overlapping fragments of the transcript weresequenced in both directions, using Energy Transfer Dye Primer(Amersham), and analyzed for the presence of heterozygous mutations. Genomic sequences flanking exon 23 were PCR-amplified
and sequenced. (For intron/exon junctions, see Xie et al., 1999.)
Exons 10, 21, and 22, which are alternatively spliced, were also
amplified from genomic DNA and sequenced. To determine whether
mutations in DNMT3B were present in a control population, RT–PCR
products from EBV immortalized lymphoblasts derived from 50
healthy individuals were analyzed using DHPLC chromatography(Transgenomic) (Liu et al., 1998).
Acknowledgments
We thank H. Lei and L. Yu for excellent technical assistance and
Dr. D. Barlow, Dr. M. Bartolomei, Dr. A. Bird, Dr. P. Laird, Dr. W.
Reik, Dr. J. Rossant, Dr. A. Smith, and Dr. S. Tilghman for various
plasmid DNA and probes, Dr. T. Bestor for anti-Dnmt1 antibody,and Dr. R. Chen for puromycin-resistant MEF cells. This work is
supported by grants from National Institutes of Health (GM52106,
CA82389) and Bristol Myers-Squibb (E. L.) and by a grant from theCenter for Cancer Risk Analysis (D. H.), Massachusetts General
Hospital. M. O. was supported by the Japan Society for the Promotion of Science and is a special fellow of Leukemia Society ofAmerica.
Received September 27, 1999; revised October 12, 1999.
References
Bestor, T.H. (1992). Activation of mammalian DNA methyltransferase
by cleavage of a Zn binding regulatory domain. EMBO J. 11, 2611–
2617.
Bestor, T.H., Laudano, A.P., Mattaliano, R., and Ingram, V.M. (1988).
Cloning and sequencing of a cDNA encoding DNA methyltransferaseof mouse cells. The carboxyl-terminal domain of the mammalian
enzymes is related to bacterial restriction methyltransferases. J.
Mol. Biol. 203, 971–983.
Brockdorff, N. (1997). Convergent themes in X chromosome inactivation and autosomal imprinting. In Genomic Imprinting: Frontiersin Molecular Biology, W. Reik and A. Sorani, eds. (Oxford: IRL Press),
pp. 191–210.
Carpenter, N.J., Filipovich, A., Blaese, R.M., Carey, T.L., and Berkel,
A.I. (1988). Variable immunodeficiency with abnormal condensationof the heterochromatin of chromosomes 1, 9, and 16. J. Pediatr.
112, 757–760.
Chaillet, J.R., Vogt, T.F., Beier, D.R., and Leder, P. (1991). Parentalspecific 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., and Rossant, J.

(1984). Cell lineage-specific undermethylation of mouse repetitive
DNA. Nature 307, 284–286.
Colot, V., and Rossignol, J.L. (1999). Eukaryotic DNA methylation
as an evolutionary device. Bioessays 21, 402–411.
Ehrlich, M. (1999). DNA hypomethylation and cancer. In DNA Alterations in Cancer: Genetic and Epigenetic Changes, M. Ehrlich, ed.
(Natick, MA: BioTechniques Books).
Feil, R., Walter, J., Allen, N.D., and Reik, W. (1994). Developmental
control of allelic methylation in the imprinted mouse Igf2 and H19
genes. Development 120, 2933–2943.
Hastie, N.D. (1989). Highly repeated DNA families in the genome of
Mus musculus. In Genetic Variants and Strains of the Laboratory
Mouse, Second Edition, M.F. Lyon and A.G. Searle, eds. (Oxford:
Oxford University Press), pp. 559–573.
Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994). Manipulating the Mouse Embryo, Second Edition (Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press).
Holliday, R., and Pugh, J.E. (1975). DNA modification mechanisms
and gene activity during development. Science 187, 226–232.
Howlett, S.K., and Reik, W. (1991). Methylation levels of maternaland paternal genomes during preimplantation development. Development 113, 119–127.
Jähner, D., and Jaenisch, R. (1984). DNA methylation in early mammalian development. In DNA Methylation: Biochemistry and Biological Significance, A. Razin, H. Cedar, and A. Riggs, eds. (New York:
Springer-Verlag), pp. 189–219.
Jähner, D., Stuhlmann, H., Stewart, C.L., Harbers, K., Löhler, J.,
Simon, I., and Jaenisch, R. (1982). De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature
298, 623–628.
Jeanpierre, M., Turleau, C., Aurias, A., Prieur, M., Ledeist, F., Fischer,A., and Viegas-Péquignot, E. (1993). An embryonic-like methylation
pattern of classical satellite DNA is observed in ICF syndrome. Hum.
Mol. Genet. 2, 731–735.
Ji, W., Hernandez, R., Zhang, X.Y., Qu, G.Z., Frady, A., Varela, M.,
and Ehrlich, M. (1997). DNA demethylation and pericentromeric rearrangements of chromosome 1. Mutat. Res. 379, 33–41.
Jones, P.A., and Laird, P.W. (1999). Cancer epigenetics comes ofage. Nat. Genet 21, 163–167.
Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey,J., Cedar, H., and Razin, A. (1992). Developmental pattern of genespecific DNA methylation in the mouse embryo and germ line. Genes
Dev. 6, 705–714.
Kumar, S., Cheng, X., Klimasauskas, S., Mi, S., Posfai, J., Roberts, R.,
and Wilson, G.G. (1994). The DNA (cytosine-5) methyltransferases.
Nucleic Acids Res. 22, 1–10.
Lei, H., Oh, S.P., Okano, M., Jüttermann, R., Goss, K.A., Jaenisch,R., and Li, E. (1996). De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122, 3195–3205.
Leonhardt, H., Page, A.W., Weier, H.-U., and Bestor, T.H. (1992). Atargeting sequence directs DNA methyltransferase to sites of DNA
replication in mammalian nuclei. Cell 71, 865–873.
Li, E. (1997). Role of DNA methylation in development. In Genomic
Imprinting: Frontiers in Molecular Biology, W. Reik and A. Surani,eds. (Oxford: IRL Press), pp. 1–20.
Li, E., Bestor, T.H., and Jaenisch, R. (1992). Targeted mutation of
the DNA methyltransferase gene results in embryonic lethality. Cell69, 915–926.
Liu, W., Smith, D.I., Rechtzigel, K.J., Thibodeau, S.N., and James,
C.D. (1998). Denaturing high performance liquid chromatography(DHPLC) used in the detection of germline and somatic mutations.
Nucleic Acids Res. 26, 1396–1400.
Monk, M., Boubelik, M., and 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.
Mountford, P., Zevnik, B., Duwel, A., Nichols, J., Li, M., Dani, C.,
Robertson, M., Chambers, I., and Smith, A. (1994). Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci. USA 91, 4303–4307.

Mammalian De Novo Methyltransferases
257

Ng, H.H., and Bird, A. (1999). DNA methylation and chromatin modification. Curr. Opin. Genet. Dev. 9, 158–163.
Okano, M., Xie, S., and Li, E. (1998a). Dnmt2 is not required for denovo and maintenance methylation of viral DNA in embryonic stem
cells. Nucleic Acids Res. 26, 2536–2540.
Okano, M., Xie, S., and Li, E. (1998b). Cloning and characterizationof a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219–220.
Palmiter, R.D., Chen, H.Y., and Brinster, R.L. (1982). Differential regulation of metallothionein-thymidine kinase fusion genes intransgenic mice and their offspring. Cell 29, 701–710.
Razin, A., and Cedar, H. (1993). DNA methylation and embryogenesis. In DNA Methylation: Molecular Biology and Biological Significance, J.P. Jost and H.P. Saluz, eds. (Basel: Birkhäuser Verlag), pp.
343–357.
Riggs, A.D. (1975). X inactivation, differentiation, and DNA methylation. Cytogenet. Cell Genet. 14, 9–25.
Robertson, K.D., Uzvolgyi, E., Liang, G., Talmadge, C., Sumegi, J.,
Gonzales, F.A., and Jones, P.A. (1999). The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res. 27,
2291–2298.
Sanford, J.P., Clark, H.J., Chapman, V.M., and Rossant, J. (1987).
Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse.
Genes Dev. 1, 1039–1046.
Smeets, D.F., Moog, U., Weemaes, C.M., Vaes-Peeters, G., Merkx,
G.F., Niehof, J.P., and Hamers, G. (1994). ICF syndrome: a new caseand review of the literature. Hum. Genet. 94, 240–246.
Stein, R., Gruenbaum, Y., Pollack, Y., Razin, A., and Cedar, H. (1982).
Clonal inheritance of the pattern of DNA methylation in mouse cells.
Proc. Natl. Acad. Sci. USA 79, 61–65.
Stewart, C., Stuhlmann, H., Jähner, D., and 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., andBarlow, D.P. (1993). Maternal-specific methylation of the imprinted
mouse Igf2r locus identifies the expressed locus as carrying theimprinting signal. Cell 73, 61–71.
Surani, M.A. (1998). Imprinting and the initiation of gene silencing
in the germline. Cell 93, 309–312.
Tremblay, K.D., Saam, J.R., Ingram, R.S., Tilghman, S.M., and Bartolomei, M.S.A. (1995). paternal-specific methylation imprinting marks
the alleles of the mouse H19 gene. Nat. Genet. 9, 407–413.
Tucker, K.L., Beard, C., Dausmann, J., Jackson-Grusby, L., Laird,
P.W., Lei, H., Li, E., and Jaenisch, R. (1996). Germ-line passage isrequired for establishment of methylation and expression patterns ofimprinted but not of nonimprinted genes. Genes Dev. 10, 1008–1020.
Walsh, C.P., Chaillet, J.R., and Bestor, T.H. (1998). Transcription of
IAP endogenous retroviruses is constrained by cytosine methylation. Nat. Genet. 20, 116–117.
Wigler, M., Levy, D., and Perucho, M. (1981). The somatic replicationof DNA methylation. Cell 24, 33–40.
Wijmenga, C., van den Heuvel, L.P., Strengman, E., Luyten, J.A.,
van der Burgt, I.J., de Groot, R., Smeets, D.F., Draaisma, J.M., vanDongen, J.J., De Abreu, R.A., et al. (1998). Localization of the ICF
syndrome to chromosome 20 by homozygosity mapping. Am. J.
Hum. Genet. 63, 803–809.
Xie, S., Wang, Z., Okano, M., Nogami, M., Li, Y., He, W.W., Okumura,K., and Li, E. (1999). Cloning, expression and chromosome locationsof the human DNMT3 gene family. Gene 236, 87–95.
Yoder, J.A., Soman, N.S., Verdine, G.L., and Bestor, T.H. (1997).
DNA (cytosine-5)-methyltransferases in mouse cells and tissues.
Studies with a mechanism-based probe. J. Mol. Biol. 270, 385–395.