Maintenance of self-renewal ability of mouse embryonicstem cells in the absence of DNA methyltransferases Dnmt1,
Dnmt3a and Dnmt3b

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Dnmt1/3a/3b
2006
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Akiko Tsumura1,4, Tomohiro Hayakawa2, Yuichi Kumaki3,6, Shin-ichiro Takebayashi1,
Morito Sakaue1, Chisa Matsuoka1, Kunitada Shimotohno4, Fuyuki Ishikawa5, En Li7,
Hiroki R. Ueda3, Jun-ichi Nakayama2 and Masaki Okano1,*
1

Laboratory for Mammalian Epigenetic Studies, 2Laboratory for Chromatin Dynamics, and 3Laboratory for Systems Biology, Center for
Developmental Biology, RIKEN, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
4
Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8501, Japan
5
Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502,
Japan
6
INTEC Web and Genome Informatics Corp., 1-3-3 Shinsuna, Koto-ku, Tokyo 136-8637, Japan
7
Epigenetics Program, Novartis Institute for Biomedical Research, 250 Massachusetts Ave., Cambridge, MA 02139, USA

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylationin CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing
and maintenance of genome integrity. Proper CpG methylation is required for the normal growthof various somatic cell types, indicating its essential role in the basic cellular function of mammaliancells. Previous studies using Dnmt1–/– or Dnmt3a–/–Dnmt3b–/– ES cells, however, have shown thatundifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation.
In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function,
we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite theabsence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple
knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics.
TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at
Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome
numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal
stability in the absence of CpG methylation and CpG DNA methyltransferases.

Introduction
Vertebrate genomes are methylated on cytosines at levels
much higher than those of other eukaryotic organisms, andare almost exclusively methylated at CpG dinucleotides(Bird 2002). CpG methylation plays an important role inepigenetic gene silencing and maintenance of genome
stability, and is involved in a broad range of physiologicaland pathological processes in mammals, including embryogenesis, genome imprinting and tumorigenesis ( Jones &Baylin 2002; Li 2002; Robertson 2005). In mice, three
Communicated by: Fumio Hanaoka*Correspondence: E-mail: okano@cdb.riken.jp

CpG DNA methyltransferases, Dnmt1, Dnmt3a andDnmt3b, coordinately regulate CpG methylation in
the genome (Li et al. 1992; Okano et al. 1999). Reduction of CpG methylation by inactivating these enzymes
or related molecules in various mammalian cells results ingrowth defects, cell death, activation of retrotransposons
and genome instability (Walsh et al. 1998; Xu et al. 1999;
Jackson-Grusby et al. 2001; Rhee et al. 2002; Eden et al.
2003; Gaudet et al. 2003; Bourc’his & Bestor 2004;
Dodge et al. 2005; Hata et al. 2006), indicating that CpG
methylation plays a fundamental role in basic cellular functions of mammalian cells. Unlike differentiated somatic
cells, undifferentiated ES cells can tolerate hypomethylation
caused by inactivation of Dnmt1 or inactivation of both

DOI: 10.1111/j.1365-2443.2006.00984.x© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Dnmt3a and Dnmt3b for their proliferation (Lei et al.
1996; Chen et al. 2003; Jackson et al. 2004), indicatingthat gene silencing and chromatin structures are regulateddifferently in ES cells.
Post-translational modifications of specific amino acidresidues of histones are also important for chromatin
dynamics and epigenetic gene regulation ( Jenuwein& Allis 2001). Functional relationships between CpG
methylation and histone modification have been demonstrated in several species (Tamaru & Selker 2001; Jackson
et al. 2002; Lehnertz et al. 2003; Tariq et al. 2003; Vireet al. 2006). Biochemical analyses have also shown thatmethyl-CpG-binding proteins interact with either histone
deacetylases or histone methyltransferases (Jones et al.
1998; Nan et al. 1998; Ng et al. 1999; Wade et al. 1999;
Zhang et al. 1999; Fujita et al. 2003; Sarraf & Stancheva
2004), further supporting the mechanistic links between
these epigenetic mechanisms. Interestingly, a previous
study using Dnmt1–/– and Dnmt3a–/–Dnmt3b–/– ES cells
showed that ES cells with a reduced level of CpG
methylation maintain proper localization of the repressive chromatin markers histone H3 methylated at lysine
9 (H3K9) and heterochromatin protein-1 (HP1) at
pericentromeric heterochromatin (Lehnertz et al. 2003),
indicating that CpG methylation may contribute little to
the regulation of higher-order chromatin structures in
ES cells. That study, however, remains inconclusive
because the mutant ES cells had residual CpG methylation and functional CpG methyltransferase activity.
In this study, we address whether mouse ES cells cansurvive in the absence of epigenetic regulation by CpG
methylation, and if so, whether higher-order chromatin
structures can be maintained in these cells.We established
ES cells deficient for all three CpG methyltransferases,
Dnmt1, Dnmt3a and Dnmt3b. We show that ES cells
without CpG methylation maintain stem cell properties,
proliferation ability, heterochromatic domains marked
with H3K9 trimethylation, and euploidy.

Results
Establishment of Dnmt1/Dnmt3a/Dnmt3b triple
knockout ES cells

We investigated the effects of the complete loss of CpG
DNA methyltransferase function by inactivating Dnmt1
via two rounds of gene targeting in Dnmt3a–/–Dnmt3b–/–
ES cells (Fig. 1A).This procedure yielded five independent
clones of viable TKO Dnmt1–/–Dnmt3a–/–Dnmt3b–/– EScells. We confirmed the accuracy of the gene targetingin these five cell lines by Southern blotting analysis(Fig. 1B and data not shown). Three of the five TKO
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cell lines were characterized in detail by Western
blotting and RT-PCR analyses, showing the loss ofgene products (Fig. 1B–D), and we used these for further
studies.
Absence of CpG methylation in TKO ES cells

We evaluated the status of genome-wide CpG methylationin the TKO cells by several independent methods. We
first examined CpG methylation of repetitive sequences
by Southern hybridization using methylation-sensitive
restriction enzymes. The methylation of two widelydispersed retroelement sequences, C-type endogenous
retrovirus and intracisternal-A particles (IAP), was much
lower in TKO cells as compared with Dnmt1–/– cells or
Dnmt1+/–Dnmt3a–/–Dnmt3b–/– clones (Fig. 2A). In TKO
cells, the digest patterns for CpG methylation-sensitive
HpaII and its CpG methylation-insensitive isoschizomer
MspI were indistinguishable, suggesting that CpG
methylation in TKO cells was below the detection limit(Fig. 2A). Analysis of minor satellite repeats at centromericregions and major satellite repeats at pericentromericregions showed similarly reduced CpG methylation
states in TKO cells (Fig. 2A).
We next examined the profiles and amounts of CpG
methylation in TKO cells using bisulfite sequencing
analysis, which can determine the exact sites of methylcytosine (Clark et al. 1994). Pericentromeric major satellite
repeats, retroelement repeats, and the single-copy imprinted
genes Snrpn and Igf2r all showed extensive loss of CpG
methylation (Fig. 2B). We also sequenced random fragments of bisulfite-treated genomic DNA via shotgun
sequencing and compared the sequences to the mouse
genome (Table 1 and <http://www.dbsb.org />). Twoexperiments using TKO cells showed that almost all
methylcytosines were lost in 786 CpG sites over about
75 kb (Table 1). We detected a very small amount ofmethylcytosine-signals both at CpG sites (0.4%) andnon-CpG sites (0.1– 0.4%), possibly reflecting residual
methylcytosines in the genome of TKO cells resulting
from mechanisms other than methylation by Dnmt1/
Dnmt3a/Dnmt3b. Such low levels, however, are nearly
equivalent to the low frequency of experimental errors,
including nucleotide misincorporation by Taq polymerase or incomplete bisulfite conversion. These datasuggest that both CpG and non-CpG methylation in the
TKO cells were below the limit of accuracy in our
experimental procedures.
We then investigated the loss of CpG methylation at
the cellular level by focusing on two cellular responses,
localization of a methyl-CpG-binding protein and silencing
of retroelements.To visualize the cytological distribution© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Dnmt1/3a/3b triple knockout ES cells

Figure 1 Generation of Dnmt1–/–Dnmt3a–/–Dnmt3b–/– ES cells. (A) Targeting of Dnmt1 in Dnmt3a–/–Dnmt3b–/– ES cells. To create anull allele, exons 31–33, encoding highly conserved Pro-Cys (PC) and Glu-Asn-Val (ENV) motifs, were replaced with the blasticidin
resistance gene flanked with loxP sites (2lox(+blas)). Introduction of Cre excised the blasticidin resistance gene (1lox(∆blas)). The
horizontal lines labeled “5′ probe” and “3′ probe” represent the two probes used for Southern blot analyses, and the arrows represent theprimers used in the RT-PCR analysis. X, XbaI; K, KpnI. (B–D) J1, wild-type line; 7aabb, Dnmt3a–/–Dnmt3b–/– ES cell line; 36, a Dnmt1–/–
ES cell line; 18 and 67, Dnmt1+/–Dnmt3a–/–Dnmt3b–/– parental lines of TKO ES cells with or without excision of the blasticidin gene by
Cre (18, 2lox(+blas); 67, 1lox(∆blas)); 19, 121 and 155, independent Dnmt1–/–Dnmt3a–/–Dnmt3b–/– TKO lines. (B) Southern blot analysisof the mutant cell lines. Genomic DNA was digested with KpnI (Dnmt1-5′probe), XbaI (Dnmt1-3′probe), HindIII (Dnmt3a probe) or
BamHI (Dnmt3b probe). wt, wild-type alleles; KO, mutant alleles; 2lox(+blas) and 1lox(∆blas), targeted Dnmt1 alleles. (C) Western blot
analysis of the mutant cell lines with anti-Dnmt1, anti-Dnmt3a or anti-Dnmt3b. Anti-tubulin was used as a control for equal protein
loading. (D) RT-PCR analysis of the mutant cell lines with primers for Dnmt1, Dnmt3a, and Dnmt3b. Glyceraldehyde 3-phosphate
dehydrogenase (Gapdh) was used as a control for equal RNA loading.

of CpG methylation in ES cells, we introduced andexpressed green fluorescent protein fused to themethyl-CpG binding domain of MBD1 (GFP-MBD,
Fujita et al. 1999). In interphase nuclei of wild-type andDnmt1–/– ES cells, GFP-MBD mainly localized to pericentromeric heterochromatin regions that were densely
stained by 4′,6′-diamino-2-phenylindole (DAPI). In
contrast, GFP-MBD distribution was diffuse in TKO
cell nuclei, and its localization was not restricted to

pericentromeric heterochromatin (Fig. 2C), in agreementwith our observation of extensive loss of CpG methylation
at pericentromeric regions in these cells (Fig. 2A,B). We
also examined the expression of two retroelements,
IAP and LINE1, which are normally silenced and are
de-repressed in severely hypomethylated embryos orgerm cells (Walsh et al. 1998; Bourc’his & Bestor 2004;
Hata et al. 2006). Consistent with the CpG methylation
status of these retroelements (Fig. 2B), transcripts of© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Figure 2 Extensive loss of CpG methylation in TKO cells. (A) DNA methylation of repetitive sequences as determined by Southern
analysis of TKO cells. Genomic DNA was digested with CpG methylation–sensitive HpaII (Hp) and MaeII (Ma) or CpG methylation–
insensitive MspI (Ms) and analyzed with probes against the following: C-type endogenous retrovirus, intracisternal-A particle
retroelements (IAP), centromeric repeats (minor satellites), and pericentromeric repeats (major satellites). Clone 11 was a non-targeted
clone during the second round of targeting of Dnmt1, which retained an intact Dnmt1 allele and underwent the same number of passages
as TKO cells. (B) DNA methylation analysis of repetitive sequences (pericentromeric major satellite repeats, IAP and LINE1; left) and
imprinted genes (Snrpn and Igf2r; right) by bisulfite sequencing analysis. The percentages of methylcytosine signal out of total CpG sites
analyzed in the sequenced clones are indicated. n.d., not detected. (C) Localization of GFP-MBD in interphase nuclei of ES cells. DNA
was visualized by DAPI. (D) Northern blot analysis of poly(A)+ RNA from the various ES cell lines using IAP and LINE1 probes. I andI∆1, full-length transcripts and variant truncated transcripts of type I IAP; II, type II IAP.

IAP were elevated in both the TKO and the Dnmt1–/– EScells, whereas transcripts of LINE1 were elevated mainly
in TKO cells (Fig. 2D). These data are consistent withthe absence of CpG methylation in the TKO cells.
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Maintenance of stem cell properties in TKO ES cells

Despite the absence of CpG methylation, TKO cells
retained the morphological features of undifferentiated© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Dnmt1/3a/3b triple knockout ES cells
Table 1 Random bisulfite sequencing analysis. Random fragments of bisulfite-treated genomic DNA from the ES cell lines weresequenced, and the methylation status of each cytosine was determined by alignment with the mouse genome. Methylation status ofclones that mapped to multiple loci in the mouse genome was based on the highest probability alignment with the genomic sequence.
No. of methylcytosines/total no. of sites

Wild-type
Dnmt3a–/–3b–/–
Dnmt1–/–TKO (#19)
TKO (#121)

No. ofclones

Total
(bp)

CpG

CpA

CpC

CpT

119
63
112
383
127

16327
8541
15450
57404
17448

112/136 (82%)
11/72 (15%)
26/143 (18%)*2/559 (0.4%)*1/227 (0.4%)

7/11173/661
8/1106
8/4125
3/1296

3/779
5/415
2/765
11/2725
4/10096/1142
3/588
1/10575/4011
1/1201*Three cytosines at CpG sites remained unconverted to uracil after the bisulfite reaction in the TKO cells, and two of the three cytosines
were derived from clones that contained more than three unconverted cytosines at non-CpG sites. In these cases, all unconverted cytosines
were clustered within a 20-bp palindromic sequence, indicating the possibility of incomplete conversion by the bisulfite reaction.

Figure 3 Maintenance of undifferentiated features in TKO cells. (A) Phase-contrast microscopic images (top) and alkaline phosphatasestaining (bottom) of wild-type (wt) and TKO cells. (B) RT-PCR analysis of four representative undifferentiated ES cell markers, Oct4,
Rex1, Fgf4 and Nanog. No signal was observed in the negative control lacking reverse transcriptase (RT). (C) Growth competition in a
mixture of wt and TKO cells under culture conditions that maintain the undifferentiated state (undiff, top) or promote differentiation(diff, bottom). Changes in the fraction of wild-type and TKO cells in the cell population at the indicated days were determined bySouthern analysis using the Dnmt3b probe.

ES cells and stained positive for alkaline phosphataseactivity, indicative of a pluripotent, undifferentiated state(Fig. 3A). TKO cells also expressed four other typicalmarkers of undifferentiated cells (Oct4, Rex1, Fgf4 andNanog) at the same or higher levels than wild-type ES
cells (Fig. 3B). Growth curve experiments suggest thatproliferation of undifferentiated TKO cells was comparable
to, although slightly slower than, that of wild-type ES cells(Supplementary Fig. S1). We also estimated the growthrate of TKO cells by growth competition analysis, in which

wild-type and TKO cells were cocultured, and the change
in the fraction of wild-type and TKO cells in the cell
population during the culture period was determined bySouthern hybridization. The growth of undifferentiatedTKO cells was comparable to that of wild-type cells, whereas
their growth was delayed upon differentiation by formationof embryoid bodies (Fig. 3C), consistent with previous
findings using Dnmt1–/– ES cells (Lei et al. 1996). These
results suggest that CpG methylation is not essential forthe self-renewal of ES cells in an undifferentiated state.

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Figure 4 Maintenance of global chromatin structure in TKO cells. (A) Western blot analysis of global histone acetylation andmethylation levels. H3Ac, acetylated histone H3; H4Ac, acetylated histone H4; H4K5Ac, H4K8Ac, H4K12Ac and H4K16Ac,
acetylation at Lys5, Lys8, Lys12, or Lys16 of histone H4, respectively; H3K4diMe and H3K9diMe, dimethylation at Lys4 or Lys9 ofhistone H3, respectively; H3K4triMe and H3K9triMe, trimethylation at Lys4 or Lys9 of histone H3, respectively; H4, histone H4.
Anti-histone H4 was used as a loading control. (B) Immunofluorescence analysis of interphase chromatin from wild-type (wt) and TKO
cells using antibodies against trimethylated H3K9, trimethylated H3K4, HP1-β and HP1-γ. DNA was visualized with DAPI. Merged
images represent overlays of immunofluorescence signal (green or red) and DAPI (blue) as indicated. “Low” and “high” indicate low andhigh magnification, respectively.

Maintenance of global chromatin structures in TKO
ES cells

We next examined whether the absence of CpG methylation affects the post-translational modification ofhistone termini in ES cells. We assessed global histone
acetylation and methylation by Western blotting using
modification- and site-specific antibodies against histones
H3 and H4. Acetylation at lysines of histones H3 and H4
and methylation at Lys4 of histone H3 (H3K4) are generally
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associated with transcriptionally active chromatin, whereas
methylation at Lys9 of histone H3 (H3K9) is associatedwith transcriptionally repressed chromatin. Overall, theamounts of histone acetylation and methylation in TKO
cells at most sites detected by the antibodies were similar
to those in wild-type cells (Fig. 4A). We did not detect asignificant increase in the acetylation of histone H4 at
Lys5 in TKO cells, which was observed in Dnmt3a–/–Dnmt3b–/– ES cells of high passage numbers ( Jackson
et al. 2004).

© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Dnmt1/3a/3b triple knockout ES cells

We also investigated whether the absence of CpG
methylation affects higher-order chromatin structure in
ES cells. Trimethylation of H3K9 and co-localization of
HP1 are characteristic features of pericentromeric heterochromatin, whereas methylation of H3K4 is normallyabsent in this region (Lehnertz et al. 2003; Maison &Almouzni 2004). Immunofluorescence analysis of TKO
cells showed that the focal staining patterns of trimethylated H3K9 and HP1 overlapped with DAPI-dense
heterochromatin regions, similar to the results forwild-type cells (Fig. 4B). The diffuse speckled patternsof trimethylated H3K4 found in TKO cell nuclei werealso indistinguishable from those of wild-type cells (Fig. 4B).
We did not observe a redistribution of H3K4 methylation
to pericentromeric heterochromatin in TKO cells, as
observed in hypomethylated mouse fibroblasts deficientfor Lsh (Yan et al. 2003), a member of the SNF/helicase
family involved in the control of global CpG methylation.
Furthermore, analysis of metaphase chromosomes showedthat TKO cells retained normal chromosomal number
and banding patterns (Supplementary Fig. S2 and data notshown), indicating that condensation and segregationof chromosomes during mitosis occur properly in theabsence of CpG methylation in ES cells.

Discussion
We have established the first mammalian cells deficientfor the three known CpG DNA methyltransferases; CpG
methylation is apparently absent in these cells. Our results
provide direct genetic evidence that CpG methylation isdispensable for ES cell growth in an undifferentiatedstate, as suggested previously (Lei et al. 1996; Chen et al.
2003; Jackson et al. 2004).This stands in clear contrast to
cases involving other cell types. Loss of Dnmt1 in mouseembryonic fibroblasts leads to growth arrest associatedwith increased expression of p21Waf1/Cip1 and p57Kip2,
and p53-dependent cell death ( Jackson-Grusby et al.
2001); moreover, the loss of both DNMT1 and DNMT3B
in human colon cancer cell lines causes growth impairment that is dependent on re-expression of p16INK4A(Rhee et al. 2002; Bachman et al. 2003). The unique cell
cycle regulation of mouse ES cells (Hong & Stambrook2004) may explain the robust growth and viability of our
TKO cells.
Reduction of Dnmt1 activity in mice has been shownto induce tumors and chromosomal instability (Edenet al. 2003; Gaudet et al. 2003), and global hypomethylation in male germ cells results in meiotic abnormality(Bourc’his & Bestor 2004; Hata et al. 2006). Furthermore, inactivation of Dnmt3b in primary mouse embryonic fibroblast cells causes aneuploidy and chromosomal

breaks and fusions (Dodge et al. 2005). These results
suggest that proper CpG methylation level is importantfor chromosomal stability, at least in certain cell types. It
remains unknown why undifferentiated ES cells retain
chromosomal stability without CpG methylation and
CpG DNA methyltransferases. One possibility is that ES
cells maintain stable heterochromatin and chromosomesby an epigenetic mechanism that is independent of CpG
methylation. Previous studies have shown that ES cells
have both unique global chromatin modifications andheterochromatin arrangement, and that chromatin proteins bind more loosely to chromatin in ES cells than indifferentiated cells, indicating the existence of specificepigenetic regulatory mechanisms in ES cells (Kimura
et al. 2004; Meshorer et al. 2006). The absence or reduction of such mechanisms may be attributed to thechromosomal abnormality in differentiated cell types
with decreased levels of CpG methylation.Taken together,
these results suggest that the relative contribution of CpG
methylation to both chromosomal stability and epigenetic gene silencing varies in a cell type-specific manner.
Our results suggest that at least some functional
properties of heterochromatic domains are retained in
the absence of CpG methylation in ES cells. However,
it is possible that some unrecognized changes in heterochromatin occur in TKO cells. Indeed, the redistributionof the histone variant macroH2A into pericentromericheterochromatin in hypomethylated Dnmt1–/– ES cells
has recently been reported (Ma et al. 2005), indicatingthat CpG methylation modulates some properties ofheterochromatic domains. Although the apparent
contribution of CpG methylation to the steady-state
heterochromatin structure in ES cells may be small, it ispossible that CpG methylation might serve as a mark foranchoring heterochromatin during dynamic chromatin
remodeling or during multiple replication processes.
Further studies in this line are required for understanding
the exact role of CpG methylation in higher-orderchromatin structures.
The genomes of primordial germ cells and preimplantation embryos are extensively hypomethylated during
normal embryogenesis (Hajkova et al. 2002; Li 2002),
and global reduction of CpG methylation is a common
feature of neoplasia (Feinberg et al. 2006). The generation of these triple methyltransferase-deficient ES cells
will be useful in studying the role of CpG methylationin cell type-specific epigenetic regulation during normal
and pathological processes.These cells will be also usefulin investigating the function of chromatin modifications
and remodeling in the absence of CpG methylation andendogenous CpG DNA methyltransferases in mammaliancellular conditions.

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Experimental procedures
ES cell culture
ES cells were maintained in Glasgow modified Eagle medium(Sigma) supplemented with 15% foetal bovine serum, 0.1 mm
nonessential amino acids (Invitrogen), 1 mm sodium pyruvate,
0.1 mm 2-mercaptoethanol and 2000 U/mL of leukemia inhibitory factor (LIF) and were grown on gelatinized culture dishes
without feeder cells. For growth curve experiments, 0.9 × 105 ES
cells were plated on gelatinized 10-cm culture dishes on the firstday. Then, ES cells were passaged every 2 days with 6- to 12-folddilution. For induction of differentiation by embryoid bodyformation, ES cells were seeded on low cell-binding dishes(Nunc) in medium without LIF. For growth competition analysis,wild-type and TKO cells were mixed at a 1 : 1 ratio and cultured
either as undifferentiated ES cells or as embryoid bodies, as previously described (Lei et al. 1996). The change in cell population
between wild-type and TKO cells was determined by Southern
analysis using the Dnmt3b probe, which distinguishes between
wild-type and TKO cells.

Generation of Dnmt1–/–Dnmt3a–/–Dnmt3b–/–
ES cells
The following nucleotide positions of Dnmt1 genomic DNA all
refer to AC073775.2. The Dnmt1 targeting vector, pTA009, wasconstructed by subcloning the 3-kb 5’ arm (nt 66903–70025) andthe 4-kb 3’ arm (nt 71625–76570) of Dnmt1 genomic DNA anda splicing acceptor–internal ribosomal entry site–blasticidin resistant gene cassette flanked by loxP sites (loxP-SAiresBlas-loxP) into
pBluescript II SK. The Dnmt1 genomic fragments were amplified
by PCR from genomic DNA of ES cells (Supplementary
Table S1). For targeting of the first Dnmt1 allele, the Dnmt1
targeting vector was transfected in Dnmt3a–/–Dnmt3b–/– ES cells(Okano et al. 1999) via electroporation, and transfected cells wereselected with 5 µg/mL blasticidin. Drug-resistant clones werescreened by Southern hybridization using a 5′ probe (nt 65650–
66729) and a 3′ probe (nt 76647–77192) external to the targeting
construct (Fig. 1A). The targeting frequency was 14% (3/22). To
generate blasticidin-sensitive ES cells, Cre recombinase wastransiently expressed in blasticidin-resistant Dnmt1+/–Dnmt3a–/–Dnmt3b–/– ES cells (clone 18) by lipofection of a circular Cre
expression plasmid. For targeting of the second wild-type Dnmt1
allele, the same targeting vector was transfected into blasticidinsensitive Dnmt1+/–Dnmt3a–/–Dnmt3b–/– ES cells (clone 67), and thetransfected cells were selected with 3.5 µg/mL blasticidin.
Targeting frequency was 4% (5/113).

DNA methylation analysis
For Southern analysis, genomic DNA was digested with the
CpG methylation-sensitive restriction enzymes HpaII or MaeII(Roche), blotted and hybridized with probes specific for C-type
endogenous retroviruses (pMO), IAP, minor satellite repeats, or
major satellite repeats (Okano et al. 1999). For bisulfite sequencing

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analysis of specific sequences, 2 µg of genomic DNA digested
with HindIII was treated with bisulfite followed by deamination(Clark et al. 1994). Deaminated fragments were amplified withprimers for IAP, LINE1, major satellite repeats, Igf2r or Snrpn(Supplementary Table S1).We sequenced 15–30 clones for repetitive
sequences and 9–15 clones for single-copy genes. Bisulfite
sequencing of random genomic fragments was carried out as previously described (Ramsahoye et al. 2000). The sequences werealigned to the mouse genome sequence, NCBI Mouse Build
33 (Y.K. and H.R.U., unpublished data). Sequenced clones
containing five non-CpG cytosines per sequenced clone thatremained unconverted to uracil by the bisulfite reaction were
excluded from the analysis. Overall, 80% of the sequenced clones
longer than 51 bp could be mapped to the mouse genome.
For localization of the methyl-CpG-binding protein, the GFPMBD1(MBD+NLS) plasmid, which encodes an enhanced green
fluorescent protein (EGFP) fused to the amino terminus of human
MBD1 containing both a methyl-CpG-binding domain and anuclear localization signal (Fujita et al. 1999; gift from M. Nakao),
was transfected into the ES cells by Lipofectamine (Invitrogen).
After 16 h of culture, the transfected cells were fixed with 4%paraformaldehyde and 0.5% [v/v] Triton X-100 in phosphatebuffered saline (PBS) and were stained with DAPI.

RT-PCR and Northern blotting
RNA samples were isolated from subconfluent cells using
the Trizol RNA extraction reagent (Invitrogen). Primer sets for
Dnmt1, Dnmt3a, Dnmt3b, Oct3/4, Rex1, Nanog, Fgf4 and Gapdh
were used for RT-PCR (Supplementary Table S1). Poly(A)+ RNA
was prepared using the Poly(A) Tract purification system (Promega).
Poly(A)+ RNA (1 µg) was used for Northern analysis.The LINE1
probe was amplified by PCR (Supplementary Table S1).

Antibodies, Western blotting andimmunofluorescence analysis
The following antibodies were used: anti-Dnmt1 (sc-20701) andanti-[acetyl-Lys5]H4 (sc8659-R) from Santa Cruz Biotechnology; anti-Dnmt3a (IMG-268) and anti-Dnmt3b (IMG-184) fromIMGENEX; anti-α-tubulin (CP06) from Oncogene; anti-acetylated
histone H3 (Lys9, Lys14; 06–599), anti-acetylated histone H4(Lys5, Lys8, Lys12, Lys16; 06– 866), anti-[acetyl-Lys8]H4 (17-211),
anti-[acetyl-Lys12]H4 (17-211), anti-[acetyl-Lys16]H4 (06-762
and 07-329), anti-[dimethyl-Lys4]H3 (07–030), anti-[dimethylLys9]H3 (07-212) and anti-histone H3 (06-755) from Upstate;anti-[acetyl-Lys16]H4 (ab1762), anti-[trimethyl-Lys4]H3 (ab8580)
and anti-[trimethyl-Lys9]H3 (ab8898) from Abcam; and antiHP1β (1MOD 1A9) and anti-HP1γ (2MOD 1G6) fromEUROMEDEX. Alexa 488- and Alexa 546-conjugated secondary
antibodies were purchased from Molecular Probes. A polyclonal
antibody for Dnmt1 was raised against a modified linear peptide,
CRESASAAVKAKEEAATKD, corresponding to the carboxy
terminus of mouse Dnmt1. For Western blot analysis, whole-cell
extracts were prepared by sonication in SDS-PAGE sample buffer,
fractionated on an 8% SDS-PAGE gel and blotted using standard© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Dnmt1/3a/3b triple knockout ES cells
procedures. Histones from ES cell nuclei were extracted with
0.2 mol/L H2SO4, precipitated with trichloroacetic acid (final concentration 25%), fractionated on an 18% SDS-PAGE gel and blottedin 10 mm CAPS (pH 11.0)/20% methanol. Indirect immunofluorescence staining was performed essentially as described (Lehnertz
et al. 2003), with some modifications as follows. Wild-type andknockout ES cells were cultured in 35-mm glass-bottom dishes(MatTek) precoated with 0.2% gelatin in PBS.The cells were fixedwith 2% formaldehyde in PBS, permeabilized with 0.1% sodium
citrate containing 0.1% [v/v] Triton X-100 followed by incubation in blocking buffer (PBS, 2.5% BSA, 0.1% [v/v] Tween-20,
10% fetal bovine serum). Primary antibodies (diluted in blocking
buffer) were added to fixed cells. After washing with PBS, 0.25%
BSA, 0.1% [v/v] Tween-20, cells were re-fixed with 2% formaldehyde in PBS and then incubated with secondary antibody. Afterseveral washes with PBS, 0.1% [v/v] Tween-20, the stained cells
were processed sequentially with 20%, 40%, 60% and 80% [v/v]
glycerol in PBS containing 2.5% 1,4-diazobicyclo-(2,2,2)-octane(DABCO) for mounting. The 20% and 40% glycerol contained
0.5 µg/mL DAPI. Finally, cells were mounted in 90% glycerol
in distilled water for microscopic observation. Fluorescencemicroscope images were acquired using a DeltaVision microscope
system (Applied Precision). Three-dimensional optical section
images were taken at 0.5-µm focus intervals and computationallyprocessed using SoftWoRx software (Applied Precision).

Acknowledgements
We thank H. Niwa for the original SAiresBlas cassette, M. Nakao
for the GFP-MBD plasmid, M. Oda for suggestions regarding ES
cell characterization, and D. Sipp, M. Royle and S. Hayashi for
critical reading.This work was supported in part by Grants-in-Aid
from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan.

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Received: 10 February 2006
Accepted: 12 April 2006

Supplementary material
The following supplementary material is available for this article
online:Figure S1 Growth curve analysis for TKO cells.
Figure S2 Chromosome analysis by Giemsa staining for TKO
cells.
Table S1 Oligonucleotides used in this study.

© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.