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First published online February 24, 2006
doi: 10.1242/10.1242/dev.02293
1 Cardiovascular Research Center, Massachusetts General Hospital, Harvard
Medical School, 149 13th Street, Charlestown, MA 02129, USA.
2 Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard
Medical School, 149 13th Street, Charlestown, MA 02129, USA.
3 Center for Developmental Biology, RIKEN, Kobe Hyogo 650-0047, Japan.
4 Epigenetics Program, Novartis Institutes for Biomedical Research, 250
Massachusetts Avenue, Cambridge, MA 02139, USA.
* Author for correspondence (e-mail: en.li{at}pharma.novartis.com)
Accepted 18 January 2006
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: DNA methylation, Dnmt3b, ICF syndrome, T cell, Apoptosis
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Wijmenga et al., 2000).
Individuals with this disease show combined immunodeficiency, including
absence or severe reduction of at least two classes of immunoglobulin and a
reduced number of T cells, making them prone to infections and death before
adulthood. Centromeric instability is characterized by the formation of
radiated chromosomes (chromosome 1, 9 and 16) due to demethylation at cytosine
residues in classical satellites 2 and 3 at juxtacentromeric regions of these
chromosomes. Facial anomalies include hypertelorism, flat nasal bridge, low
set ears, protrusion of the tongue, epicanthal folds, micrognathia and high
forehead. Most individuals also exhibit growth and mental retardation. Several
studies have demonstrated that ICF syndrome is caused by homozygous or
compound heterozygous mutations of the DNA methyltransferase 3B
(DNMT3B) gene (Hansen et al.,
1999; Okano et al.,
1999; Xu et al.,
1999).
Dnmt3b is one of the three active DNA cytosine methyltransferases
identified in human and mouse (Okano et
al., 1998; Xie et al.,
1999). Dnmt3a and Dnmt3b have high structural similarity, and both
can carry out de novo methylation in embryonic stem cells and during embryonic
development (Okano et al.,
1999). Although these two enzymes exhibit overlapping functions
during early development, each has distinct expression patterns, genomic
targets and functions (Chen et al.,
2003; Okano et al.,
1999). Although Dnmt3a deficient mice develop to term and
appear to be normal at birth, Dnmt3b deficient mice are embryonic
lethal. Recently, Dnmt3a, but not Dnmt3b, has been shown to be essential for
the establishment of methylation imprints during gametogenesis
(Kaneda et al., 2004). Genetic
analysis of Dnmt3a and Dnmt3b function in embryonic stem
cells has revealed that Dnmt3a and Dnmt3b, and their different variants, have
shared as well as distinct DNA targets
(Chen et al., 2003). However,
the specific functions of Dnmt3a or Dnmt3b in vivo have not been fully
analyzed yet.
To investigate the function of Dnmt3b in mouse development and to determine whether Dnmt3b mutations result in phenotypes similar to those of individuals with ICF syndrome, we generated mice with point mutations in Dnmt3b corresponding to the mutations found in human patients (ICF mice). Our studies of Dnmt3b null and ICF mutant mice show that Dnmt3b is essential for mouse embryonic development, and that the ICF mice exhibit phenotypes that resemble some of the symptoms of the human ICF syndrome. We also demonstrate that Dnmt3b is essential for the survival of T cells in the thymus of newborn mice.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Hata et al., 2002). Human
DNMT3B cDNA was synthesized by RT-PCR from total RNA of NT-2 cells,
by using the oligonucleotides 5'-ATGAAGGGAGACACCAGGC-3' and
5'-GCCTGGCTGGAACTATTCAC-3' as primers, and subcloned into
pCAG-IRESblast vector. The cDNAs of ICF mutants were generated by a PCR-based
site-directed mutagenesis experiment, by using mouse Dnmt3b1 or human
DNMT3B cDNA as template, and subcloned into the pEGFP-C1 (CLONTECH)
and/or the pCAG-IRESblast vector. The Dnmt3b A609T and D823G knock-in
vectors were generated by sequentially subcloning Dnmt3b fragments
and a floxed IRES-ßgeo cassette, in which IRES-ßgeo is flanked by
loxP sites, into pBluescript II SK. The fragments containing A609T and D823G
mutations were generated by PCR using a bacterial artificial chromosome clone
as a template and the following oligonucleotides as primers:
5'-CTGGAGCTGCTATATGTGCC-3',
5'-GGAAAAGTACATTACCTCCGA-3',
5'-CACAGACTTCGGAGGTAATG-3' and
5'-TTGGTGATTTTCCGGACGTC-3' for A609T; and
5'-CAGACAGGGCAAAAACCAGC-3' and
5'-CCGCGGCCCATGTTGGACACGCC-3' for D823G. The other Dnmt3b
fragments were obtained from a bacterial artificial chromosome clone. The
identities of all constructs were verified by DNA sequencing.
Protein expression and analysis
Transient transfection was carried out in COS-7 cells for
immunoprecipitation assay and NIH3T3 cells for fluorescence microscopy
analysis, using LipofectAMINE PLUS reagent (Invitrogen). Immunoprecipitation,
immunoblotting and fluorescence microscopy analyses were performed as
previously described (Chen et al.,
2002; Hata et al.,
2002). Stable transfection of Dnmt3b-expression vectors
in ES cells was performed according to a procedure described previously
(Chen et al., 2003). The
antibodies used in these experiments were anti-myc (Roche), anti-GFP (Roche),
anti-Dnmt3b, anti-Dnmt3a (clone 64B1446; Imgenex), and anti-
-tubulin
(Ab-1; Oncogene Research Products).
Generation of ICF mutant mice
The Dnmt3b A609T and D823G targeting vectors were transfected into
ES cells via electroporation, and transfected cells were selected with G418,
as previously described (Li et al.,
1992). Clones with homologous recombination were identified by
Southern blot analysis. Genomic DNA from ES cell clones was digested with
BamHI, blotted and hybridized to an external probe. The wild-type,
null, A609T and D823G alleles give fragments of 17 kb, 5.7 kb, 6.5 kb, and 14
kb, respectively (Fig. 2D).
Chimeric mice and F1 heterozygotes were generated from multiple A609T or D823G
mutant ES cell lines as described (Li et
al., 1992). Mutant mice were maintained on a
129SvJaexC57BL/6 hybrid background and analyzed.
DNA methylation analysis
Genomic DNA isolated from ES cells, E12.5 embryos, tissues of newborns, and
tails of adult mice was digested with methylation-sensitive restriction
enzymes and analyzed by Southern hybridization as previously described
(Chen et al., 2003;
Lei et al., 1996). The probes
used for methylation analysis were: pMR150 for minor satellite repeats, pMO
for endogenous C-type retroviruses, and an oligonucleotide probe for major
satellite repeats.
Histological analysis, TUNEL assay, and skeletal staining
For histological analysis and TUNEL assay, tissues were fixed in 10%
buffered formalin and processed into paraffin wax-embedded sections using
routine procedures. For general morphology, deparaffinized sections were
stained with Hematoxylin and Eosin using standard procedures. TUNEL assay was
performed using the ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit
(Serologicals Corporation), by following the manufacturer's protocol. For
skeletal staining, mice were skinned, placed in 1.5% KOH and macerated. After
4-5 days, the mice were stained in Alizarin Red for 3 days, and then placed in
1.5% KOH for 1 day to remove the excess stain. The stained bones were then
transferred to glycerol.
Flow cytometric analysis
Cells from the thymus, spleen, and bone marrow were prepared and analyzed
for the expression of surface differentiation antigens as described
(Georgopoulos et al., 1994;
Winandy et al., 1995). Flow
cytometric analysis was performed using a FACScan flow cytometer (BD). All
antibodies used for staining were from PharMingen.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Wijmenga et al.,
2000; Xu et al.,
1999). STP813ins is a 9 bp insertion at codon 813 encoding three
amino acids, serine, threonine and proline, corresponding to the change found
in a patient with ICF syndrome (Okano et
al., 1999).
First, the ability of Dnmt3b mutant proteins to interact with Dnmt3a was
examined. Immunoprecipitation analysis was performed using GFP-Dnmt3b fusion
proteins and myc-tagged Dnmt3a (Fig.
1B). As previously reported, Dnmt3b1 was co-immunoprecipitated
with Dnmt3a, as well as with Dnmt3b1 itself
(Kim et al., 2002). We also
showed the binding of endogenous Dnmt3a to endogenous Dnmt3b in ES cells by
co-immunoprecipitation assay (see Fig. S1 in the supplementary material). The
A609T mutation disrupted the interaction with Dnmt3a, as well as with Dnmt3b1.
The other ICF mutations did not affect this interaction. Further mutagenesis
analysis revealed that the region of Dnmt3b required for its interaction with
Dnmt3a is in the amino-terminal region of the catalytic domain, including
alanine 609 (see Fig. S1 in the supplementary material), suggesting that the
A609T mutation alters the conformation of the Dnmt3a interaction domain.
We next examined the subcellular localization of these mutant proteins
using GFP fusion proteins. As described before
(Bachman et al., 2001;
Chen et al., 2004), wild-type
Dnmt3b1 and Dnmt3b2 displayed punctate nuclear localization patterns with two
major types of nuclear foci: large foci, which corresponded to pericentric
heterochromatin; and small foci, which were distributed throughout the
nucleus, excluding the nucleoli. Both types of foci were present in the
majority of transfected cells (pattern A); however, only the small foci were
visible in some transfected cells (pattern B). By contrast, Dnmt3b3, which
lacks part of motif IX in the catalytic domain and has no enzymatic activity,
displayed diffuse nuclear localization patterns with or without accumulation
in pericentric heterochromatin (patterns C and D, respectively). Four of the
ICF mutations, A609T, V732G, STP813 and D823G, exhibited obvious changes in
localization patterns when compared with wild-type Dnmt3b1. A609T showed no
accumulation in pericentric heterochromatin (
75% of transfected cells
showed pattern B and the rest showed pattern D), suggesting that Dnmt3a-Dnmt3b
heterodimerization and/or Dnmt3b homodimerization may be required for
targeting Dnmt3b to pericentric heterochromatin. V732G, STP813 and D823G
showed diffuse patterns similar to those of Dnmt3b3, indicating that these
mutations disrupt the association of Dnmt3b with the small type of nuclear
foci. Although the identity of these foci remains to be determined, it is
possible that these structures correspond to heterochromatin regions, which
usually consist of repetitive DNA sequences, including satellite repeats.
Failure to target Dnmt3b to heterochromatin may thus contribute to
demethylation of satellite DNA, a hallmark of ICF syndrome. The other two
mutations, L670T and A772P, did not affect the localization patterns of
Dnmt3b, although a minor increase in the number of cells exhibiting pattern B
was observed with the L670T mutation (Fig.
1C).
We then examined the effects of the ICF mutations on the methyltransferase
activity of Dnmt3b. Mouse Dnmt3b1, A609T, D823G and PC
(Dnmt3b1 with its PC motif mutated), as well as human
DNMT3B1, A603T and D817G, cDNAs were introduced into highly
demethylated Dnmt3a-/-Dnmt3b-/- ES
cells. Individual clones that expressed different levels of these proteins, as
determined by immunoblotting analysis, were obtained
(Fig. 1D). DNA methylation
patterns were examined using genomic DNA isolated from these clones. As
reported previously (Chen et al.,
2003), expression of wild-type Dnmt3b1 substantially restored the
methylation levels of all the repetitive sequences examined. We found that
human DNMT3B1 could also restore the methylation levels of the mouse
endogenous C-type retroviral DNA (Fig.
1E), and of the major and minor satellite repeats (data not
shown). By contrast, mouse A609T and D823G, as well as human A603T and D817G,
proteins failed to restore the DNA methylation of these repetitive sequences,
suggesting that these ICF mutants have little or no methyltransferase
activity.
Taken together, our results suggest that different ICF mutations result in a loss of function via different mechanisms; some by disrupting protein-protein interactions and others by altering protein localization.
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). We have
previously generated a Dnmt3b null allele in which the region of the
catalytic domain that encompassed the highly conserved PC and ENV motifs was
deleted (Okano et al., 1999).
The three different mutant alleles of Dnmt3b are shown in
Fig. 2B. These alleles could be
distinguished by Southern blot analysis
(Fig. 2C), and the point
mutations were confirmed by sequence analysis of genomic DNA extracted from
the tails of the heterozygous mice (data not shown). Mice carrying A609T and
D823G mutations produced full-length proteins at levels similar to those of
wild-type mice, whereas no Dnmt3b protein was detected in
Dnmt3b-/- mice, as determined by immunoblotting of E12.5
embryo extracts with Dnmt3b antibody (Fig.
2D). The expression of Dnmt3a and Dnmt3a2 proteins was not
affected by the Dnmt3b mutations
(Fig. 2D). We tried to confirm
the effects of the A609T and D823G mutations on Dnmt3b subcellular
localization in vivo. Immunostaining of wild-type and mutant embryos at E8.5
and E12.5, as well as of wild-type embryonic fibroblasts, only detected weak
signals of a diffuse nuclear pattern (data not shown), consistent with the
observations that the level of Dnmt3b is low (as compared with that in ES
cells) and that the major isoform is Dnmt3b3 at these developmental stages
(Fig. 2D).
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), so no homozygous newborn mice were found
(Fig. 3A). By contrast, normal
Mendelian frequencies of mice with T/-, G/-, T/T, G/G and T/G genotypes were
found at birth, but most of the mutant mice died within 24 hours after birth.
A few mice survived longer than 24 hours, and only four mice (three T/T mice
and one T/G mice) were found at weaning age (about 3 weeks). These differences
in lethality between Dnmt3b-/- and the ICF mutants
indicate that the ICF mutations are not complete loss-of-function alleles.
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The Dnmt3b null mutation results in embryonic lethality with multiple tissue defects
To investigate the role of Dnmt3b in mouse development, we first analyzed
Dnmt3b-/- embryos. Dissection of embryos revealed that
most of the Dnmt3b-/- mice were lethal between 13.5 days
post-coitum (dpc) and 16.5 dpc (Fig.
4A), showing progressive necrosis. All of the live
Dnmt3b-/- embryos that were recovered at this stage showed
subcutaneous edema and liver atrophy (Fig.
4B,C,F), which became apparent at 13.5 dpc. Some of the
Dnmt3b-/- embryos showed ectopic hemorrhage in the head
region (Fig. 4B). Furthermore,
genotyping revealed that Dnmt3b-/- embryos were slightly
underrepresented from the expected Mendelian ratio at 11.5-13.5 dpc
[Fig. 4A,
Dnmt3b+/+:Dnmt3b+/-:Dnmt3b-/-
= 57(27%): 100(48%): 33(14%)], suggesting an earlier lethality of
Dnmt3b-/- embryos in a small population (about 30%).
Occasionally, we recovered abnormal Dnmt3b-/- embryos
between 9.5 and 12.5 dpc (Okano et al.,
1999), but the cause of lethality was unclear.
Serial sections of the thoracic region of Dnmt3b-/-
embryos revealed that the ventricular septum was not closed in the heart of
Dnmt3b-/- embryos at 14.5 and 15.5 dpc
(Fig. 4D, and data not shown),
although ventricular septum closure is normally completed by 13.5 dpc in mice
(Webb et al., 1998). Other
histological features of the heart, such as trabeculation, were normal in
Dnmt3b-/- embryos. We also found hemorrhaging in the
middle of the dorsal root ganglia and in the limb region
(Fig. 4E, and data not shown),
which suggests defects of blood vessel formation or maintenance in
Dnmt3b-/- embryos. We hypothesize that the ventricular
septum defect and blood vessel abnormalities account for the subcutaneous
edema of Dnmt3b-/- embryos.
The fetal liver of Dnmt3b-/- embryos was much smaller (about one fifth) than that of normal littermates (Fig. 4F). Because the fetal liver is a major hematopoietic organ at this stage, we examined expression of the late erythroid marker TER-119 and the hepatocyte marker albumin in the liver of Dnmt3b-/- embryos by immunohistochemistry (data not shown). However, we did not see a dramatic change in the erythroblast or hepatocyte populations, suggesting that a defect in hematopoiesis was not the sole reason for this phenotype. We also did not observe significant differences in BrdU incorporation or TUNEL staining in the mutant fetal liver (data not shown), suggesting that the proliferation and apoptosis of hepatocytes was normal.
Mice with ICF mutations develop to term
In contrast to Dnmt3b-/- animals, mice with ICF
mutations (T/T, G/G, T/G, T/- and G/-) developed to term and were alive at
birth. These mice showed no sign of edema or liver atrophy, although some
exhibited hemorrhage in the head region. The gross anatomy of the mutant mice
appeared to be normal, although the body size of the mutant mice was much
smaller than that of the control mice (Fig.
5A,B). The body weight of the mutant mice was less than two-thirds
of that of the normal littermates at birth, and the adult mutant mice that
survived remained smaller (Fig.
5C).
All four types of adult ICF mice showed characteristic facial anomalies, such as shorter nose and wider nasal bridge, as shown in Fig. 5D. These features are similar to the hypertelorism and the flat nasal bridge that are frequently seen in individuals with ICF syndrome. The craniofacial defects were further characterized after skeletal preparations were made (Fig. 5E). The calvarium of adult T/T mice showed abnormally shaped frontal bones. The frontal bones of ICF mice were wider than those of normal littermates, resulting in a wider distance between the eyes. The lateral view revealed that the frontal and parietal bones were round shaped (data not shown). The nasal bone of T/T mice was significantly shorter, whereas the axial length of the calvarium of the mutant mice was the same as that of the control mice. The skeletons of newborn ICF mice showed a similar defect to that of the adults (Fig. 5F). An enlarged frontal fontanel was present in the mutant, indicating that ossification was delayed at the newborn stage. These craniofacial defects were observed in all newborn and adult mice that had T/T, G/G, T/G, T/- or G/- mutations.
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DISCUSSION
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). However,
the function of Dnmt3a or Dnmt3b alone in development remains largely unknown.
In this study, we showed that mice homozygous for a null mutation of
Dnmt3b die at the late gastrula stage, whereas mice carrying missense
mutations of two human ICF syndrome alleles develop to term. Analysis of
Dnmt3b mutant mice has revealed that Dnmt3b is essential for the
development of the fetal heart, liver and craniofacial structures, and for the
survival of T cells at the newborn stage.
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;
Wijmenga et al., 2000).
Because Dnmt3b null mutant mice die during fetal development
(Okano et al., 1999), it is
believed that individuals with ICF syndrome must have residual DNMT3B activity
and that a complete loss of function of DNMT3B is probably incompatible with
fetal development in humans. Indeed, most ICF patients carry missense
mutations in or near the catalytic domain of DNMT3B and none were homozygous
for nonsense alleles (Hansen et al.,
1999; Okano et al.,
1999; Shirohzu et al.,
2002; Wijmenga et al.,
2000; Xu et al.,
1999), suggesting that the complete loss of function of DNMT3B is
lethal. This also implies that most missense mutations in individuals with ICF
may not be complete loss-of-function alleles. This notion was supported by the
in vitro analysis of six different missense mutations in the catalytic domain
of Dnmt3b (Gowher and Jeltsch,
2002). The catalytic activities of the mutant Dnmt3b was shown to
be reduced 10- to 50-fold, but was still detectable. However, in vivo studies
using cultured mammalian cells expressing Dnmt3b missense mutants
characteristic of ICF syndrome failed to detect de novo methylation of
episomal DNA (Xu et al.,
1999). In this study, we tested the function of several Dnmt3b mutations in mouse ES cells. We showed that Dnmt3b cDNA containing characteristic ICF mutations failed to induce detectable de novo methylation of repetitive sequences after being introduced into mouse ES cells lacking endogenous Dnmt3a and Dnmt3b (Fig. 1E). However, introduction of two of the ICF mutations into the mouse Dnmt3b gene did not result in embryonic lethality. The fact that these mice survive to term and adult stages, and show significantly higher levels of genomic methylation than do Dnmt3b null embryos, strongly indicates that these ICF mutations are not null, but are hypomorphic alleles. The reason for the difference between the cell culture and in vivo studies is unknown. It is possible that the introduction of the ICF mutations into the endogenous Dnmt3b gene by homologous recombination may allow more precise regulation of Dnmt3b than the introduction of mutant cDNA into ES cells. Dnmt3b may also function more efficiently in whole embryos than in cultured cells.
Developmental defects in Dnmt3b-deficient mice
We showed that all Dnmt3b-/- mice die during embryonic
development between 14.5 and 16.5 dpc as a result of multiple developmental
defects, whereas mice expressing point mutations in one or both alleles
survive to term. The residual Dnmt3b activity in the ICF mutants is therefore
sufficient to allow mutant mice to escape from some of the early developmental
defects shown in Dnmt3b null embryos.
The embryonic lethality of Dnmt3b-/- mice might be
caused by several different factors. Dnmt3b may normally repress the
expression of genes that control cell growth, and dysregulated activation of
these genes may lead to inappropriate growth arrest. We have shown that MEF
cells derived from Dnmt3b-/- embryos undergo premature
senescence (Dodge et al.,
2005). It is also possible that the multiple cardiovascular
defects observed in Dnmt3b-/- mice might lead to fetal
death, although the ventricular septal defect alone does not cause embryonic
lethality. Dnmt3b is highly expressed in the placental tissues and disruption
of Dnmt3b may lead to placental defects and fetal death.
The Dnmt3b hypomorphic mutations (ICF mutations) lead to a partial loss of
de novo methyltransferase activity and a decrease in overall methylation of
genomic DNA. Most of the Dnmt3b hypomorphic mutants die shortly after birth
but the cause of the newborn lethality remains unknown. We found hemorrhages
in the head region in some mice, similar to what was observed in
Dnmt3b-/- embryos. Unlike Dnmt3b-/-
embryos, no ventricular septum malformation was detected, although some mice
showed thickening of the myocardium. Other organs, including brain, lung,
liver, kidney, stomach, gut and spleen, appeared to be histologically
indistinguishable from their normal littermates. Several knockout mice with
global alterations of DNA methylation die shortly after birth. Conditional
deletion of Dnmt1 in neural progenitor cells (nestin promoter Cre) leads to
perinatal lethality due to respiratory failure in the pups
(Fan et al., 2001). Targeted
disruption of the Lsh gene, which encodes an SNF2 family protein,
also causes global hypomethylation and perinatal lethality
(Dennis et al., 2001;
Geiman and Muegge, 2000). Lsh
has been shown to regulate DNA methylation and histone modification
(Dennis et al., 2001;
Yan et al., 2003a;
Yan et al., 2003b). It remains
to be determined how Lsh and DNA methylation may regulate developmental
processes that are essential for postnatal survival.
Immune defects in Dnmt3b hypomorphic mutants
The two hypomorphic Dnmt3b mutant strains also showed defects in lymphocyte
homeostasis. We observed extensive apoptosis of T cells in the thymus of P1
newborn pups. T cell apoptosis appears to start after birth, as the thymocyte
profiles were normal in embryonic and newborn (P0) mice. This suggests that de
novo DNA methylation plays a crucial role in suppressing T cell apoptosis in
the newborn. As Dnmt3b functions primarily as a de novo methyltransferase, and
DNA methylation has been shown to regulate the expression of genes including
the T cell cytokine genes IL2, IL4 and interferon (INF)
(Bruniquel and Schwartz, 2003;
Fitzpatrick et al., 1998;
Makar et al., 2003), it is
possible that alterations in the expression of these genes may trigger cell
death.
Recent studies in mice have suggested an important role for DNA methylation
in lymphocytes. Mice carrying a hypomorphic Dnmt1 mutation, which causes
genome-wide hypomethylation in all tissues, have been shown to develop T cell
lymphomas that exhibit chromosomal instabilities
(Gaudet et al., 2003).
Conditional deletion of Dnmt1 in early double-negative thymocytes leads to
impaired survival of TCRß+ cells
(Lee et al., 2001), suggesting
that Dnmt1 is required for the maintenance of mature T cells. The importance
of DNA methylation in lymphocyte survival is also suggested by the phenotype
of Lsh deficient mice. Targeted deletion of Lsh results in global genomic
hypomethylation and perinatal lethality
(Dennis et al., 2001;
Geiman et al., 2001).
Injection of Lsh-/- fetal liver cells into Rag2-/- mice
to reconstitute lymphoid development caused a reduction in T cells and B cells
compared with controls (Geiman and Muegge,
2000). These results suggest that DNA methylation plays a crucial
role in the maintenance and normal function of lymphocytes. The specific
target genes regulated by DNA methylation during T cell differentiation,
however, remain largely unknown.
Mouse models of ICF syndrome
DNMT3B mutations primarily affect the lymphocytes of individuals with ICF
syndrome. Characteristic symptoms include agammaglobulinemia and combined
immunodeficiency (Ehrlich,
2003). ICF syndrome patients also exhibit hypomethylation of
satellite 2 repeats in the pericentromeric heterochromatin, and rearrangements
of chromosomes 1, 9 and 16 via hypomethylated satellite 2 in lymphocytes to
form multiradiate chromosomes (Jeanpierre
et al., 1993; Xu et al.,
1999). It should be noted that chromosomal rearrangements have
been observed in bone marrow cells from only one of four patients studied
(Fasth et al., 1990;
Hulten, 1978;
Smeets et al., 1994;
Turleau et al., 1989), and
have never been detected in fibroblast cells derived from four ICF syndrome
patients (Brown et al., 1995;
Carpenter et al., 1988;
Maraschio et al., 1988;
Tiepolo et al., 1979). Facial
anomaly is another characteristic symptom afflicting individuals with ICF.
However, many of the ICF syndrome symptoms are rare and difficult to study
because of a limitation in the number of patients. The development of mouse
models are thus of great importance.
In this study, we describe mice with two independent ICF point mutations
(A609T and D823G) that show developmental defects including T cell defects,
facial anomaly and low body weight, which are common symptoms of human ICF
syndrome. In addition, genome-wide hypomethylation was observed in both the
ICF-like mice and human patients. In the Dnmt3b mutant mice, all
repetitive sequences tested were found to be hypomethylated, although the
methylation status of single genes has not been analyzed. In ICF patients,
hypomethylation of satellite 2, satellite 3, and non-satellite repeats D4Z4
and NBL2 has been observed (Jeanpierre et
al., 1993; Kondo et al.,
2000; Xu et al.,
1999). The striking resemblance of ICF-like Dnmt3b mutant
mice to individuals with ICF suggests that these mice will serve as good
models for understanding the etiology of ICF syndrome and will aid in the
identification of target genes that are regulated by DNA methylation during
development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/1183/DC1
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Bachman, K. E., Rountree, M. R. and Baylin, S. B.
(2001). Dnmt3a and Dnmt3b are transcriptional repressors that
exhibit unique localization properties to heterochromatin. J. Biol.
Chem. 276,32282
-32287.
Brown, D. C., Grace, E., Sumner, A. T., Edmunds, A. T. and
Ellis, P. M. (1995). ICF syndrome (immunodeficiency,
centromeric instability and facial anomalies): investigation of
heterochromatin abnormalities and review of clinical outcome. Hum.
Genet. 96,411
-416.[Medline]
Bruniquel, D. and Schwartz, R. H. (2003).
Selective, stable demethylation of the interleukin-2 gene enhances
transcription by an active process. Nat. Immunol.
4, 235-240.[CrossRef][Medline]
Carpenter, N. J., Filipovich, A., Blaese, R. M., Carey, T. L.
and Berkel, A. I. (1988). Variable immunodeficiency with
abnormal condensation of the heterochromatin of chromosomes 1, 9, and 16.
J. Pediatr. 112,757
-760.[CrossRef][Medline]
Chen, T., Ueda, Y., Xie, S. and Li, E. (2002).
A novel Dnmt3a isoform produced from an alternative promoter localizes to
euchromatin and its expression correlates with active de novo methylation.
J. Biol. Chem. 277,38746
-38754.
Chen, T., Ueda, Y., Dodge, J. E., Wang, Z. and Li, E.
(2003). Establishment and maintenance of genomic methylation
patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol.
Cell. Biol. 23,5594
-5605.
Chen, T., Tsujimoto, N. and Li, E. (2004). The
PWWP domain of Dnmt3a and Dnmt3b is required for directing DNA methylation to
the major satellite repeats at pericentric heterochromatin. Mol.
Cell. Biol. 24,9048
-9058.
Dennis, K., Fan, T., Geiman, T., Yan, Q. and Muegge, K.
(2001). Lsh, a member of the SNF2 family, is required for
genome-wide methylation. Genes Dev.
15,2940
-2944.
Dodge, J. E., Okano, M., Dick, F., Tsujimoto, N., Chen, T.,
Wang, S., Ueda, Y., Dyson, N. and Li, E. (2005). Inactivation
of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation,
chromosomal instability, and spontaneous immortalization. J. Biol.
Chem. 280,17986
-17991.
Ehrlich, M. (2003). The ICF syndrome, a DNA
methyltransferase 3B deficiency and immunodeficiency disease. Clin.
Immunol. 109,17
-28.[CrossRef][Medline]
Fan, G., Beard, C., Chen, R. Z., Csankovszki, G., Sun, Y.,
Siniaia, M., Biniszkiewicz, D., Bates, B., Lee, P. P., Kuhn, R. et al.
(2001). DNA hypomethylation perturbs the function and survival of
CNS neurons in postnatal animals. J. Neurosci.
21,788
-797.
Fasth, A., Forestier, E., Holmberg, E., Holmgren, G., Nordenson,
I., Soderstrom, T. and Wahlstrom, J. (1990). Fragility of the
centromeric region of chromosome 1 associated with combined immunodeficiency
in siblings. A recessively inherited entity? Acta Paediatr.
Scand. 79,605
-612.[Medline]
Fitzpatrick, D. R., Shirley, K. M., McDonald, L. E.,
Bielefeldt-Ohmann, H., Kay, G. F. and Kelso, A. (1998).
Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3
(IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma
promoter demethylation and mRNA expression are heritable in CD44 (high) CD8+ T
cells. J. Exp. Med. 188,103
-117.
Gaudet, F., Hodgson, J. G., Eden, A., Jackson-Grusby, L.,
Dausman, J., Gray, J. W., Leonhardt, H. and Jaenisch, R.
(2003). Induction of tumors in mice by genomic hypomethylation.
Science 300,489
-492.
Geiman, T. M. and Muegge, K. (2000). Lsh, an
SNF2/helicase family member, is required for proliferation of mature T
lymphocytes. Proc. Natl. Acad. Sci. USA
97,4772
-4777.
Geiman, T. M., Tessarollo, L., Anver, M. R., Kopp, J. B., Ward,
J. M. and Muegge, K. (2001). Lsh, a SNF2 family member, is
required for normal murine development. Biochim. Biophys.
Acta 1526,211
-220.[Medline]
Georgopoulos, K., Bigby, M., Wang, J. H., Molnar, A., Wu, P.,
Winandy, S. and Sharpe, A. (1994). The Ikaros gene is
required for the development of all lymphoid lineages.
Cell 79,143
-156.[CrossRef][Medline]
Gowher, H. and Jeltsch, A. (2002). Molecular
enzymology of the catalytic domains of the Dnmt3a and Dnmt3b DNA
methyltransferases. J. Biol. Chem.
277,20409
-20414.
Hansen, R. S., Wijmenga, C., Luo, P., Stanek, A. M., Canfield,
T. K., Weemaes, C. M. and Gartler, S. M. (1999). The DNMT3B
DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome.
Proc. Natl. Acad. Sci. USA
96,14412
-14417.
Hata, K., Okano, M., Lei, H. and Li, E. (2002).
Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to
establish maternal imprints in mice. Development
129,1983
-1993.[Medline]
Hulten, M. (1978). Selective somatic pairing
and fragility at 1q12 in a boy with common variable immunodeficiency.
Clin. Genet. 14,294
.
Jeanpierre, M., Turleau, C., Aurias, A., Prieur, M., Ledeist,
F., Fischer, A. and Viegas-Pequignot, E. (1993). An
embryonic-like methylation pattern of classical satellite DNA is observed in
ICF syndrome. Hum. Mol. Genet.
2, 731-735.
Kaneda, M., Okano, M., Hata, K., Sado, T., Tsujimoto, N., Li, E.
and Sasaki, H. (2004). Essential role for de novo DNA
methyltransferase Dnmt3a in paternal and maternal imprinting.
Nature 429,900
-903.[CrossRef][Medline]
Kim, G. D., Ni, J., Kelesoglu, N., Roberts, R. J. and Pradhan,
S. (2002). Co-operation and communication between the human
maintenance and de novo DNA (cytosine-5) methyltransferases. EMBO
J. 21,4183
-4195.[CrossRef][Medline]
Kondo, T., Bobek, M. P., Kuick, R., Lamb, B., Zhu, X., Narayan,
A., Bourc'his, D., Viegas-Pequignot, E., Ehrlich, M. and Hanash, S. M.
(2000). Whole-genome methylation scan in ICF syndrome:
hypomethylation of non-satellite DNA repeats D4Z4 and NBL2. Hum.
Mol. Genet. 9,597
-604.
Lakso, M., Pichel, J. G., Gorman, J. R., Sauer, B., Okamoto, Y.,
Lee, E., Alt, F. W. and Westphal, H. (1996). Efficient in
vivo manipulation of mouse genomic sequences at the zygote stage.
Proc. Natl. Acad. Sci. USA
93,5860
-5865.
Lee, P. P., Fitzpatrick, D. R., Beard, C., Jessup, H. K., Lehar,
S., Makar, K. W., Perez-Melgosa, M., Sweetser, M. T., Schlissel, M. S.,
Nguyen, S. et al. (2001). A critical role for Dnmt1 and DNA
methylation in T cell development, function, and survival.
Immunity 15,763
-774.[CrossRef][Medline]
Lei, H., Oh, S. P., Okano, M., Juttermann, 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.[Abstract]
Li, E., Bestor, T. H. and Jaenisch, R. (1992).
Targeted mutation of the DNA methyltransferase gene results in embryonic
lethality. Cell 69,915
-926.[CrossRef][Medline]
Makar, K. W., Perez-Melgosa, M., Shnyreva, M., Weaver, W. M.,
Fitzpatrick, D. R. and Wilson, C. B. (2003). Active
recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes
and T cells. Nat. Immunol.
4,1183
-1190.[CrossRef][Medline]
Maraschio, P., Zuffardi, O., Dalla Fior, T. and Tiepolo, L.
(1988). Immunodeficiency, centromeric heterochromatin instability
of chromosomes 1, 9, and 16, and facial anomalies: the ICF syndrome.
J. Med. Genet. 25,173
-180.
Okano, M., Xie, S. and Li, E. (1998). Cloning
and characterization of a family of novel mammalian DNA (cytosine-5)
methyltransferases. Nat. Genet.
19,219
-220.[CrossRef][Medline]
Okano, M., Bell, D. W., Haber, D. A. and Li, E.
(1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential
for de novo methylation and mammalian development.
Cell 99,247
-257.[CrossRef][Medline]
Shirohzu, H., Kubota, T., Kumazawa, A., Sado, T., Chijiwa, T.,
Inagaki, K., Suetake, I., Tajima, S., Wakui, K., Miki, Y. et al.
(2002). Three novel DNMT3B mutations in Japanese patients with
ICF syndrome. Am. J. Med. Genet.
112, 31-37.[CrossRef][Medline]
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 case and review of the literature. Hum.
Genet. 94,240
-246.[Medline]
Tiepolo, L., Maraschio, P., Gimelli, G., Cuoco, C., Gargani, G.
F. and Romano, C. (1979). Multibranched chromosomes 1, 9, and
16 in a patient with combined IgA and IgE deficiency. Hum.
Genet. 51,127
-137.[CrossRef][Medline]
Turleau, C., Cabanis, M. O., Girault, D., Ledeist, F., Mettey,
R., Puissant, H., Prieur, M. and de Grouchy, J. (1989).
Multibranched chromosomes in the ICF syndrome: immunodeficiency, centromeric
instability, and facial anomalies. Am. J. Med. Genet.
32,420
-424.[CrossRef][Medline]
Webb, S., Brown, N. A. and Anderson, R. H.
(1998). Formation of the atrioventricular septal structures in
the normal mouse. Circ. Res.
82,645
-656.
Wijmenga, C., Hansen, R. S., Gimelli, G., Bjorck, E. J., Davies,
E. G., Valentine, D., Belohradsky, B. H., van Dongen, J. J., Smeets, D. F.,
van den Heuvel, L. P. et al. (2000). Genetic variation in ICF
syndrome: evidence for genetic heterogeneity. Hum.
Mutat. 16,509
-517.[CrossRef][Medline]
Winandy, S., Wu, P. and Georgopoulos, K.
(1995). A dominant mutation in the Ikaros gene leads to rapid
development of leukemia and lymphoma. Cell
83,289
-299.[CrossRef][Medline]
Xie, S., Wang, Z., Okano, M., Nogami, M., Li, Y., He, W. W.,
Okumura, K. and Li, E. (1999). Cloning, expression and
chromosome locations of the human DNMT3 gene family.
Gene 236,87
-95.[CrossRef][Medline]
Xu, G. L., Bestor, T. H., Bourc'his, D., Hsieh, C. L., Tommerup,
N., Bugge, M., Hulten, M., Qu, X., Russo, J. J. and Viegas-Pequignot, E.
(1999). Chromosome instability and immunodeficiency syndrome
caused by mutations in a DNA methyltransferase gene.
Nature 402,187
-191.[CrossRef][Medline]
Yan, Q., Cho, E., Lockett, S. and Muegge, K.
(2003a). Association of Lsh, a regulator of DNA methylation, with
pericentromeric heterochromatin is dependent on intact heterochromatin.
Mol. Cell. Biol. 23,8416
-8428.
Yan, Q., Huang, J., Fan, T., Zhu, H. and Muegge, K.
(2003b). Lsh, a modulator of CpG methylation, is crucial for
normal histone methylation. EMBO J.
22,5154
-5162.[CrossRef][Medline]
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and 
indicated in the panel is shown below. (F) Dorsal (upper) and lateral
(lower) views of bone staining of a newborn G/- (left) and a G/+ (right)
skull. The lines demonstrate the foremen.