First published online February 24, 2006
doi: 10.1242/10.1242/dev.02293
Development 133, 1183-1192 (2006)
Published by The Company of Biologists 2006
Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome
Yoshihide Ueda1,
Masaki Okano1,3,
Christine Williams2,
Taiping Chen1,4,
Katia Georgopoulos2 and
En Li1,4,*
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.
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Fig. 1. Biochemical characterization of ICF mutations. (A) Schematic
diagram of the mouse Dnmt3b protein structure. The conserved PWWP and ATRX
domains, the methyltransferase motifs (I, IV, VI, IX and X), and the ICF
mutations that we introduced into mouse Dnmt3b cDNA are indicated.
The location of the interaction domain with Dnmt3a (see Fig. S1 in the
supplementary material) is also shown. (B) Interaction of ICF mutants
with wild-type Dnmt3a and Dnmt3b. COS-7 cells were transfected with two
expression vectors, one for myc-Dnmt3a (left panel) or myc-Dnmt3b1 (right
panel), and another for GFP-Dnmt3b1 or GFP-ICF mutants, as indicated.
GFP-tagged proteins were immunoprecipitated from cell extracts using an
anti-GFP antibody. Immunoblotting analysis of the immunoprecipitates was
carried out using anti-myc antibody (top panel). The middle and bottom panels
show the results of immunoblotting of the total cell extract (TCE) from
transfected cells with anti-myc and anti-GFP antibodies, respectively.
(C) Subcellular localization of Dnmt3b isoforms and ICF mutants.
GFP-tagged Dnmt3b isoforms or ICF mutants were expressed in NIH3T3 cells, and
the cells were fixed and analyzed by fluorescence microscopy. For each
construct, 200-300 transfected (green) cells were counted and the percentages
of cells showing different localization patterns are indicated. (D)
Stable expression of wild-type and mutant Dnmt3b in
Dnmt3a-/-, Dnmt3b-/- ES cells.
Expression vectors encoding mouse Dnmt3b1 (m3b1), A609T, D823G and PC (Dnmt3b
with its PC motif mutated) (top panel), and human DNMT3B (h3B1), A603T and
D817G (lower panel), were individually (or in a combination of two ICF
mutants) electroporated into late-passage 7aabb cells and selected in
blasticidin-containing medium. Blasticidin-resistant clones were analyzed by
immunoblotting using anti-Dnmt3b and anti- -tubulin antibodies.
(E) DNA methylation analysis. Genomic DNA from the indicated ES cell
lines was digested with HpaII and analyzed by Southern hybridization
using a probe (pMO) for the endogenous C-type retrovirus repeats. DNA from
wild-type ES cells (J1) digested with MspI (M) was used as a control
for complete digestion.
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Fig. 2. Generation of mouse models for ICF syndrome. (A) Maps of the
Dnmt3b genomic locus, targeting vector, targeted locus, and
recombined locus after exposure to Cre. The vertical bars represent the exons;
arrowheads indicate the ICF mutations introduced. B, BamHI; E5,
EcoRV; S1, SacI; Xb, XbaI; Sal, SalI; Sm,
SmaI. (B) Maps of Dnmt3b wild-type and mutant
(n=3) alleles used in this study. (C) Southern blot analysis
of the different Dnmt3b alleles. Genomic DNA was digested with
BamHI and hybridized with a 5' external probe indicated in A
and B. (D) Immunoblot analysis of protein samples prepared from whole
E12.5 embryos and ES cells with anti-Dnmt3b (upper panel), anti-Dnmt3a (middle
panel) and anti--tubulin (lower panel) antibodies. The migration of
Dnmt3a and Dnmt3b isoforms is indicated (arrowheads).
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Fig. 3. ICF mutations result in a partial loss of function of Dnmt3b.
(A) Progeny derived from intercrosses of Dnmt3b mutants.
(B-D) DNA methylation analysis of Dnmt3b mutant mice. Genomic
DNA from E12.5 embryos (B), tails of adult mice (C) and various tissues of
newborns (D) was digested with HpaII or MaeII (left panel of
B) and hybridized to probes for major satellite repeats (left panel of B),
minor satellite repeats (middle panel of B;C,D), and endogenous C-type
retrovirus repeats (pMO; right panel of B). DNA digested with MspI
(M) was used as a control for a complete lack of DNA methylation.
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Fig. 4. Developmental defects of Dnmt3b-/- embryos.
(A) Analysis of embryos derived from intercrosses of
Dnmt3b+/- mice. Numbers in parentheses indicate the number
of abnormal embryos. (B) Gross morphology of
Dnmt3b-/- embryos and a wild-type littermate at 13.5 dpc.
Some Dnmt3b-/- embryos showed a smaller and paler fetal
liver than their wild-type littermates, which could be recognized from outside
(middle embryo, arrow). Some Dnmt3b-/- embryos showed
bleeding at the head region (right embryo, arrow). (C) Posterior view
of the Dnmt3b-/- embryo shown in B. Most of the
Dnmt3b-/- embryo showed subcutaneous edema (arrows) at
13.5 dpc. (D) Ventricular septum defect in the heart of a
Dnmt3b-/- embryo at 14.5 dpc. The right panel shows a
higher magnification of the inset shown in D. (E) Ectopic hemorrhage at
the dorsal root ganglion of a Dnmt3b-/- embryo at 12.5
dpc. (F) Malformation of the fetal liver in
Dnmt3b-/- embryos at 13.5 dpc.
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Fig. 6. Dnmt3b mutations lead to the apoptosis of thymocytes.
(A) Representative FACS profiles showing staining patterns of
thymocytes in P1 ICF mice and control mice. Cells were stained with
anti-CD4-PE and anti-CD8-FITC for upper panels, and Annexin V-FITC (AnV) and
propidium iodide (p.i.) for lower panels. Numbers shown in FACS profiles
denote the percentage of cells that fall into each quadrant. (B)
Absolute numbers of total thymocytes (total), CD4-CD8-
double-negative cells (DN), CD4+CD8+ double-positive
cells (DP), CD4+ single-positive cells (CD4 SP), and
CD8+ single-positive cells (CD8 SP) in a T/- mouse and a wild-type
littermate. (C) TUNEL staining (upper panels) and DAPI staining (lower
panels) of sections of thymus and spleen from a T/- mutant mouse and a T/+
littermate control at P1. (D) Fragmentation of DNA extracted from the
thymus, spleen, liver, kidney and brain of ICF mutant mice and their
littermate controls at P0 or P1. DNA was analyzed by electrophoresis on a 1%
agarose gel and stained with ethidium bromide.
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© The Company of Biologists Ltd 2006
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.