First published online February 18, 2004
doi: 10.1242/10.1242/dev.00995
Development 131, 975-982 (2004)
Published by The Company of Biologists 2004
De novo DNA methylation is dispensable for the initiation and propagation of X chromosome inactivation
Takashi Sado1,2,3,*,
Masaki Okano4,
En Li4 and
Hiroyuki Sasaki1,5
1 Division of Human Genetics, National Institute of Genetics, 1111 Yata,
Mishima, 411-8540, Japan
2 Department of Biosystems Science, The Graduate School for Advanced Studies
(SOKENDAI)
3 PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi,
Saitama, Japan
4 Cardiovascular Research Center, Massachusetts General Hospital, Department of
Medicine, Harvard Medical School, 149 13th Street, Charlestown, MA 02129,
USA
5 Department of Genetics, The Graduate School for Advanced Studies
(SOKENDAI)
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Fig. 1. Methylation profiles of Xist in [Dnmt3a-/-,
Dnmt3b-/-] embryos. (A) Gross morphology of
[Dnmt3a-/-, Dnmt3b-/-] embryo (left)
and a wild-type littermate (right) at E9.5. Scale bar: 1 mm. (B) Bisulfite
sequencing of the promoter region of Xist and Hprt in
[Dnmt3a-/-, Dnmt3b-/-] female embryos
at E9.5. Three [Dnmt3a-/-, Dnmt3b-/-]
female embryos were analyzed in a comparison with wild-type females.
Methylated and unmethylated CpG sites are indicated by closed and open
circles, respectively. The arrows indicate the transcription start sites.
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Fig. 2. Expression of Xist in [Dnmt3a-/-,
Dnmt3b-/-] embryos. (A-F) Expression of Xist
examined by RNA-FISH in [Dnmt3a-/-,
Dnmt3b-/-] embryos at E9.5. Although a subset of cells in
both male and female embryos expressed ectopic Xist (arrow heads),
the majority of the cells maintained normal pattern of expression in both
sexes. (A) Wild-type female, (B,C) [Dnmt3a-/-,
Dnmt3b-/-] female, (D) wild-type male and (E,F)
[Dnmt3a-/-, Dnmt3b-/-] male. The
chromosomes coated by Xist RNA in C and F were confirmed to be X
chromosomes using a painting probe (data not shown). (G) Percentage of nuclei
containing the indicated numbers of the Xist domain. Ectopic
accumulation detected in males (#1 and 2) and females (#3-6) is shown in
red.
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Fig. 3. Cytological evidence that one of the two X chromosomes is inactivated in
[Dnmt3a-/-, Dnmt3b-/-] female embryos.
(A) A late replicating X chromosome found in [Dnmt3a-/-,
Dnmt3b-/-] female embryos at E9.5 (arrow). An active
counterpart is indicated by an arrowhead. (B) The number of cells with early
or late replicating X chromosomes in male (#1 and 2) and female (#3-6)
embryos. ERX, early replicating X chromosome; LRX, late replicating X
chromosome. (C) Immunostaining of metaphase chromosomes in
[Dnmt3a-/-, Dnmt3b-/-] female embryos.
Metaphase chromosomes were stained with an antibody against acetylated histone
H4. Acetylated histone H4 was excluded from one (Xi) of the two X chromosomes
in wild-type and [Dnmt3a-/-,
Dnmt3b-/-] female embryos at E9.5.
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Fig. 4. Quantitative RT-PCR of X-linked genes. Expression levels of each gene
(Rps4, Pgk1, G6pd, and Hprt) relative to the abundance of
Gapd were compared between males and females in
[Dnmt3a-/-, Dnmt3b-/-] embryos.
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Fig. 5. [Dnmt3a-/-, Dnmt3b-/-] male ES
cells were induced to differentiate. (A) RNA-FISH was performed on
[Dnmt3a-/-, Dnmt3b-/-] male ES cells
at 12 days of differentiation. Ectopic expression of Xist was
evident, which colocalized with the X chromosome visualized by X (red) and Y
(green) chromosome painting. (B) Percentage of cells with ectopic
Xist accumulation in J1 (wild type), Dnmt3a-/-,
Dnmt3b-/- and [Dnmt3a-/-,
Dnmt3b-/-] ES cells at 0, 5 and 12 days of
differentiation. Similar results were observed in 2 or 3 different
experiments.
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Fig. 6. Ectopic expression of Xist does not induce X-inactivation in
[Dnmt3a-/-, Dnmt3b-/-] male ES cells.
(A) No selective loss of [Dnmt3a-/-,
Dnmt3b-/-] male ES cells upon differentiation in a 1:1
mixture of wild type and [Dnmt3a-/-,
Dnmt3b-/-] male ES cells. (B) RT-PCR amplifying X-linked
genes on cDNA prepared from wild-type and [Dnmt3a-/-,
Dnmt3b-/-] male ES cells, and their differentiated
derivatives as embryoid bodies. Tsix and Xist were amplified
from a nascent and a processed transcript, respectively, with a common primer
pair. Differentiation was monitored by Oct3/4. No amplification from
genomic DNA was confirmed by PCR on a reaction of cDNA synthesis without
reverse transcriptase (data not shown).
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Fig. 7. A model that underlies the induction of differential expression of
Xist at the onset of X-inactivation. Distinct chromatin structure
above seems to be sufficient for inducing differential expression of
Xist, which may be constructed by modification of histone tail such
as methylation of histone H3 tail. DNA methylation and the exclusion of
acetylated histones play a role in stabilizing the transcriptionally
repressive state.
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© The Company of Biologists Ltd 2004
