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)
* Author for correspondence (e-mail: tsado{at}lab.nig.ac.jp)
Accepted 12 November 2003
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SUMMARY |
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Key words: X chromosome inactivation, De novo DNA methyltransferases, Xist
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Introduction |
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The dose difference of the X-linked genes between males and females is
functionally equalized by inactivating one of the two X chromosomes in female
somatic cells during early development
(Lyon, 1961). Although X
chromosomes inherited from father and mother are both active in
undifferentiated cells of preimplantation embryos, one of them becomes
transcriptionally silenced upon cellular differentiation. This process is
regulated by a cytogenetically defined region on the X chromosome called X
chromosome inactivation center (Xic) (reviewed by
Avner and Heard, 2001
). Xic is
essential for X-inactivation to occur in cis and involved in both `choice' of
X chromosome to be inactivated and subsequent `initiation' of chromosomal
inactivation. The inactivated state then spreads both proximally and distally
from the Xic. Non-coding RNAs, Xist and its antisense Tsix,
which are mapped in Xic, play a crucial role in this process
(Brown et al., 1991
;
Brockdorff et al., 1991
;
Borsani et al., 1991
;
Penny et al., 1996
;
Marahrens et al., 1997
;
Lee et al., 1999
;
Lee and Lu, 1999
;
Lee, 2000
;
Sado et al., 2001
). At the
onset of X-inactivation, Xist is upregulated on the future inactive X
chromosome and subsequently accumulates on it in cis. However, expression of
Xist is repressed on the X chromosome that remains active.
Tsix is a negative regulator of Xist in cis. Extinction of
Tsix induces Xist accumulation on the X chromosome being
subject to inactivation, whereas continued expression of Tsix
prevents upregulation of Xist.
Although the mechanism how Xist mediates chromosomal silencing is
not fully understood, specific association of Xist RNA along the
entire region of the inactive X chromosome implies that it may recruit
proteins required for heterochromatinization (Brockdorff, 2002). Recent
studies showed that a polycomb group (PcG) protein complex containing Eed and
Enx1 (also known as Ezh2), which harbors an activity of methylating histone H3
at lysine 9 and 27 (Cao et al.,
2002; Kuzmichev et al.,
2002
; Müller et al.,
2002
; Czermin et al.,
2002
), is localized to X chromosome in Xist-dependent
manner in the early phase of inactivation
(Mak et al., 2002
;
Silva et al., 2003
;
Plath et al., 2003
;
Erhardt et al., 2003
).
DNA methylation has been implicated in the regulation of Xist in
differentiated cells (Beard et al.,
1995). The promoter region of the transcriptionally active
Xist allele on the inactive X chromosome is unmethylated, whereas
that of the transcriptionally inactive Xist allele on the active X
chromosome is highly methylated (Norris et
al., 1994
). Studies on Dnmt1-deficient ES cells and
embryos suggested that although X-inactivation can occur in the absence of DNA
methylation, maintenance of the methylation at the Xist promoter is
necessary for its stable repression in differentiated cells
(Beard et al., 1995
;
Panning and Jeanisch, 1996
).
It is possible, however, that the intact de novo methyltransferases present in
Dnmt1-deficient embryos generate minimal methylation patterns
required for differential activation of Xist. We previously showed
that the Xist locus is extensively demethylated in ES cells deficient
for both Dnmt3a and Dnmt3b (Okano et al.,
1999
). This suggests that the locus is one of the targets for
these de novo methyltransferases, which create the differential methylation
pattern at the Xist promoter after the blastocyst stage
(McDonald et al., 1998
). It is
of particular interest whether the differential methylation underlies the
mechanism of monoallelic expression of Xist at the onset of
X-inactivation.
We have studied X-inactivation in mouse embryos deficient for de novo
methyltransferases Dnmt3a and Dnmt3b. Although the promoter of Xist
is extremely hypomethylated in these embryos, expression of Xist was
not affected in the majority of cells. Cytogenetic and molecular analyses
indicated that one of the two X chromosomes was properly inactivated,
suggesting that de novo methylation is dispensable for the initiation and
propagation of X-inactivation. We further demonstrate that delayed
upregulation of Xist caused by hypomethylation at the promoter does
not induce X-inactivation, supporting the importance of the developmental
window for the chromosomal inactivation
(Wutz and Jeanisch, 2000).
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Materials and methods |
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The conditional alleles of Dnmt3a and Dnmt3b were
introduced into mice, which harbored 2 loxP sites on both sides of
the catalytic domain (M.O. and E.L., unpublished; a detailed description of
these mice will appear elsewhere). These mice were crossed with CAG-cre
transgenic mice to uniformly excise the catalytic domain of each
methyltransferase. The null mutant alleles thus produced were functionally
equivalent to the disrupted alleles previously reported
(Okano et al., 1999).
[Dnmt3a-/-, Dnmt3b-/-] embryos were
obtained at E9.5 from intercrosses between double heterozygotes. For
genotyping of the embryos, DNA was prepared from the yolk sac and all the
analyses were performed on the embryos proper.
Bisulfite genomic sequencing
Genomic DNA prepared from the embryos proper was digested with
EcoRI and treated with urea/bisulfite essentially as previously
described (Paulin et al.,
1998). Two round PCR was performed for amplification of both
Xist and Hprt. For the first cycle, a Pr1/Pr2 pair and an
Hprt-bs1/Hprt-bs3 pair were used for Xist and
Hprt, respectively. For the second round, Pr2 and Pr3, and
Hprt-bs2 and Hprt-bs3 were used, accordingly. Amplified
products were cloned using TOPO-TA-cloning (Invitrogen). The thermal
conditions for PCR was 94°C for 5 minutes, followed by 25 cycles of
94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and
final extension at 72°C for 5 minutes. Primers for Xist, Pr1, Pr2
and Pr3, are described elsewhere (McDonald
et al., 1998
). Primers for Hprt are as follows:
Hprt-bs1, 5'-gtg att att tgg gaa ttt ttt ggg aga-3';
Hprt-bs2, 5'-gta tgg tta gta tta ttt ttt tta gaa-3'; and
Hprt-bs3, 5'-act cta cta aaa tcc cct taa ctc acc-3'.
RNA-FISH and replication timing analysis
RNA-FISH was performed as described previously
(Sado et al., 2001). BrdU was
incorporated into E9.5 embryos for 7-8 hours and cytological preparations were
made in the same manner as RNA-FISH and stained with Acridine Orange.
Immunostaining
Embryos incubated for 3-4 hours in the presence of colcemid were
trypsinized and treated with 0.075 M KCl for 8 minutes at room temperature.
Cells were fixed by an addition of equal volume of 4% paraformaldehyde, which
were then subjected to cytospin for 10 minutes at 800 rpm (Cytospin 2,
Shandon). The specimens thus prepared were permeabilized in KCM buffer (120 mM
KCl, 10 mM NaCl, 20 mM Tris-HCl pH 7.7) containing 0.1% Triton X-100 for 10
minutes at room temperature. The first antibody against acetylated histone H4
(Upstate) and the second antibody against rabbit IgG conjugated with FITC were
diluted 100-fold with KCM containing 0.1% Triton X-100.
RT-PCR
Total RNA (2.5 µg) was converted to cDNA using random primers. For
quantitative study, real-time PCR was carried out using LightCycler-FastStart
DNA Master SYBR Green I (Roche). Concentration of MgCl2 was
determined at 3 mM for all primer pair sets. Standard curves for each primer
pair were drawn using a series of dilution of cDNA generated from wild-type
male ES cells. Expression levels of each message were determined as a value
relative to the abundance of Gapd. The thermal conditions of
LightCycler were as follows: for Hprt, Rps4 and Gapd,
94°C for 10 minutes, followed by 34 cycles of 94°C for 10 seconds,
55°C for 10 seconds, 72°C for 24 seconds; for G6pd and
Pgk1, 94°C for 10 minutes, followed by 32 cycles of 94°C for
10 seconds, 58°C for 10 seconds, 72°C for 24 seconds. Primer sequences
are as follows: Pgk1F, 5'-gtt gca gac aag atc cag ac-3',
Pgk1R, 5'-aca ttg ctg aga gca tcc ac-3'; G6pdF,
5'-atc atc gtg gag aag ccc tt-3', G6pdR, 5'-ttc ttc
aca tag agg aca gc-3'; Rps4F, 5'-tca tca gca ttg aca aga
cc-3', Rps4R, 5'-ggattc ctt ttc ctc tgg ga-3';
Gapd/F, 5'-atg gcc ttc cgt gtt cct ac-3', and
Gapd/R, 5'-tgt gag gga gat gct cag tg-3'.
MusHprtF and MusHprtR have been previously described
(Sado et al., 1996).
Amplifications of Xist and Tsix were performed on 1/50 of
the cDNA prepared from ES cells and embryoid bodies using Mx23b and MIX20 as
described previously (Kay et al.,
1993; Sado et al.,
1996
). Oct3/4 sequence was amplified using the following
primers: Oct3/4F, 5'-tgg gtg gat tct cga acc tg;
Oct3/4R, 5'-cct tct gca ggg ctt tca tg-3'.
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Results |
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To explore the role for de novo DNA methylation in X-inactivation and also
in the regulation of Xist at the onset of X-inactivation, we analyzed
mouse embryos homozygous for the disruptions of Dnmt3a and
Dnmt3b. All the embryos examined were recovered at embryonic day (E)
9.5, when [Dnmt3a-/-, Dnmt3b-/-]
embryos are readily identified by gross morphology
(Fig. 1A)
(Okano et al., 1999). The
methylation profile at the Xist promoter was analyzed in
[Dnmt3a-/-, Dnmt3b-/-] female embryos
by bisulfite genomic sequencing. As expected, the region differentially
methylated in wild-type female embryos was extremely hypomethylated in
[Dnmt3a-/-, Dnmt3b-/-] females
(Fig. 1B). Similarly, the CpG
island containing the Hprt promoter was almost completely
unmethylated in these embryos, whereas in wild-type female embryos, it showed
variable levels of methylation in about half of the molecules
(Fig. 1B). The differential
methylation at this locus is still being established at E9.5
(Lock et al., 1987
).
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Finally we performed real time PCR to compare the expression levels of X-linked genes, Rps4, Pgk1, G6pd and Hprt, all of which are subject to X-inactivation, in [Dnmt3a-/-, Dnmt3b-/-] embryos. The expression level of each gene relative to that of Gapd did not show a significant difference between males and females (Fig. 4), suggesting that one of the two alleles at each locus were silenced in [Dnmt3a-/-, Dnmt3b-/-] females. It is worth mentioning that a relative expression level of Pgk1, although appropriately compensated between males and females, was significantly higher in double mutant than wild-type embryos. It is likely that the regulation of basal expression of Pgk1 has been affected by a loss of functional de novo DNA methyltransferases.
|
Prolonged culture of [Dnmt3a-/-, Dnmt3b-/-] male ES cells causes derepression of Xist
In the above experiments, however, a small proportion of cells in
[Dnmt3a-/-, Dnmt3b-/-] embryos did
show ectopic accumulation of Xist
(Fig. 2A-F). As previous
studies on Dnmt1-deficient ES cells suggested that DNA methylation is
required for stable repression of Xist
(Panning and Jeanisch, 1996),
the ectopic Xist accumulation in the [Dnmt3a-/-,
Dnmt3b-/-] embryos may have arisen from delayed
activation, not from failure in initial repression. To address this issue, we
made use of male ES cells deficient for both Dnmt3a and
Dnmt3b (Okano et al.,
1999
). RNA-FISH was performed on these male ES cells before and
after induction of differentiation. Although the single X chromosome was never
coated with Xist RNA in undifferentiated state, ectopic Xist
accumulation was observed in about 3.2% and 16.8% of cells at day 2 and day 5
of differentiation, respectively (Fig.
5B; data not shown). This is in agreement with the results
obtained with the E9.5 [Dnmt3a-/-,
Dnmt3b-/-] embryos (0-17.7%). At day 12 of
differentiation, however, a surprisingly high percentage (68%) of cells from
[Dnmt3a-/-, Dnmt3b-/-] embryoid bodies
showed ectopic Xist accumulation
(Fig. 5A,B), suggesting
progressive activation of the unmethylated Xist locus in the
differentiated [Dnmt3a-/-, Dnmt3b-/-]
cell population. In control experiments, Xist accumulation was never
detected in either Dnmt3a-/-,
Dnmt3b-/- or parental wild-type ES cells, but a subset of
Dnmt1-/- cells showed ectopic Xist accumulation
(Fig. 5B), in agreement with
the previous report (Panning and Jeanisch,
1996
). These observations confirm the importance of DNA
methylation in stable repression of Xist
(Panning and Jeanisch,
1996
).
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Discussion |
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Previous studies on Dnmt1-/- embryos and ES cells
showed that initiation of monoallelic Xist expression and
X-inactivation can occur in the absence of maintenance-type DNA methylation
(Beard et al., 1995;
Panning and Jeanisch, 1996
).
They did not, however, address whether de novo methylation at the
Xist promoter, which should occur in the mutant cells, contributes to
the initiation of X-inactivation. Some residual methylation was, in fact,
detected at the Xist promoter in Dnmt1-/- embryos
(Sado et al., 2000
), which was
most probably mediated by the de novo DNA methyltransferases. In
[Dnmt3a-/-, Dnmt3b-/-] female embryos,
despite the lack of de novo methylation at the Xist promoter,
accumulation of Xist RNA was confined to a single X chromosome in the
great majority of nuclei. The present study, therefore, provides the first
evidence that a mechanism(s) other than DNA methylation is responsible for
causing the differential expression of Xist. It is clear,
nevertheless, that DNA methylation is important for the stable repression of
Xist on the active X chromosome in differentiated cells. In agreement
with the previous reports on Dnmt1-deficient cells
(Beard et al., 1995
;
Panning and Jeanisch, 1996
),
the present study indicated that upon differentiation, hypomethylation at the
Xist promoter caused by a functional loss of de novo DNA
methyltransferases resulted in progressive derepression of Xist. It
should be noted, however, that our finding was at variance with the study by
Panning and Jaenisch (Panning and
Jaenisch, 1996
) in that ectopically expressed Xist did
not lead to silencing of X-linked genes. While available evidence suggests
that Xist expressed in differentiated cells does not cause
X-inactivation (Tinker and Brown,
1998
; Wutz and Jaenisch,
2000
) (this study), Hall et al.
(Hall et al., 2002
) recently
demonstrated that a human XIST transgene could induce chromosome
inactivation in postdifferentiation human HT-1080 cells. Perhaps, ectopic
expression of Xist might exert its effect on silencing in some
particular cell types.
Our results suggested that X-inactivation was not induced despite the
cis-association of Xist on the X chromosome. Taking advantage of an
inducible expression system of Xist in transgenic ES cells, Wutz and
Jaenisch (Wutz and Jaenisch,
2000) previously showed that Xist can initiate
chromosomal silencing only during an early phase of ES cell differentiation
(up to 48 hours after induction of differentiation), which indicates that
there is a crucial developmental window for X-inactivation. Ectopic expression
of Xist, therefore, most probably occurs later than this window in
[Dnmt3a-/-, Dnmt3b-/-] embryos and
male ES cells, thereby resulting in no induction of X-inactivation.
It has been suggested that DNA methylation is important for the stable
repression of genes on the inactivated X chromosome. Our previous study on
Dnmt1-deficient mouse embryos suggests that substantial loss of DNA
methylation leads to partial reactivation of X-linked lacZ transgenes
in the embryonic lineage of E9.5 embryos
(Sado et al., 2000). By
contrast, hypomethylation caused by the failure of de novo DNA methylation did
not appear to reactivate the silent copy of the four endogenous X-linked genes
in [Dnmt3a-/-, Dnmt3b-/-] females at
E9.5. This may be ascribed to different susceptibility to hypomethylation
between the endogenous genes and the transgenes. It is possible that the
regulation of multicopy exogenous sequences such as a tandem array of the
lacZ transgenes is more sensitive to the loss of DNA methylation than
the endogenous genes. It will be important to address whether or not the
maintenance mechanism of the X-inactivated genes is affected in
[Dnmt3a-/-, Dnmt3b-/-] background.
It is known that Xist is exclusively expressed from the paternal
allele in each blastomere of early preimplantation embryos
(Kay et al., 1993;
Sheardown et al., 1997
),
although the promoter region of Xist shows a low level of CpG
methylation on both parental alleles
(McDonald et al., 1998
). It
seems, therefore, possible that distinctive chromatin structures inherited
from the parents are responsible for the upregulation of the paternal allele
and stable repression of the maternal allele in the preimplantation embryos.
It is therefore likely that the modification of chromatin is capable of
inducing the differential expression of Xist on its own. Methylation
of histone H3 at lysine 9 and 27 and histone H4 at lysine 20 is implicated in
repression of gene expression in yeast and fruit fly
(Nakayama et al., 2001
;
Noma et al., 2001
;
Cao et al., 2002
;
Nishioka et al., 2002a
;
Fang et al., 2002
), both of
which essentially lack DNA methylation. It is tempting to speculate that these
histone modifications in combination with methylation of histone H3 at lysine
4, which is involved in transcriptional activation
(Strahl et al., 1999
;
Noma et al., 2001
;
Wang et al., 2001
;
Nishioka et al., 2002b
),
primarily regulate the differential expression of Xist
(Fig. 7), which would not be
affected in [Dnmt3a-/-, Dnmt3b-/-]
embryos. CpG methylation and hypoacetylation
(Gilbert and Sharp, 1999
) then
follow these events to fix the established epigenetic states. As Tsix
appears to regulate the expression of Xist negatively at the onset of
X-inactivation (Lee, 2000
;
Sado et al., 2001
), it should
also play a role in monoallelic upregulation of Xist. It should be
noted that Tsix became downregulated in the absence of de novo
methyltransferases in differentiating ES cells. The CpG island found in the
vicinity of the major transcription start site of Tsix, which became
methylated upon differentiation in male ES cells, stayed unmethylated after
differentiation in [Dnmt3a-/-,
Dnmt3b-/-] male ES cells, suggesting that regulation of
Tsix is also independent of DNA methylation (data not shown). Further
studies on the epigenetic regulation and function of Tsix may clarify
the initial event that triggers X-inactivation.
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ACKNOWLEDGMENTS |
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