Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan
Author for correspondence (e-mail:
aokif{at}k.u-tokyo.ac.jp)
Accepted 3 February 2004
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SUMMARY |
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Key words: Embryo, Oocyte, Histone H3, Lysine 9, Methylation, Nuclear transfer
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Introduction |
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After fertilization, the genomes from the two parents are separated in the
embryos into male and female pronuclei, before being re-unified during the M
phase at the one-cell stage. Although these pronuclei reside in the same
cytoplasm, they are heterogeneous in many aspects. The temporal and spatial
distribution of the sites of DNA replication are asynchronous between the two
pronuclei, in that the female pronucleus requires a longer time to complete
replication in the intranuclear region but not in the peripheral regions
(Aoki and Schultz, 1999).
Transcriptional regulation also differs; the male pronucleus supports a higher
level of transcription than the female pronucleus
(Bounial et al., 1995
;
Aoki et al., 1997
), because the
chromatin of the female pronucleus (but not that of the male pronucleus) is in
a transcriptionally repressed state
(Wiekowski et al., 1993
;
Majumder et al., 1997
;
Cho et al., 2002
). The two
pronuclei also show asymmetric DNA methylation, which may be responsible for
functional differences between the parental genomes during development. As
early as 4 hours after fertilization, the male pronucleus is almost completely
demethylated, whereas the female pronucleus remains methylated during the
one-cell stage and undergoes gradual demethylation until the blastocyst stage
(Mayer et al., 2000a
;
Reik et al., 2001
). As the
male and female pronuclei show different features in the same cytoplasmic
environment, it appears that the differences in their chromosomes are
distinguished by cytoplasmic factors after fertilization.
The differences in the properties of paternal and maternal genomes in early
embryos may be attributable to differences in the epigenetic modifications to
their genomes. Recent studies have shown that modifications of the
chromatin-packaging proteins, histones, play important roles in the regulation
of gene expression. Covalent modifications of histones, such as acetylation,
methylation and phosphorylation, contribute to a mechanism that can alter
chromatin structure, thereby causing inheritable differences in
transcriptional `on-off' states (Jenuwein
and Allis, 2001; Goll and
Bestor, 2002
; Turner,
2002
). It seems likely that histone modifications differ in the
parental genomes in one-cell embryos, as the reconstruction of chromatin by
protamine-histone exchange occurs in the paternal chromatin soon after
fertilization. The chromosomal histones, which are acquired from the
cytoplasmic pool of the oocyte, may undergo different modifications in the
male pronucleus than in the female pronucleus, the latter of which is acquired
from the oocyte chromosome. The different histone modifications are probably
implicated in the differential properties of the parental genomes.
An interesting, recent finding suggests that histone H3 methylation at
lysine 9 (H3/K9) is involved in the formation of the constitutive
heterochromatin, as well as the facultative heterochromatin of the inactive X
chromosome (Heard et al.,
2001; Jacobs et al.,
2001
; Noma et al.,
2001
; Boggs et al.,
2002
; Peters et al.,
2002
). Methylation of H3/K9 has also been shown to be associated
with the silencing of euchromatic genes
(Hwang et al., 2001
;
Nielsen et al., 2001
;
Peters et al., 2002
). In
addition, H3/K9 methylation has been correlated with DNA cytosine methylation,
and it has been suggested that DNA methylation acts downstream of H3/K9
methylation (Tamaru and Selker,
2001
; Gendrel et al.,
2002
; Jackson et al.,
2002
).
In this study, we investigated H3/K9 methylation in oocytes and early pre-implantation embryos to understand the mechanism by which the genomes of different parental origin are distinguished. Our results show that the asymmetric H3/K9 methylation pattern between parental genomes is generated soon after fertilization, and persists during early preimplantation development. The different methylation patterns are generated by changes in the properties of the cytoplasm after fertilization, and not by a specific chromatin structure. The mechanism that maintains the paternal genome in the undermethylated state is an active process, as inhibition of protein synthesis or gene expression increased methylation in the male pronucleus to a level that was similar to that of the female pronucleus. Finally, correspondent methylation of H3/K9 and DNA occurred in male pronuclei that were transplanted into oocytes, which suggests that asymmetric H3/K9 methylation is associated with asymmetric DNA methylation in genomes of different parental origin during preimplantation development.
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Materials and methods |
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To inhibit the synthesis of protein or mRNA, 50 µg/ml cycloheximide or
25 µg/ml -amanitin (Sigma, St Louis, MO, USA), respectively, was
added to the culture medium at the time of insemination. These concentrations
of the inhibitors were sufficient to inhibit completely the synthesis of
protein and mRNA, as 20 µg/ml cycloheximide was previously demonstrated to
abolish protein synthesis (Manejwala et
al., 1991
). Furthermore, we confirmed previously that 25 µg/ml
-amanitin completely inhibited transcription, which was detected by
bromouridine (BrU) incorporation in embryos that were loaded with BrUTP (F.A.,
unpublished).
Parthenogenesis and androgenesis
Androgenetic embryos were prepared by fertilizing enucleated oocytes.
Cumulus-oocyte complexes were collected from the ampullae of oviducts 14-15
hours after hCG injection, and placed in KSOM
(Lawitts and Biggers, 1993)
that contained 0.3 mg/ml bovine testicular hyaluronidase. After complete
removal of the cumulus cells, the oocytes were transferred to a
micromanipulation drop, which contained HEPES-buffered KSOM that was
supplemented with 5 µg/ml cytochalasin B. The zona pellucida was cored by a
Piezo-impact-driven micromanipulator (Prime Tech Ltd, Ibaraki, Japan), and the
MII chromosomes were aspirated with a minimal volume of oocyte cytoplasm using
a pipette. The enucleated oocytes were transferred to acidic MEMCO for a short
time to dissolve the zona pellucida. Zona-free oocytes were cultured in
Whitten's medium for 1 hour, to allow them to recover after zona pellucida
removal, and were then subjected to in vitro fertilization. Thirty minutes
after insemination, the oocytes were washed and cultured in CZB medium.
Parthenogenetically activated oocytes were prepared by exposure of MII-stage oocytes to 7% ethanol for 6 minutes or to 10 mM Sr2+ for 10 minutes in CZB. In order to obtain diploid parthenogenetic embryos, the oocytes were cultured in CZB that contained 5 µg/ml cytochalasin B for 6 hours, and then cultured in CZB.
Pronuclear transplantation
Enucleated germinal vesicle (GV) and MII-stage oocytes were used as
recipients for nuclear transfer. Enucleation of MII oocytes was conducted as
described above. The fully-grown GV oocytes were collected from 4-week-old ddY
mice by puncturing the follicles with a sharp needle in Whitten's medium that
was supplemented with 20 mM HEPES and 0.2 mM 3-isobutyl-1-methylxanthine
(IBMX). Only those oocytes with a diameter >70 µm were sorted for
further use. The cumulus cells were removed from cumulus-oocyte complexes by
gentle pipetting through a narrow-bore glass pipette. The enucleation protocol
for GV oocytes was similar to that for MII oocytes. GV-stage oocytes were
cultured in HEPES-buffered KSOM that contained 5 µg/ml cytochalasin B and
0.2 mM IBMX for 30 minutes before GV aspiration. The zona pellucida was cored
using a Piezo-impact-driven micromanipulator, and the GV, which was surrounded
by a small amount of cytoplasm, was removed with a pipette of inner diameter
15 µm. To inhibit spontaneous meiosis resumption, 0.2 mM IBMX was added to
the micromanipulation medium. The enucleated oocytes were cultured in CZB (for
MII oocytes), or CZB that contained 0.2 mM IBMX (for GV oocytes), for 1 hour
before use.
The male pronucleus at 6 hours after insemination was used as the nuclear donor. The zygotes were treated with 5 µg/ml cytochalasin B for 20 minutes before pronucleus aspiration. The zona pellucida was cored using a Piezo-impact-driven micromanipulator, and the male pronucleus, which was surrounded by a small amount of cytoplasm, was removed with a pipette of inner diameter 15 µm. The male pronucleus was then inserted into the perivitelline space of the enucleated oocyte. The fusion of donor-recipient pairs was induced by a DC pulse of 1500 V/cm for 20 µs in 300 mM mannitol that contained 0.1 mM MgSO4, 0.1 mg/ml polyvinyl alcohol and 3 mg/ml bovine serum albumin. The oocytes were evaluated 30 minutes after application of the electropulses to ensure fusion. Successfully fused reconstructed oocytes were cultured for 3 hours in Whitten's medium with or without 0.2 mM IBMX. When MII-stage oocytes were used as recipients, the reconstructed oocytes were cultured with 0.5 µg/ml nocodazole to prevent spontaneous activation.
Immunocytochemistry
Oocytes and embryos were washed in PBS that contained 3 mg/ml
polyvinylpyrrolidone (PBS/PVP), fixed for 1 hour in 3.7% paraformaldehyde in
PBS, and permeabilized with 0.5% Triton X-100 in PBS for 20 minutes at room
temperature. The cells were incubated for 1 hour with a 1:200 dilution of the
rabbit polyclonal antibody that recognizes dimethyl-lysine 9 on histone H3
(Upstate Biotechnology, Lake Placid, NY, USA; catalogue number 07-212), and a
secondary FITC-conjugated antibody (Jackson Immunoresearch, West Grove, PA,
USA). For the detection of 5-methl-cytosine (5-MeC), the cells were treated
with 2N HCl at room temperature for 30 minutes, and neutralized subsequently
for 10 minutes with 100 mM Tris-HCl buffer (pH 8.5), after permeabilization.
After extensive washing with 0.05% Tween-20 in PBS, the cells were incubated
with anti-5-MeC antibodies (Eurogentec, Seraine, Belgium), followed by
incubation with secondary FITC-conjugated antibody (Jackson Immunoresearch).
Double-antibody staining was accomplished by successive incubation with the
antibodies against 5-MeC and methylated H3/K9. DNA was stained with 3 µg/ml
4,6-diamidino-2-phenylindole (DAPI) for 20 minutes, and the cells were mounted
on a glass slide in Vectashield anti-bleaching solution (Vector Laboratories,
Burlingame, CA, USA). Fluorescence was detected using a Leica TCS SP2
laser-scanning confocal microscope.
Semi-quantitative analysis of the fluorescence signals was conducted using
the NIH Image program (National Institute of Health, Bethesda, MD, USA), as
described previously (Kim et al.,
2002). Briefly, the pixel value/unit area was measured for the
nucleus, and the value for the cytoplasm was subtracted as background. The
derived value was multiplied by the nuclear area to yield the total amount of
fluorescence in the nucleus.
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Results |
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During the first M phase, the maternal and paternal chromosomes move
together but remain compartmentalized. Even after cleavage to form the
two-cell stage, the chromosomes are still separated topologically, which
suggests that differential epigenetic reprogramming occurs in the parental
genomes (Mayer et al., 2000a;
Mayer et al., 2000b
;
Haaf, 2001
). If H3/K9
methylation is an epigenetic marker, it should also be localized within the
compartmentalized areas of the nuclei of the two-cell embryos.
Immunocytochemistry revealed methylated H3/K9 fluorescence present in only
half of the nuclear area (Fig.
2A). This topological separation was due to the different genome
origins, as uniform fluorescence was observed in the whole nuclei of two-cell
embryos that were produced by parthenogenesis, and no fluorescence was seen in
two-cell embryos that were produced by androgenesis
(Fig. 2B). Thus, asymmetric
methylation of H3/K9 in parents of different origin was maintained until the
late two-cell stage.
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Methylation of DNA cytosine in the male pronucleus after transfer to an enucleated oocyte
DNA methylation of CpG dinucleotides, which is a major epigenetic
modification of the genome, plays an important role in the regulation of gene
expression and is essential for mammalian embryogenesis
(Ferguson-Smith and Surani,
2001; Reik et al.,
2001
). Studies using fungi and plants have revealed a relationship
between DNA methylation and H3/K9 methylation, and have suggested that DNA
methylation acts downstream of H3/K9 methylation
(Tamaru and Selker, 2001
;
Gendrel et al., 2002
;
Jackson et al., 2002
).
However, there is little evidence to support a similar scenario in mammals. To
investigate the relationship between DNA and H3/K9 methylation, male pronuclei
were transferred into enucleated oocytes and changes in the methylation
patterns were examined. Before nuclear transfer, we examined the DNA
methylation patterns of the donor pronuclei and verified the previous
observation that active DNA demethylation was confined to the paternal
pronucleus (Mayer et al.,
2000a
). No cytosine methylation fluorescence signals were detected
in the male pronuclei 6 hours after insemination (data not shown).
Reconstructed oocytes were produced by transferring the male pronuclei to
enucleated GV-stage or MII-stage oocytes. When GV-stage oocytes were used as
recipients, the reconstructed oocytes were arrested at prophase of the first
meiosis in the presence of IBMX, which is an inhibitor of meiotic resumption,
whereas they spontaneously resumed meiosis and underwent germinal vesicle
breakdown (GVB) 3 hours after nuclear transfer in the absence of IBMX.
Regardless of the occurrence of GVB, de novo DNA methylation occurred in the
transplanted nuclei, and was accompanied by global histone H3/K9 methylation
(Fig. 7). By contrast, DNA
methylation did not occur in the male pronuclei that were transferred into
MII-stage oocytes, although H3/K9 methylation did occur in this case.
|
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Discussion |
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Histones in the oocyte cytoplasmic pool vary in terms of modifications with
respect to different histones and different lysine residues. For instance,
histone H4 is acetylated at lysines 5 and 12 by the cytoplasmic
acetyltransferase, transported to the nucleus, and assembled into chromatin
(Sobel et al., 1995;
Verreault et al., 1996
;
Adenot et al., 1997
). After
assembly, H4 is deacetylated, and then acetylated in an appropriate manner by
the nuclear deacetylases and acetyltransferases. In one-cell embryos, the male
pronuclei exhibit higher levels of hyperacetylated H4 than the female
pronuclei at G1 phase, but the levels are similar in both pronuclei during the
S and G2 phases (Adenot et al.,
1997
). Thus, the H4 acetylation pattern changes dramatically
during the one-cell stage. In contrast to histone H4 acetylation, the
asymmetric methylation pattern of H3 in the parent genomes was stably
maintained until the two-cell stage. H3 was methylated in the chromatin, but
undermethylated in the oocyte cytoplasmic pool. After fertilization, the sperm
chromatin exchanged protamines for histones. Thus, the paternal chromatin
incorporated hypomethylated H3, and could be discriminated from the maternal
genome, which consisted of methylated H3. This difference in methylation
pattern was maintained stably, as the H3 methylase did not function after
fertilization. These results suggest that H3/K9 methylation serves as an
inherited epigenetic code to distinguish parental genomes during early
pre-implantation development.
Asymmetric H3/K9 methylation may function as a precursor for asymmetric DNA
methylation, which is involved in genomic imprinting or asymmetric X
chromosome inactivation in trophectoderm cells. Studies have shown that
parent-specific imprints are established on some endogenous genes and
exogenous transgenes during or after fertilization
(El-Maarri et al., 2001;
Pickard et al., 2001
). After
fertilization, however, the DNA methylation level decreases until the
blastocyst stage, and de novo DNA methylation occurs around the time of
implantation (Reik et al.,
2001
). For instance, the imprinting control region of the
Lit1 gene is completely demethylated until the two-cell stage and
then is methylated only in the genome of maternal origin at the blastocyst
stage (Yatsuki et al., 2002
).
Furthermore, we have shown that the DNA methyltransferase does not function in
unfertilized oocytes (Fig. 7),
which indicates that DNA methylation is not sufficient for epigenetic marking
during and after fertilization. Therefore, the epigenetic marking appears to
involve an epigenetic modification other than DNA methylation. This suggests
the existence of an epigenetic marking process that occurs in the cytoplasm of
oocytes and targets the DNA for methylation at later stages of development.
The de novo cytosine methyltransferase seems to take cues from this type of
epigenetic marker (El-Maarri et al.,
2001
; Pickard et al.,
2001
). It is unclear how the two parental genomes are
distinguished in embryos. The asymmetric H3/K9 methylation we observed, in
which only the histone that was taken over from the oocyte chromosome was
methylated, could be a simple yet secure mechanism for distinguishing parental
genomes in embryos.
Studies with Neurospora and Arabidopsis suggest that DNA
methylation takes its cue from histone H3/K9 methylation
(Bartee et al., 2001;
Tamaru and Selker, 2001
;
Gendrel et al., 2002
;
Jackson et al., 2002
;
Richards and Elgin, 2002
). In
Neurospora, mutation of the histone H3 methyltransferase appears to
abolish all cytosine methylation (Tamaru
and Selker, 2001
). In Arabidopsis, recent research has
shown that chromomethylase 3 (CMT3) interacts with the Arabidopsis
homologue of HP1 (Cbx5 - Mouse Genome Informatics), which, in turn, interacts
with methylated histones, suggesting that CpNpG DNA methylation is controlled
by histone H3/K9 methylation via the interaction of CMT3 with methylated
chromatin (Jackson et al.,
2002
). Furthermore, the Suv39h histone methylase in mammalian
cells is required for DNA methylation
(Lehnertz et al., 2003
). Thus,
H3/K9 methylation appears to direct DNA methylation, although the opposing
view that CpG methylation directs H3/K9 methylation has also been suggested
for Arabidopsis (Soppe et al.,
2002
). In mouse zygotes, the methylation states of DNA cytosine
are consistent with those of H3/K9 (Arney
et al., 2002
). The absence of H3/K9 methylation in the paternal
pronucleus is consistent with the preferential demethylation of paternal DNA,
which occurs within 4 hours of fertilization
(Santos et al., 2002
). In
bovine embryos, the methylation level of H3/K9 has been found to increase in
parallel with that of DNA after the eight-cell stage
(Santos et al., 2003
). Our
results also reveal corresponding de novo methylation of H3/K9 and DNA in
paternal pronuclei that were transferred into enucleated GV-stage oocytes
(Fig. 7). Furthermore, our
finding that the levels of H3/K9 methylation, but not those of DNA
methylation, increased in pronuclei that were transferred into MII-stage
oocytes (Fig. 7) suggests that
H3/K9 methylation does not depend on DNA methylation, as these results would
not have been obtained if DNA methylation directed H3/K9 methylation.
Similarly, the level of DNA methylation does not increase in embryos treated
with
-amanitin or cycloheximide (F.A. and J.-M.K., unpublished),
although the level of H3K9 methylation increased
(Fig. 6). The DNA
methyltransferase would be inactivated during meiotic maturation, resulting in
unchanged levels of DNA methylation in the nuclei of two-cell embryos treated
with
-amanitin or cycloheximide, or in pronuclei transferred into
MII-stage oocytes, despite increased H3/K9 methylation. Our finding that the
levels of DNA methylation were unchanged in paternal pronuclei that were
transferred into MII-stage oocytes is consistent with the successful
production of cloned animals by the transfer of somatic nuclei, and the
observation that genome imprinting is inherited faithfully by cloned mice
(Inoue et al., 2002
).
A recent report has suggested that trimethyl-, but not dimethyl-, H3/K9, is
involved in DNA methylation (Tamaru et
al., 2003). We have examined the changes in the levels of H3/K9
trimethylation during meiotic maturation and early preimplantation
development. Immunocytochemistry using an antibody that specifically
recognizes trimethyl-H3/K9 revealed that the level of trimethylation changes
in a manner similar to that observed for dimethylation in this study (F.A. and
J.-M.K., unpublished). As the anti-dimethyl-H3/K9 antibody used in our study
did not cross-react with trimethyl-H3/K9 (see Fig. S1 at
http://dev.biologists.org/supplemental/),
both the dimethylated and trimethylated forms of H3/K9 are present, and their
levels appear to be under the control of a single mechanism. It remains to be
clarified whether these two epigenetic modifications play different roles in
the regulation of genome function.
In mouse embryos, transcription is initiated during the one-cell stage, at
which stage transcriptional activity is much higher in the male pronucleus
than in the female pronucleus (Aoki et al.,
1997). Studies have suggested that this difference is caused by
differences in the repressive states of chromatin; the chromatin in the male
pronucleus is not repressed, whereas that in the female pronucleus is
partially repressed (Wiekowski et al.,
1993
; Majumder et al.,
1997
). However, the mechanism regulating this differential
repression is not clear. It has been suggested that H3/K9 methylation is
involved in the repression of gene expression in both euchromatic and
heterochromatic regions (Hwang et al.,
2001
; Nielsen et al.,
2001
; Peters et al.,
2002
; Saccani and Natoli,
2002
). It is possible that the asymmetric methylation of H3/K9 is
involved in the different repressive states of the two parental genomes.
During the one-cell stage, DNA replication occurs asynchronously between
the two pronuclei, in that the female pronucleus requires a longer time to
complete replication (Aoki and Schultz,
1999). This suggests that heterochromatin is asymmetrically
constituted in the two pronuclei, as H3K9 methylation is involved in the
formation of heterochromatin, which is late-replicating
(O'Keefe et al., 1992
;
Spector, 1993
). A higher level
of H3K9 methylation would promote the formation of heterochromatin in the
female pronucleus, resulting in late DNA replication. Supporting this
hypothesis are the facts that methylated H3K9 recruits heterochromatin protein
1 (HP1), an essential protein constituting heterochromatin
(Bannister et al., 2001
;
Schultz et al., 2002
), and
that this protein is accumulated only in the female pronucleus before S phase
of the one-cell stage (Arney et al.,
2002
). The biological significance of this asymmetric
heterochromatin formation is unclear. It does not directly involve asymmetric
X chromosome inactivation, as HP1 does not accumulate with the inactive X
chromosome (Peters et al.,
2002
).
The maintenance of differential H3/K9 methylation patterns between the
paternal and maternal genomes is an active process that depends on de novo
protein synthesis and gene expression. De novo H3/K9 methylation occurred in
the male pronuclei after transfer into enucleated oocytes
(Fig. 5), which indicates that
H3/K9 methylase activity exists during meiosis. The absence of histone
methylase activity after fertilization was not due to degradation of the
enzyme, as the enzyme functioned when protein synthesis was inhibited by
cycloheximide (Fig. 6A).
Although the histone methylase exists in both oocytes and embryos after
fertilization, the activity of the enzyme is inhibited by its inhibitor(s),
which may be synthesized as early as male pronucleus formation, i.e. 4 hours
after insemination. Fertilization of oocytes is accompanied by changes in the
pattern of protein synthesis (Xu et al.,
1997). These changes occur within 4 hours, at which time gene
expression has not yet been initiated
(Bounial et al., 1995
;
Aoki et al., 1997
); thus, these
changes are due to the recruitment of maternal mRNAs. It has been reported
that several maternal mRNAs are recruited for protein synthesis after
fertilization, including
-catenin, Ptp4a1, Spin
(Oh et al., 2000
) and cyclin
A2 (Fuchimoto et al., 2001
).
The maternal mRNA for the inhibitor of methylase may also be recruited for
translation after fertilization. Alternatively, it is possible that the
inhibitor protein is already present, albeit in an inactive form, in the
oocyte cytoplasm before fertilization. After fertilization, this protein is
activated and functions as an inhibitor of H3/K9 methylase. When the inhibitor
is labile, the inhibition of protein synthesis by cycloheximide brings about
the abolition of the inhibitor protein, which results in increased levels of
H3/K9 methylation in the male pronuclei. In any case, it seems likely that
there is an inhibitor protein(s) and that inhibition of its synthesis results
in the increase in the H3/K9 level in the male pronucleus after fertilization.
This suggests that the inhibitor protein(s) inactivates H3/K9 methylase,
thereby generating an asymmetric H3/K9 methylation pattern after
fertilization.
The inhibition of zygotic transcription resulted in de novo histone
methylation of the paternal genome (Fig.
6B). The transition from maternal to embryonic control of
development occurs in the two-cell stage of mouse embryos. During the two-cell
stage, a burst of transcriptional activation occurs in the embryonic genome.
The transcripts from the embryonic genome are used soon after their synthesis,
and most of the maternal transcripts used before this timepoint are degraded
(Flach, 1982;
Schultz, 1993
). The asymmetric
H3/K9 methylation pattern between parental genomes persists during the
transition from maternal to embryonic control, and gene expression from the
embryonic genomes is required for the maintenance of the asymmetric pattern.
After the degradation of maternal mRNA, the inhibitor of histone methylase is
probably produced via a transcript from the embryonic genome.
Asymmetric H3/K9 methylation may function as the mechanism that
distinguishes the genomes of different parental origin. The genomes are simply
identified as being marked or unmarked, based on whether they are in oocytes
before fertilization; only maternal genomes are present in the oocytes before
fertilization. This mechanism does not require differences between the genomes
themselves, as genomic distinction is maintained because the H3/K9 methylase
does not function, and de novo methylation does not occur after fertilization.
In addition, active H3/K9 demethylation does not seem to occur, as no histone
demethylase has been found in any organism. However, the methylation level of
the maternal genome decreases without de novo methylation. During embryonic
development, the chromatin is replicated, and methylated H3 is diluted with
unmethylated H3 from the cytoplasmic or nucleoplasmic pools. The H3/K9
methylase is activated at the four-cell stage, and the genomes of both
parental origins are methylated symmetrically. Thus, after the two-cell stage,
some other mechanism takes over from H3/K9 methylation to maintain distinction
of the parental genomes. Indeed, Xist begins to be expressed from the two-cell
stage and is associated with the X chromosome of paternal origin
(Huynh and Lee, 2003). In the
mechanism of paternal X chromosome inactivation during pre-implantation
development, Xist may take-over from H3/K9 methylation as the mechanism for
discriminating paternal genomes. Our hypothesis for the mechanism that
generates different H3/K9 methylation patterns, and thereby distinguishes the
different parental origins of genomes, is summarized in
Fig. 8.
|
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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* Present address: College of Animal Science and Technology, Nanjing
Agricultural University, Nanjing 210095, China
These authors contributed equally to this work
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