Role of Histone Methyltransferase G9a in CpG
Methylation of the Prader-Willi Syndrome Imprinting Center*
Zhenghan
Xin
,
Makoto
Tachibana§,
Michele
Guggiari¶,
Edith
Heard¶,
Yoichi
Shinkai§, and
Joseph
Wagstaff
**
From the Departments of
Biochemistry and
Molecular Genetics and
Pediatrics, University of Virginia,
Charlottesville, Virginia 22908-0733, § Department of Cell
Biology, Institute for Virus Research, Kyoto University, Kyoto
606-8507, Japan, and the ¶ Curie Institute, 75248 Paris, France
Received for publication, November 18, 2002, and in revised form, February 12, 2003
 |
ABSTRACT |
Imprinted genes in mammals are often located in
clusters whose imprinting is subject to long range regulation by
cis-acting sequences known as imprinting centers (ICs). The mechanisms
by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an ~2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here
we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in
mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27
methyltransferase and that maintenance of CpG methylation of the PWS-IC
in mouse ES cells requires the function of G9a. We show by RNA
fluorescence in situ hybridization (FISH) that expression
of Snrpn, an imprinted gene regulated by the PWS-IC, is
biallelic in G9a
/
ES cells, indicating loss of
imprinting. By contrast, Dnmt1
/
ES cells lack CpG
methylation of the PWS-IC but have normal levels of H3 Lys-9
methylation of the PWS-IC and show normal monoallelic Snrpn
expression. Our results demonstrate a role for histone methylation in
the maintenance of parent-specific CpG methylation of imprinting
regulatory regions and suggest a possible role of histone methylation
in establishment of these CpG methylation patterns.
 |
INTRODUCTION |
Although the number of mammalian genes identified as showing
parent-of-origin-specific expression patterns is growing, the mechanisms leading to their imprinted expression patterns are still
poorly understood (1). For many imprinted genes, there is a correlation
between parent-specific CpG methylation of 5' regulatory regions and
parent-specific expression. Targeted inactivation of the
Dnmt1 DNA methyltransferase gene leads to disruption of imprinted expression for many imprinted genes (2). Imprinted genes are
often located in clusters containing both maternally expressed and
paternally expressed genes whose imprinting is subject to long range
regulation by cis-acting sequences known as imprinting centers
(ICs)1 (3-5).
One of the best characterized imprinting centers is the Prader-Willi
syndrome (PWS) IC, which is required for establishment and maintenance
of the paternal pattern of gene expression and CpG methylation in the
Prader-Willi syndrome/Angelman syndrome (PWS/AS) region of human
chromosome 15 and mouse chromosome 7 (3, 6, 7). The PWS-IC in human and
mouse contains the promoter region of the imprinted Snrpn
gene, and in both species, somatic cells show CpG methylation specific
for the maternal copy of the PWS-IC. In mouse, this CpG methylation of
the maternal PWS-IC occurs during oogenesis, and this methylation has
been hypothesized to be the gametic imprint for the PWS/AS region in mouse (8). However, recent studies have shown that the PWS-IC is
completely unmethylated in human oocytes (9); therefore, maternal-specific CpG methylation of this region must occur after fertilization, and some other epigenetic mark must constitute the
gametic imprint.
We have shown previously in human somatic cells that histone H3 is
modified by Lys-9 methylation on the maternal copy of the PWS-IC and
that H3 is Lys-4-methylated on the paternal copy of the PWS-IC (10). We
have proposed histone H3 methylation on Lys-9 in the PWS-IC as a
candidate gametic imprint for the human PWS/AS region, with CpG
methylation of the IC and of other promoter regions occurring as a
consequence of the primary histone-based imprint. Recent studies by
Tamaru and Selker in Neurospora (11) and by Jackson et
al. in Arabidopsis (12) have demonstrated dependence of
cytosine methylation on histone H3 Lys-9 methyltransferases. In this
study, we examine the hypothesis that CpG methylation of the mouse
PWS-IC requires the histone H3 Lys-9/Lys-27 methyltransferase G9a.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
Undifferentiated G9a +/+,
/
, and
/
with G9a transgene ES cell lines (13), as
well as a Dnmt1
/
ES cell line (14), were maintained in
ES cell medium containing 10% fetal calf serum and 1000 units/ml ESGRO (Chemicon).
Chromatin Immunoprecipitation (ChIP)--
Chromatin was prepared
from ES cells as described (15) and was sonicated to an average
size of ~0.5 kb. Spleens from F1 mice (C57Bl/6J x CAST/Ei and CAST/Ei
x C57Bl/6J) were dissociated in Hanks' balanced salt solution buffer
and cross-linked with 1% formaldehyde for 10 min at room temperature.
The fixed cells were collected by centrifugation at 700 × g for 10 min, resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0), and then
sonicated to produce chromatin of an average size of ~0.5 kb.
Chromatin was immunoprecipitated as described (15) with rabbit
polyclonal antibody to dimethyl Lys-9 H3 or with rabbit polyclonal
antibody to dimethyl Lys-4 H3. DNA recovered from the immunoprecipitated material was amplified by PCR with the following primers: PWS-IC (Snrpn 5' region, Msnp1,
5'-ACACGCTCAAATTTCCGCAG-3' and Msnp2, 5'-TAGTCTTGCCGCAATGGCTC-3'), and
D13Mit55 (D13Mit-F, 5'-TCAATATTAACTGCTAGCATGGTT-3', and
D13Mit-R, 5'-GCTTTTCCTCCCCAAACATT-3'). Amounts of ChIP DNA per reaction
were normalized to give equal amounts of product with
D13Mit55 control primers, which amplify a locus near the
centromere of mouse chromosome 13; PCR yield with D13Mit55
primers on ChIP DNA is independent of G9a genotype for ChIP
with antibodies to methyl Lys-9 H3 and methyl Lys-4 H3 (data not
shown). Annealing temperature was 59 °C for Msnp1/Msnp2 and 55 °C
for D13Mit-F/D13Mit-R. Products in Fig. 2, a and
c, were visualized by ethidium bromide staining.
Quantitative analysis of ChIP DNA (see Fig. 2b) was carried
out by PCR in the presence of [
-32P]dCTP for 28 cycles, where product yield is a linear function of cycle number for
both primer sets. PCR products were separated on 12% nondenaturing
polyacrylamide gels and visualized by autoradiography, and films were
scanned with a Amersham Biosciences densitometer and analyzed
with ImageQuant software. ChIP DNA PCR ratios for the two
G9a
/
ES cell lines were calculated as [(PWS-IC product from
/
)/(D13Mit55 product from
/
)]/[(PWS-IC
product from +/+)/(D13Mit55 product from +/+)].
Bisulfite Genomic Sequencing--
Bisulfite treatment of genomic
DNA was carried out as described by Clark et al. (16).
Bisulfite-modified DNA was amplified by nested PCR with the
following primers: Snrpn 5' region (SNB1, 5'-GTGATGTTTGTAATTATTTGGGAG-3' + SNB2, 5'-TTCACTACTAAAATCCACAAACC-3', and SNB3N, 5'-ATTATTTTAGATTGATAGTG-3' + SNB4,
5'-AATCCACAAACCCAACTAAC-3'). PCR products were cloned into a PCR4-TOPO
vector (Invitrogen) and sequenced.
RNA FISH--
RNA FISH was performed as described (17). CITB
mouse BAC 289D17 (clone obtained from Research Genetics) was
used to detect Snrpn RNA;
clone 510 was used to detect
Xist RNA.
 |
RESULTS |
To determine whether parent-specific H3 Lys-9 methylation at the
PWS-IC is present in somatic cells of the mouse, we studied a single
base pair variation between C57Bl/6J and Mus musculus castaneus (Cast-Ei) mice, located 104 bp 5' to the major site of
Snrpn transcription initiation. Somatic tissues from F1
hybrids between C57Bl/6J and Cast-Ei were subjected to ChIP with
antibody to methyl Lys-9 H3; sequencing of genomic DNA from reciprocal hybrids showed equal peaks corresponding to each of the parental alleles, but sequencing of PCR-amplified ChIP DNA showed only the
maternal allele from each of the crosses (Fig.
1). Therefore, the maternal-specific
association of methyl Lys-9 H3 with the PWS-IC region is conserved
between human and mouse.

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Fig. 1.
Maternal-specific association of
methyl Lys-9 H3 with the PWS-IC. Genomic DNA from F1 offspring of
reciprocal crosses between C57Bl/6J and M. musculus
castaneus-Ei (Cast-Ei) shows equal peaks at the variant
site 104 bp 5' to the major start site of Snrpn
transcription (arrow). DNA obtained by ChIP of spleen cells
from the F1 mice with antibody specific for methyl Lys-9 H3 contains
exclusively the maternal allele from both crosses. In the sequence,
N indicates two coincident peaks.
|
|
To determine which histone methyltransferase(s) is required for H3
Lys-9 methylation of the PWS-IC region in mouse, we examined ES cells
homozygous for a targeted deletion of the gene encoding the G9a histone
H3 Lys-9/Lys-27 methyltransferase (18). Loss of G9a function causes
loss of H3 Lys-9 methylation primarily in euchromatic regions, rather
than in regions of centromeric heterochromatin, and mice homozygous for
the G9a deletion die in midgestation (13). ES cells
homozygous for the G9a deletion are viable (13), and two
independent G9a
/
ES cell lines both showed reduction of
H3 Lys-9 methylation at the PWS-IC to ~30% of G9a +/+
levels (Fig. 2). Total H3 Lys-9
methylation of the PWS-IC in G9a
/
ES cells with a
G9a transgene was similar to that in G9a +/+ ES
cells (Fig. 2). G9a inactivation did not have any
significant effect on the level of H3 Lys-4 methylation in the PWS-IC
(Fig. 2).

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Fig. 2.
Association of PWS-IC with methylated H3 in
G9a +/+ and / ES cell lines. Lane 1,
G9a +/+; lanes 2 and 3, G9a
/ ; lane 4, G9a / with G9a
transgene. a, analysis of DNA obtained by ChIP with antibody
to methyl Lys-9 H3. Upper panel, PCR analysis of PWS-IC
showing decreased PCR product visualized by ethidium bromide staining
in G9a / lanes; lower panel, PCR
analysis of control locus D13Mit 55. b, analysis
of DNA obtained by ChIP with antibody to methyl Lys-9 H3 with PCR
products visualized by incorporation of [ -32P]dCTP and
autoradiography. The ratio of PWS-IC PCR product to D13Mit55
product for each G9a / ES cell line was normalized to
the ratio for the G9a +/+ cell line. c, analysis
of DNA obtained by ChIP with antibody to methyl Lys-4 H3. Upper
panel, PCR analysis of PWS-IC; lower panel, PCR
analysis of control locus D13Mit55.
|
|
DNA from G9a +/+, +/
, and
/
ES cells was analyzed by
bisulfite genomic sequencing (16) to determine the methylation status of CpG dinucleotides in the PWS-IC region. Seven out of ten clones from
+/+ cells showed evidence of methylation at all or almost all CpG sites
(Fig. 3), and five out of nine clones
from +/
cells showed methylation at all or almost all sites (data not
shown); by contrast, 32 clones from 3 G9a
/
cell lines
showed no evidence of methylation at any CpG site (Fig. 3).
G9a
/
cells with a G9a transgene showed no
evidence of methylation at any CpG site (data not shown).

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Fig. 3.
G9a / ES cells lack CpG
methylation of the PWS-IC. Bisulfite genomic sequencing of the
PWS-IC region in G9a +/+ and / ES cells shows dependence
of CpG methylation on G9a function. Each row of
circles connected by a horizontal line represents
one PCR product from bisulfite-treated DNA that has been cloned and
sequenced. The amplified region (GenBankTM accession
number AC026878, nucleotides 135219-135499) contains 16 CpG
dinucleotides, each represented by a circle. Closed
circles indicate methylated cytosine residues (not converted to
uracil by bisulfite treatment), and open circles indicate
unmethylated cytosine residues that have been converted to uracil by
bisulfite treatment. (Snrpn transcription starts at
nucleotide 135416.) Results are shown for the parental G9a
+/+ ES cell line and three G9a / ES cell lines
(independent homozygous derivatives of a G9a +/ ES cell
line).
|
|
We examined the role of G9a in control of imprinted Snrpn
expression by RNA FISH analysis of ES cell lines. Simultaneous
Xist RNA FISH was performed as a control as this transcript
can normally be detected in almost 100% of undifferentiated male ES
cells. Greater than 80% of G9a +/+ or +/
ES cells showed
either a single Snrpn signal or one strong and one very weak
G9a signal per nucleus (Fig.
4, a and b); by
contrast, >80% of G9a
/
ES cells from two lines showed
two strong Snrpn hybridization signals, indicating biallelic
expression (Fig. 4, c and d). The observation of
one strong and one very weak Snrpn RNA FISH signal in
G9a wild-type nuclei suggests that Snrpn
imprinting in ES cells is slightly leaky, unlike the situation in
somatic tissues; results consistent with this observation have been
reported by Szabo and Mann (19). G9a
/
ES cells with a
G9a transgene also showed two Snrpn signals in
>80% of cells, although the intensity of the two signals within each
nucleus tended to be less equal than in the G9a
/
ES
cells (data not shown).

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Fig. 4.
G9a / ES cells show loss of
imprinting of Snrpn. RNA FISH analysis of
Snrpn expression was performed in male ES cells. Red
signal, Snrpn; green signal,
Xist. G9a +/+ (a) and +/
(b) ES cells show either a single strong Snrpn
signal or one strong Snrpn signal and one very weak signal
per nucleus. G9a / ES cells (c and
d) show two strong Snrpn signals in >80% of
cells. All cells have a single copy of Xist that is either
unreplicated (a, c, and d) or
replicated (b).
|
|
We also tested methylation of CpG sites in the PWS-IC region in
embryonic day 9.5 G9a +/+ and
/
embryos arising from
heterozygote intercrosses. We found no significant difference between
the CpG methylation patterns in G9a +/+ and
/
embryos
(Fig. 5).

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Fig. 5.
Bisulfite genomic sequencing of the PWS-IC
region from G9a +/+ and / embryonic day 9.5 embryos. Analysis was carried out in DNA from one +/+ embryo and
two / embryos as described in the legend for Fig. 3 and shows no
significant effect of the G9a genotype on CpG
methylation.
|
|
To determine whether CpG methylation of the PWS-IC region is required
for normal levels of H3 Lys-9 methylation, we performed bisulfite
genomic sequencing and ChIP analysis of a Dnmt1
/
ES
cell line. Bisulfite genomic sequencing of 10 clones showed a complete
absence of CpG methylation of the PWS-IC region (data not shown). ChIP
analysis showed that the association of the PWS-IC region with methyl
Lys-9 H3 was unaffected by targeted inactivation of the Dnmt1
maintenance DNA methyltransferase (Fig.
6). RNA FISH analysis of the
Dnmt1
/
ES cells showed that >80% of cells contained either a single Snrpn signal or one strong and one very weak
G9a signal per nucleus, as in wild-type ES cells (Fig.
7).

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Fig. 6.
Association of PWS-IC with Lys-9-methylated
H3 in Dnmt1 +/+ and / ES cell lines. DNA
obtained by ChIP with antibody to methyl Lys-9 H3 from Dnmt1
+/+ and / ES cells was analyzed by PCR for the PWS-IC (top
panel) and for the control locus D13Mit55 (lower
panel).
|
|

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Fig. 7.
Dnmt1 / ES cells show normal
monoallelic expression of Snrpn. RNA FISH
analysis of Snrpn expression was performed in male ES cells.
Red signal, Snrpn; green signal,
Xist. Dnmt1 / ES cells show either a single
strong Snrpn signal or one strong Snrpn signal
and one very weak signal per nucleus, the same result observed in
wild-type (G9a +/+) ES cells (Fig. 4). All cells have a
single copy of Xist.
|
|
 |
DISCUSSION |
Our data show that the PWS-IC is H3 Lys-9-methylated specifically
on the maternal allele, that H3 Lys-9 methylation of the PWS-IC region
is reduced in G9a
/
ES cells, and that maternal-specific CpG methylation of the PWS-IC is lost in G9a
/
ES cells.
Previous authors have suggested that CpG methylation of the maternal
copies of some imprinting centers, established in the female germline, serves as the gametic imprint for coordinately regulated clusters of
imprinted genes such as those in the PWS/AS region and in the Beckwith-Wiedemann syndrome (BWS) region (1). Although mouse data
showing CpG methylation of the PWS-IC and the BWS-IC2 in oocytes but
not in sperm are consistent with this hypothesis (4, 8), the recent
demonstration that the PWS-IC is completely unmethylated on CpG
dinucleotides in human oocytes (9) implies that parent-specific CpG
methylation differences at the human PWS-IC occur after fertilization
and that CpG methylation of the human PWS-IC cannot be the gametic
imprint for the human AS/PWS region.
Tamaru and Selker (11) have shown that CpG methylation in
Neurospora requires the Dim-5 H3 Lys-9 methyltransferase,
and Jackson et al. (12) have demonstrated that CpNpG
methylation in Arabidopsis is dependent on the H3 Lys-9
methyltransferase KYP. We have demonstrated that maintenance of
CpG methylation at the PWS-IC in mouse ES cells is also dependent on an
H3 Lys-9/Lys-27 methyltransferase, G9a. The mechanisms leading to
dependence of cytosine methylation on histone H3 Lys-9
methyltransferases in these three systems have not yet been elucidated.
G9a +/+ and
/
ES cells show no differences in DNA
methyltransferase activities or in expression levels of
Dnmt1, Dnmt3a, or
Dnmt3b.2 The level
of H3 Lys-9 methylation of the PWS-IC in G9a
/
ES cells is reduced to ~30% of that observed in G9a +/+ ES
cells. The residual H3 Lys-9 methylation in G9a
/
ES
cells must be maintained by a histone methyltransferase other than G9a;
either the methyltransferase or the pattern of Lys-9 methylation
catalyzed by that methyltransferase must be unable to participate in
maintaining CpG methylation of the PWS-IC.
We have shown that restoration of G9a function in G9a
/
ES cells with a transgene leads to near normal levels of H3 Lys-9 methylation of the PWS-IC but does not restore CpG methylation or
monoallelic Snrpn expression. This result suggests that
although G9a is required for maintenance of CpG methylation at the
PWS-IC in ES cells, its presence is not sufficient for de
novo CpG methylation of the PWS-IC, which must require molecules
that are not present in ES cells. Biniszkiewicz et al. (20)
have shown that overexpression of Dnmt1 in Dnmt1
/
ES cells does not lead to CpG methylation of the Snrpn
promoter, and they have suggested that de novo CpG methylation of this region can occur only during gametogenesis. G9a transgene expression in G9a
/
ES cells is
also clearly not a sufficient condition for monoallelic expression of
Snrpn.
Although G9a
/
ES cells show complete loss of CpG
methylation at the PWS-IC, we have seen no effect of the G9a
mutation on CpG methylation of these regulatory regions in embryonic
day 9.5 embryos. The reason for this difference is unclear. In both cases, the observed cytosine methylation represents maintenance, rather
than de novo, methylation. Our results may indicate the existence of histone methylation-independent mechanisms for maintenance of CpG methylation at imprinting centers in embryos but not in ES
cells; alternatively, other H3 methyltransferases may compensate for
loss of G9a function in embryos but not in ES cells.
We have seen no effect of targeted mutation of the Dnmt1
maintenance cytosine methyltransferase on H3 Lys-9 methylation of the
PWS-IC. In the ES cell system, dependence of CpG methylation of the
PWS-IC on the G9a H3 Lys-9/Lys-27 methyltransferase does not appear to
be accompanied by a reciprocal dependence of H3 Lys-9 methylation on
CpG methylation. RNA FISH analysis of these Dnmt1
/
ES
cells showed normal monoallelic expression of Snrpn, indicating that CpG methylation of the PWS-IC is not required for
monoallelic expression of Snrpn in ES cells. Previous
studies of imprinted gene expression in undifferentiated
Dnmt1
/
ES cells have not distinguished monoallelic from
biallelic expression; however, Tucker et al. (21) showed
that the level of expression of Igf2r, which is
normally maternally expressed, is the same in Dnmt1 +/+ and
/
ES cells. Our results suggest that, in undifferentiated ES cells,
histone H3 Lys-9 methylation is sufficient and CpG methylation is not
required for maintenance of Snrpn monoallelic expression. We
hypothesize that the pattern of H3 Lys-9 methylation of the PWS-IC in
G9a
/
ES cells with a G9a transgene is not
the normal pattern established during gametogenesis, both in terms of
allele specificity and distribution on nucleosomes, and that this
qualitative abnormality of H3 Lys-9 methylation accounts for the
biallelic expression of Snrpn in these cells. This
hypothesis is difficult to test because the parental ES cell line used
in these studies contains very few heterozygous polymorphic sites so
that it is not possible to test for allele-specific association of
modified histones with the PWS-IC and because it is very difficult to
assess the sensitivity of ChIP to the density of histone modifications.
Previously we have proposed, based on our observations of
maternal-specific association of methyl Lys-9 H3 with the PWS-IC and on
the lack of CpG methylation of the PWS-IC in human oocytes, that H3
methylation may be the gametic imprint for the maternal allele of the
human AS/PWS region and may trigger CpG methylation of the PWS-IC and
parent-specific expression throughout the AS/PWS region after
fertilization (10). The data presented here, showing dependence of CpG
methylation at the mouse PWS-IC on the G9a histone methyltransferase,
are completely consistent with this hypothesis. Most putative germline
imprints involve repression on the maternal alleles (22), and a
histone-modification-based gametic imprint can only be carried on the
maternal allele because histones are replaced by protamines during
spermatogenesis. It is clear that the PWS-IC is CpG methylated in mouse
oocytes, unlike the situation in human oocytes. We hypothesize that
this difference does not reflect a fundamental difference between mice
and human in the imprinting process, but rather that the difference is
in the timing of histone methylation-dependent CpG methylation:
prefertilization in the mouse, postfertilization in human. Our
data do not address the question of the role of G9a and histone
methylation in the establishment of imprints in the germline; an
answer to this question will require conditional knockouts of
G9a and other histone methyltransferases limited to the germline.
 |
ACKNOWLEDGEMENTS |
We thank C. David Allis for antibodies to
methyl Lys-9 H3 and methyl Lys-4 H3 and for helpful comments. We thank
En Li for the Dnmt1
/
ES cell line. We also thank Jason
Lau for unpublished data regarding the D13Mit55 locus.
 |
FOOTNOTES |
*
This work was supported by grants from the Ward Family
Foundation and the March of Dimes (to J. W.), from the Ministry of Science, Technology, and Culture of Japan (to Y. S.), and from the
"Centre Nationale pour la Recherche Scientifique" and the "Fondation pour la Recherche Nationale" (to E. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Pediatrics and
Dept. of Biochemistry and Molecular Genetics, University of Virginia
Health System, Jordan Hall, Box 800733, Charlottesville, VA 22908-0733. Tel.: 434-243-5818; Fax: 434-924-5069; E-mail: wagstaff@virginia.edu.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M211753200
2
M. Tachibana and Y. Shinkai, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
IC, imprinting center;
PWS, Prader-Willi syndrome;
PWS/AS, Prader-Willi
syndrome/Angelman syndrome;
BWS, Beckwith-Wiedemann syndrome;
ES, embryonic stem;
ChIP, chromatin immunoprecipitation;
FISH, fluorescence
in situ hybridization.
 |
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