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J. Biol. Chem., Vol. 276, Issue 12, 8674-8680, March 23, 2001
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,From the Cancer and Developmental Biology Laboratory, Division of Basic Sciences, NCI-FCRDC, National Institutes of Health, Frederick, Maryland 21702
Received for publication, October 13, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Although most imprinted genes show allelic
differences in DNA methylation, it is not clear whether methylation
regulates the expression of some or all imprinted genes in somatic
cells. To examine the mechanisms of silencing of imprinted alleles, we
generated novel uniparental mouse embryonic fibroblasts exclusively
containing either the paternal or the maternal genome. These
fibroblasts retain parent-of-origin allele-specific expression
of 12 imprinted genes examined for more than 30 cell generations. We
show that p57Kip2 (cyclin-dependent
kinase inhibitor protein 2) and Igf2 (insulin-like growth factor 2) are induced by inhibiting histone deacetylases; however, their activated state is reversed quickly by withdrawal of
trichostatin A. In contrast, DNA demethylation results in the heritable
expression of a subset of imprinted genes including H19
(H19 fetal liver mRNA), p57Kip2,
Peg3/Pw1 (paternally expressed gene 3), and
Zac1 (zinc finger-binding protein regulating apoptosis and
cell cycle arrest). Other imprinted genes such as Grb10
(growth factor receptor-bound protein 10), Peg1/Mest
(paternally expressed gene 1/mesoderm-specific transcript), Sgce (epsilon-sarcoglycan), Snrpn (small
nuclear ribonucleoprotein polypeptide N), and U2af1 (U2
small nuclear ribonucleoprotein auxiliary factor), remain inactive,
despite their exposure to inhibitors of histone deacetylases and DNA
methylation. These results demonstrate that changes in DNA methylation
but not histone acetylation create a heritable epigenetic state at some
imprinted loci in somatic cells.
Normal development of mammalian embryos requires the genetic
contribution of both maternal and paternal genomes (1, 2). Uniparental
mouse embryos, in which the entire genome is either of maternal
(parthenotes) or paternal (androgenotes) origin, usually die during
early stages of embryogenesis (2, 3). Lethality of uniparental embryos
appears to be caused by either the lack of, or overexpression of,
specific genes that are imprinted and only expressed from the
nonimprinted parental allele. More than 35 autosomal genes in the mouse
exhibit parent-of-origin, allele-specific imprinting in embryonic and
adult tissues. The imprint, once set, is stable during mitosis but is
reversed and reset by passage through meiosis during gametogenesis (4,
5). Given the epigenetic nature of imprinting, much attention has
centered on whether DNA methylation is crucial to the establishment and
maintenance of the silent imprinted allele (6, 7). Most imprinted genes show parental differences in methylation patterns (7-10), although the
extent varies among the different genes. Furthermore, despite changes
in global levels of DNA methylation in early embryogenesis, methylation
of some imprinted genes remains constant even at the blastocyst stage,
whereas the rest of the genome is hypomethylated (8, 11, 12). After
implantation, most of the CpG sequences are progressively methylated,
except those located in the promoter region of active
"housekeeping" genes (13). These findings together with the
derivation of embryos deficient in methyltransferases, in particular
DNA methyltransferase 1, suggest that proper expression of some
imprinted genes (10, 14) requires DNA methylation. However, it is
unclear whether methylation is involved in the regulation of some or
all imprinted alleles. If methylation regulates the monoallelic
expression of imprinted genes, demethylation should reactivate silent
alleles in vitro. We tested this hypothesis by analyzing the
reactivation of imprinted alleles in a series of novel nonexpressing
uniparental mouse embryonic fibroblasts (MEFs)1 treated by
demethylating agents 5-azacytidine (AzaC) or 5-aza-2'-deoxycytidine (AzadC) alone or in combination with a histone deacetylase inhibitor, trichostatin A (TSA). Our results suggest that both DNA methylation and
histone deacetylase activities regulate the differential allelic expression of some but not all imprinted genes in somatic cells and
that the epigenetic modifications required for maintenance of
monoallelic expression vary among different imprinted loci. Significantly, the activated state induced by DNA demethylation, but
not by histone acetylation, is propagated and stably inherited during
mitosis, indicating that methylation is required for long term
repression of some imprinted genes.
Derivation of Wild-type (WT) and Uniparental MEFs--
WT and
uniparental MEFs were derived from explanted day 13 (day of plug = day 1) embryos after removing the head and internal organs.
Androgenetic (AG) MEFs were generated from chimeras made by injecting
androgenetic ES cells, constitutively expressing the Neo
gene (15, 16), into blastocysts. Parthenogenetic (PG) MEFs were
generated from PG 171 WT chimeric embryos. PG embryos were derived by
ethanol activation of eggs, with suppression of polar body formation,
and were then aggregated at the 4- or 8-cell stage with WT embryos
(17). The PG eggs were derived from mouse lines that constitutively
expressed the Neo gene (15). The AG and PG MEFs were derived
by culturing the primary explants in media supplemented with G418 for
at least 1 week which selected against the WT cells lacking the
NeoR gene.
Treatment of Cells with Inhibitors of DNA Methylation and Histone
Deacetylases--
Uniparental MEFs were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and
Pen/Strep. The day before treatment, cells were split to 50-60%
density. For the experiment shown in Fig. 2, cells were treated with
various concentrations of TSA (Wako) (0.2-5 µM) or AzaC
or AzadC (Sigma) (1-10 µM) for 24 h and total RNA
was prepared and analyzed by RT-PCR. For the experiment shown in Figs.
3 and 4, cells were incubated in media containing 0.3 µM
of TSA for 72 h. For treatment with the AzaC or AzadC (Sigma),
cells were incubated with medium containing initially 1 µM AzaC or AzadC for 24 h followed by 0.3 µM AzaC or AzadC for an additional 48 h. For the
combined treatment with AzaC/TSA or AzadC/TSA, cells were incubated
with AzadC or AzaC at the final concentration of 1 µM for
24 h followed by 0.3 µM for another 24 h, after
which 0.4 µM TSA was added in the presence of 0.3 µM AzadC or AzaC for an additional 24 h. After
treatment, cells were either used to prepare total RNA or cultured in
the absence of the drugs for up to 2 weeks.
RT-PCR Analysis of Imprinted Gene Expression--
Total RNA was
extracted from WT and uniparental MEFs using the RNeasy kit (Qiagen)
and treated with RNase-free DNase I (Promega) to eliminate residual
genomic DNA. 1.5 µg of RNA was converted to cDNA using random
primers and avian myeloblastosis virus reverse transcriptase in a
20-µl reaction for 60 min at 42 °C. PCR was performed with 1-2
µl of cDNA in 25-50 µl using Taq polymerase (Roche
Molecular Biochemicals). Amplification consisted of 25 or 30 cycles of
94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s performed in a PerkinElmer Life Sciences GeneAmp PCR machine 9600. Primers for all target genes are listed in
Table I.
For quantitative evaluation of the expression of imprinted genes in
untreated, TSA- and/or AzadC-treated AG and PG MEFS, total RNA was
converted to cDNA, and quantitative PCR was performed using serial
dilutions to assure a linear amplification of the target and control
genes (18). The levels of target gene expression were measured relative
to the housekeeping genes Hprt (hypoxanthine phosphoribosyltransferase) and Gapd
(glyceraldehyde-3-phosphte dehydrogenase) as internal controls. The
amplified products were analyzed by Southern blotting and quantified by
a PhosphorImager Storm 860 (Molecular Dynamics).
RNase Protection Assays--
Total RNA (10 µg) isolated from
untreated, TSA- and/or AzadC-treated MEFs was incubated overnight at
45 °C with radiolabeled probe (~106 cpm) in a 20-µl
reaction mixture containing hybridization buffer (Ambion, Inc). The
reaction mixtures were digested with RNases A and T1 and subsequently
analyzed by electrophoresis on polyacrylamide gel and visualized by
autoradiography. The relative intensities of the target and control
mRNAs were quantified by phosphorimaging.
Western Blotting of Acetylated Histones H3 and
H4--
Uniparental MEFs cultured for 24 h in the absence or
presence of TSA (0.2, 1, or 5 µM) were harvested and
nuclei prepared as described previously (19). Nuclei (3-5 × 106) were resuspended in lysis buffer (10 mM
HEPES, pH 7.5, 1.5 mM MgCl2, 10 mM
KCl, 1 mM dithiothreitol, 1 mM AEBSF (ICN) and
proteinase inhibitors complete (Roche)), then SDS was added to
final concentration of 1%. 20 µg of proteins from treated and
untreated AG and PG MEFs were separated by 16.5% SDS-polyacrylamide
gel electrophoresis and transferred to nitrocellulose membrane.
Hyperacetylated histones were detected by anti-acetylated H3 and H4
antibodies (Upstate Biotechnology) and were visualized by
chemiluminescence (Amersham Pharmacia Biotech). As control for the
amount of protein loading, a parallel gel was stained with Coomassie Blue.
DNA Extraction and Methylation Assay--
Genomic DNA was
extracted by proteinase K/SDS digestion, phenol/chloroform extraction,
and isopropyl alcohol precipitation. For
p57Kip2 (cyclin-dependent kinase
inhibitor protein 2) analysis, DNA was first digested with
HindIII and XbaI followed by incubation with either HpaII or MspI for at least 16 h at
37 °C. For the methylation analysis of U2af1 (U2 small
nuclear ribonucleoprotein auxiliary factor) and Snrpn (small
nuclear ribonucleoprotein polypeptide N) genes, DNA was digested first
with BamHI and HindIII followed by
HpaII or MspI. Digested DNA was analyzed by
Southern blotting as described previously (19).
Expression of Imprinted Genes in Uniparental MEFs--
We
generated uniparental diploid MEFs containing exclusively either the
paternal AG or the maternal PG genome. In these uniparental cells, the
epigenetic regulation of both alleles should be identical, with an
imprinted gene being either expressed or silent depending on the
parent-of-origin profile of imprinting. Probes to a panel of imprinted
genes showed that these cell lines stably retain parent-of-origin
allele-specific imprinting status over 30 cell generations (Fig.
1). The paternally expressed genes,
Igf2 (insulin-like growth factor 2),
Peg1/Mest (paternally expressed gene 1/mesoderm-specific transcript), Peg3/Pw1 (paternally expressed gene 3),
Snrpn, and U2af1 (20-24) as well as the two
newly identified imprinted genes Sgce (epsilon-sarcoglycan,)
and Zac1 (zinc finger-binding protein regulating apoptosis
and cell cycle arrest) (25) were detected only in AG and WT MEFs. In
contrast, the transcripts of maternally expressed H19 (H19
fetal liver mRNA), Grb10/Meg1 (growth factor receptor-bound protein 10/maternally expressed gene 1), and
p57Kip2 (26-28) were detected exclusively in PG
MEFs and WT MEFs. The one exception was the
Igf2r/M6Pr (insulin-like growth factor 2 receptor/mannose 6-phosphate receptor) (29). This gene is strongly expressed from the maternal allele (in PG and WT MEFs), but low levels
of expression were also detected in the AG MEFs, indicating that the
silencing of the paternal allele was not fully acquired in day 13 uniparental embryonic tissues. Furthermore, the tissue-specific imprinted genes Rasgrf1 (Ras protein-specific guanine
nucleotide-releasing factor 1), which is expressed exclusively from the
paternal allele in the brain, heart, and stomach (30), and
Mash2 (mammalian achaete-scute homologue 2), a
trophoblast-specific maternally expressed gene (31), were not detected
in WT and uniparental MEFs. The housekeeping genes, Gapd and
Hprt, were expressed in all MEFs. These results demonstrate
that AG and PG MEFs retain the correct expression pattern of 12 imprinted genes and provide unique and significant advantages to
analyze mechanisms leading to allele-specific silencing of these
genes.
Reactivation of Imprinted Genes in Nonexpressing Cells--
To
examine the roles that DNA methylation and histone deacetylation play
in differential expression of imprinted genes, uniparental AG and PG
MEFs were treated with increasing concentrations of either a specific
inhibitor of histone deacetylase (32), TSA (0.2-5 µM,
Fig. 2) or with inhibitors of DNA
methylation, AzaC/AzadC (1-10 µM) for 24 h. RT-PCR
was performed to determine whether imprinted genes were induced by drug
treatment in nonexpressing cells. TSA treatment resulted in a
dose-dependent induction of Igf2 and
p57Kip2 in PG and AG MEFs, respectively (Fig.
2A). A modest induction of both Igf2 and
p57Kip2 was observed in cells treated with a low
dose of TSA (0.2 µM). TSA reactivation was significantly
higher at 1 µM and decreased at 5 µM,
possibly because of its adverse effect on cell proliferation and
viability (33). Unlike Igf2 and
p57Kip2, all of the other imprinted genes listed
in Fig. 1 remained silent, despite induced accumulation of
hyperacetylated histones H3 and H4 after culturing uniparental cells in
the presence of different doses of TSA (Fig. 2B).
Acetylation of core histones, in particular H3 and H4, is believed to
play a role in chromatin unfolding and transcription regulation, as
actively transcribed genes are enriched in hyperacetylated histones H3
and H4 (33-35). Western blot analysis showed that levels of acetylated
H3 and H4 were very low (detectable only at high exposure, data not
shown) in untreated AG and PG MEFs. Incubation with 0.2-5
µM TSA resulted in the accumulation of acetylated H3 and
H4 as determined by antibodies directed against acetylated histones
(Fig. 2B; 0.2, 1, and 5 µM TSA). Under these experimental conditions, among the imprinted genes analyzed (Fig. 1),
only Igf2 and p57Kip2 were
reactivated. At this point we cannot rule out whether the induction of
these two genes is triggered by a locus-specific histone acetylation or
by a secondary effect of a global histone acetylation.
In contrast, treatment with demethylating agents AzaC or AzadC for
24 h was not sufficient to induce detectable levels of imprinted
gene mRNA in nonexpressing cells (data not shown). However, treatment with high doses of AzaC and AzadC (5-10 µM)
for over 24 h was toxic and inhibited cell proliferation. To
minimize the adverse effects of drug treatment on cell proliferation, a
protocol was elaborated in which uniparental AG and PG MEFs were
treated for 3 days with low dose of AzaC, AzadC, and/or TSA (see
"Materials and Methods"). As shown in Fig.
3, this resulted in the activation of a
subset of imprinted genes. Peg3 and Zac1 were
reactivated in PG MEFs and, p57Kip2and
H19 in AG cells after exposure of cells to either AzaC or AzadC. Igf2 was induced in PG MEFs treated with TSA
alone and was not responsive to demethylating agents, whereas both TSA
and AzaC/AzadC synergistically activated p57Kip2
in AG MEFs. In contrast, Grb10, Peg1,
Rasgrf1, Sgce, Snrpn, and U2af1 remained inactive after both treatments. The effects
of TSA and AzadC on the expression of Igf2r were not
assessed because the gene is not fully silenced in AG MEFs.
The levels of expression of a gene paternally imprinted,
p57Kip2, and two maternally imprinted genes,
Peg3 and U2af1, were evaluated further by RNase
protection assay and/or quantitative RT-PCR (Fig. 4). Measurement by phosphorimaging
analysis of changes in p57Kip2 mRNA levels
revealed a significant induction of p57Kip2
expression after treatment with either TSA or AzadC (Fig.
4A). However, the combined treatment with AzadC and TSA
synergistically activated p57Kip2 expression to
about 5-fold higher levels than treatment with either AzadC or TSA
alone. These results suggest that both histone deacetylase and DNA
methylation activities mediate silencing of the paternal allele of
p57Kip2 in cultured MEFs. Unlike
p57Kip2, Peg3 was activated in PG
MEFs treated with AzadC alone, whereas the addition of TSA
antagonized the induction by AzadC in a combined treatment (Fig.
4B), demonstrating that in this case the hyperacetylation of
histones has a negative effect on gene expression. Under the same
protocols, treatment of PG MEFs with AzadC and/or TSA did not
significantly change the expression levels of U2af1,
Hprt, or Gapd (Fig. 4, B and
C). Overall the results shown in Figs. 2-4 suggest that
only a subset of imprinted genes is activated in response to inhibitors
of histone deacetylation and/or DNA methylation.
AzaC and AzadC Treatments Correlate with DNA Demethylation--
We
analyzed the methylation status of p57Kip2
(induced by TSA and AzadC) and U2af1 (not responsive to
either TSA or AzadC treatment) by comparing the digestion of genomic
DNA from untreated, TSA-, AzaC-, or AzadC-treated AG and PG MEFs (Fig.
5). After treatment, cells were cultured
in the absence of drugs for 3-7 days, and DNA was purified and
digested with the methylation-sensitive HpaII or insensitive
MspI enzymes and analyzed by Southern blotting. Blots were
hybridized with probes derived from the promoter region of
p57Kip2 (Fig. 5A) and
U2af1 (Fig. 5B). DNA from WT MEFs, which carry both active and silent alleles of each imprinted gene, was used as a
control.
In all samples, DNA from nonexpressing cells (AG for
p57Kip2 and PG for U2af1), either
untreated or TSA-treated, was relatively resistant to HpaII
digestion, but was cut by MspI to produce heterogeneous fragments of various sizes (Fig. 5, A and B). The
results demonstrated that in nonexpressing cells,
p57Kip2 (in AG MEFs) and U2af1 (in PG
MEFs) genes are densely methylated, whereas in the expressing cells DNA
is unmethylated, as it is digested to the same extent with either
HpaII or MspI (compare Fig. 5A,
lanes 6 and 7, with Fig. 5B,
lanes 7 and 9). After treatment with AzadC,
p57Kip2 DNA from AG MEFs was readily digested
with HpaII (Fig. 5A, compare lanes 2 and 4), demonstrating that AzadC induced extensive
demethylation at this locus, particularly with regard to the appearance
of a major band at 0.4 kilobase. However, DNA from the same
cells treated with TSA (which also activated
p57Kip2) showed only minor changes in
HpaII digestion, indicating that TSA did not cause
significant demethylation as seen in AzadC-treated samples. Similar
results were obtained when blots were hybridized with U2af1
(Fig. 5B). DNA from AzaC- or AzadC-treated PG cells was
partially digested with HpaII, whereas DNA from untreated cells was fully resistant to digestion (compare lanes 2-5).
Thus, under these experimental conditions DNA is significantly
demethylated after AzaC or AzadC treatment, whereas the same sequences
in untreated cells are densely methylated. However, despite significant
levels of DNA demethylation, U2af1 and other imprinted genes
remained silent after treatment (Figs. 3 and 4), suggesting that
partial demethylation was not sufficient to reactivate these silent alleles.
Transient Inhibition of DNA Methylation, but Not Histone
Deacetylase, Promotes a Heritable State of Gene Expression--
In
mammals, propagation of the methylation state within CpG dinucleotides
by the maintenance DNA methyltransferase occurs during or
shortly after replication (36). Inhibition of DNA methylation by AzaC
or AzadC leads to the reactivation of some imprinted genes,
specifically H19, Peg3,
p57Kip2, and Zac1, whereas TSA
treatment induces two genes, p57Kip2 and
Igf2 (Fig. 3). To determine whether these induced
patterns of expression are maintained after multiple cell divisions,
uniparental MEFs initially treated for 3 days with TSA, AzaC, or AzadC
(Fig. 6, lanes P1) were
cultured for 7 days (lanes P2) or 15 days (lanes P4) after withdrawal of the inducing agents. The results shown in
Fig. 6 revealed that the expression of H19, Peg3,
p57Kip2, and Zac1 induced by
transient treatment with either AzaC or AzadC is enriched and
maintained over the course of multiple cell divisions in the absence of
either inhibitor (compare P1 with P4). However,
the TSA-induced transient expression of p57Kip2
and Igf2 was quickly reversed to a silent state after
two to four cell cycles after drug withdrawal. These results
demonstrate that changes in DNA methylation, but not levels of histone
acetylation, create a dominant and heritable epigenetic state of gene
expression at some imprinted loci.
In mammals, genomic imprinting results from the differential
epigenetic modification to a subset of genes in the germ line, leading
to parent-of-origin monoallelic expression during embryogenesis and in
adult tissues. Studying the molecular basis that distinguishes the
paternal and the maternal alleles of imprinted genes has been complicated by the simple fact that all somatic tissues contain both
maternal and paternal genomes. This necessitated the use of a variety
of techniques to distinguish between the parental alleles, such as
polymorphisms generated by intercrosses or the introduction of
mutations or deletions into different imprinted genes. Here we have
greatly simplified the analysis of imprinted gene expression by
generating novel uniparental mouse fibroblast lines retaining
parent-of-origin patterns of imprinting. We showed that the parental
specific expression is stably inherited during multiple cell
generations in vitro, demonstrating that these uniparental cells provide unique advantages for analyzing the mechanisms of silencing of imprinted alleles. Treatment of uniparental cells with
inhibitors of histone deacetylases or DNA methyltransferases resulted
in activation of only a subset of imprinted genes. In PG cells, both
Zac1 and Peg3 were reactivated in cells treated with AzaC or AzadC, whereas Igf2 was induced only by
TSA. In AG MEFs, p57Kip2 was synergistically
activated by combination of both AzadC and TSA, and H19 was
induced after AzadC treatment but not with TSA. However, under
identical experimental conditions Grb10, Peg1, Sgce, Snrpn, and U2af1, as well as the
tissue-specific imprinted genes Rasgrf1 and
Mash2, remained silent after all treatments, demonstrating
that not all imprinted genes are responsive to changes in DNA
methylation or histone acetylation levels in somatic tissues.
Recently DNA methylation and histone deacetylation have been
functionally linked to transcriptional repression. Methyl-CpG-binding proteins MeCP2 (37, 38), MBD2 (39), and MBD3 (40, 41) reside in
multiprotein complexes with histone deacetylases. These complexes
assemble on methylated DNA mediating transcriptional repression through
chromatin hypoacetylation with the silent state being partially
reversed by TSA. Consistent with this model, we have shown that
p57Kip2 is responsive to inhibitors of both DNA
methylation and histone deacetylase (Figs. 2-4). This suggests that
the silent paternal allele of p57Kip2 may be
associated with a repressor complex similar to those described above
and may contain both histone deacetylase and methyl-binding proteins
responsible for targeting the complex to methylated DNA. In contrast,
H19, Peg3, and Zac1 were only
responsive to loss of methylation but not to an inhibitor of histone
deacetylase, suggesting that complexes containing methyl-binding
proteins but not histone deacetylase may be involved in silencing at
these loci. Whether the reactivation of all these genes is caused by direct alteration of their "imprint" or by indirect changes in methylation elsewhere, remains to be determined. However, simultaneous analysis of the methylation levels of imprinted genes (Fig. 5) in both
AG and PG MEFs suggests that treatment affects global DNA methylation
rather than some gene-specific regulatory process. Genetic evidence has
implicated DNA methylation in silencing imprinted alleles of
H19, Igf2, Igf2r, and
p57Kip2 because a loss-of-function mutation of
the maintenance DNA methyltransferase gene (Dnmt1) resulted
in biallelic expression of both H19 and p57Kip2 (10, 14) but repression of
Igf2 and Igf2r expression (10). In
agreement with the genetic data, we showed that in vitro
demethylation caused reactivation of a subset of imprinted genes,
including p57Kip2 and H19 in
nonexpressing AG MEFs, and the newly identified Zac1 gene
and Peg3 from the maternal alleles in PG MEFs. Under these experimental conditions, the reactivation of Zac1 and
Peg3 suggests that methylation is also required to maintain
the silent state at these imprinted loci. The expression level of
Snrpn also appeared to increase in Dnmt1 null
mice (42); however, it is not clear whether the elevated expression is
caused by an increase in the transcription from the active paternal
allele or to the reactivation of the silent maternal allele. Our
results show that loss in DNA methylation in PG MEFs (Fig. 5) did not
result in detectable changes in expression of Snrpn (Fig.
3). Other reports (43-46) have also shown that Igf2,
H19, and p57Kip2 are responsive to
inhibitors of DNA methylation and histone deacetylases in a number of
tissues and cell culture systems. Pedone et al. (45)
suggested that Igf2 may also be induced by inhibitors
of DNA methylation, and H19 may be activated in a combined
treatment with AzaC + TSA or sodium butyrate. In a DNA
methyltransferase null background (Dnmt1 Here we showed (Figs. 3 and 6) that the same treatment that led to
reactivation of H19, p57Kip2,
Peg3, and Zac1 did not change the silent state of
Grb10, Peg1, Sgce, Snrpn,
and U2af1, despite the paternal and maternal alleles of
these genes being differentially methylated (7, 42, 47) and that
treatment with AzadC resulted in substantial demethylation of both
p57Kip2 and U2af1(Fig. 5). This
suggests that some imprinted genes (Grb10, Peg1,
Sgce, Snrpn, U2af1) are associated
with repressive heterochromatin-like structures that may be propagated
independently of DNA methylation and/or levels of histone acetylation.
Similar mechanisms were also suggested to explain the different rates
at which HPRT and PGK were reactivated on the
silent X chromosome after treatment with AzaC (48). If this is the
case, whatever the nature of the initial imprint is, once the silencing
is set, the repressive state becomes mitotically stable and reversible
only by passage through meiosis (4, 5). Partial DNA demethylation in
MEFs is therefore not sufficient to induce detectable expression of these imprinted genes. Also, the observed differential methylation between the expressed and silent alleles might be a consequence and not
the cause of silencing. In this case, DNA demethylation would not
affect the silencing. Detailed analysis of chromatin structure and
composition may help elucidate the mechanisms orchestrating allele-specific silencing of this group of imprinted genes.
Propagation of epigenetic states of expression during multiple cell
divisions is essential for the stability of imprinting. Remarkably, we
observed that the activated state of H19,
p57Kip2, Peg3, and Zac1
mediated by DNA demethylation was enriched and stably propagated
through multiple mitoses (Fig. 6). In contrast, the activated state of
Igf2 and p57Kip2, induced by
transient inhibition of histone deacetylase, was quickly reversed after
withdrawal of TSA from the culture medium, suggesting that the histone
deacetylase activity associated with silencing Igf2
and p57Kip2 is only temporarily inhibited but
not disrupted by TSA treatment. These results support a previous
observation (4) that changes in DNA methylation, but not histone
acetylation, create a heritable epigenetic state at some, but not all,
imprinted loci in somatic cells.
Here, we have shown that uniparental AG and PG MEFs provide a unique
model to study the regulation of imprinted gene expression in
vitro. In these lines, as in the whole mouse, imprinted genes are
expressed according to their parental origin. Among the imprinted genes
examined, four were responsive to partial loss of methylation (i.e. H19, p57Kip2,
Peg3, and Zac1), suggesting that DNA methylation
is the primary silencing mechanism for this set of imprinted genes and
that additional mechanisms may regulate silencing of other imprinted
genes in somatic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Primers and RT-PCR products
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Relative expression of imprinted genes in
wild-type and uniparental MEFs. Total RNA isolated from WT, PG,
and AG MEFs at successive passages (P2-P28) was analyzed by RT-PCR.
The authenticity of the amplified PCR products was confirmed by either
DNA sequencing or Southern blotting.
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Fig. 2.
TSA induces Igf2 and
p57Kip2 and causes accumulation of acetylated
histones in uniparental MEFs. A, RNA isolated from PG
and AG MEFs untreated (0) or treated with TSA (0.2, 1, or 5 µM) was analyzed by RT-PCR. The expression of
Hprt was analyzed as an internal control, and RNA from
untreated WT MEFs was used as a positive control. B, AG and
PG MEFs were cultured in absence (0) or presence of TSA (0.2, 1, and 5 µM) for 24 h, and nuclear extract was analyzed by
Western blot using anti-acetylated histone H3 and H4 antibodies as
indicated. A parallel gel was stained with Coomassie Blue for loading
control.
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Fig. 3.
Treatment of AG and PG MEFs with AzaC, AzadC,
and/or TSA results in reactivation of a subset of imprinted genes.
RNA isolated from untreated ( 
) or treated (+) AG and PG MEFs was
analyzed by RT-PCR. RNA from untreated WT MEFs was used as a positive
control for all imprinted genes except in Rasgrf1, the RNA
was from adult mouse brain.
View larger version (27K):
[in a new window]
Fig. 4.
Quantitative evaluation of
p57Kip2,
Peg3, and U2af1
expression in uniparental MEFs treated with AzadC and/or
TSA. Total RNA from uniparental MEFs untreated ( ) or treated (+)
with TSA, AzadC, or combination of both AzadC + TSA was analyzed by
RNase protection assay or quantitative RT-PCR and Southern blotting.
Panel A, expression of p57Kip2 in AG
and WT MEFs. The graph (right) results from a
phosphorimaging analysis of the RNase protection experiment
(left). The numbers indicate the percent of
p57Kip2 expression in treated AG MEFs compared
with WT, which is set to 100. Levels of expression of Peg3
(B) as well as U2af1 (C) mRNAs in
both untreated () and treated (+) MEFS were evaluated by quantitative
RT-PCR. In each reaction cDNA samples were analyzed in quadruplet
as undiluted or diluted 5, 25, or 100 times and quantified relative to
the housekeeping genes Gapd and Hprt. The graphs
result from a phosphorimaging analysis of the data from two
experiments.
View larger version (61K):
[in a new window]
Fig. 5.
DNA is demethylated in AG and PG MEFS treated
with AzaC or AzadC. A, analysis of DNA methylation of
the p57Kip2 gene in AG, PG, and WT MEFs. Genomic
DNA from untreated ( ) or treated (+) with AzadC or TSA was isolated
and digested with HpaII (lanes 2-4,
7, 8, and 10) or MspI
(lanes 5, 6, and 11) or left
undigested (u, lanes 1 and 9) as
indicated on the top of the panel. All DNAs in
panel A were digested with
HindIII/XbaI and analyzed by Southern blotting
using a probe that hybridizes to the promoter region of
p57Kip2. In the lower panel, the
genomic structure of the mouse p57Kip2 is shown.
The closed boxes numbered E1-E4 are exons 1-4.
The horizontal arrow indicates the putative transcription
start site. The positions of CpG islands and HpaII sites are
indicated. B, evaluation of DNA methylation of
U2af1 gene after treatment with AzaC or AzadC. Genomic DNA
from untreated () or treated (+) MEFs was either undigested
(u) or digested with HpaII or MspI as
indicated on the top of the panel. DNA samples
were also digested with HindIII/BamHI, and the
blot was hybridized with a probe spanning the U2af1 promoter
region. In the lower panel, the genomic structure of the
mouse U2af1, the positions of CpG islands and
HpaII sites are indicated.
View larger version (18K):
[in a new window]
Fig. 6.
Propagation of a heritable active state of
H19,
p57Kip2,
Peg3, and Zac1
induced by inhibitors of DNA methylation. AG and PG cells
initially treated for 3 days with AzaC, AzadC, or TSA (lanes
P1) were cultured for 7 days (lanes P2) or 15 days
(lanes P4) after withdrawal of the inhibitors. RNA samples
from passages P1, P2, and P4 were analyzed by RT-PCR. The control RNA
from untreated uniparental (U) and wild-type (WT)
MEFs is indicated. The induction of H19,
p57Kip2, and Mash2 was examined in AG
MEFs and that of Igf2, Peg3, Zac1,
Peg1, Sgce, and Hprt in PG MEFs. The
RNA from wild-type (WT) MEFs was used as a positive control
for all imprinted genes except in Mash2 the RNA was from
placenta.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/)
Igf2 is not expressed, with both the maternal and
paternal alleles being silent and H19 being expressed from
both alleles. These conflicting results suggest that the relaxation of
the coregulated Igf2 and H19 imprinting is
complex and that epigenetic changes elsewhere in the genome may also
affect their expression under certain conditions.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Lidia Hernandez and Lori Sewell for excellent technical assistance; Camilynn I. Brannan and Shoichi Sunahara for Snrpn and U2af1 probes; Matthieu Gerard for stimulating discussions; and Amar Klar, Jacob Z Dalgaard, and Peter Johnson for suggestions and comments on the manuscript.
| |
FOOTNOTES |
|---|
* 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: CDBL, NCI-FCRDC, Bldg.
539, Rm. 135, 1050 Boyles St., P. O. Box B, Frederick, MD
21702. Tel.: 301-846-5158; Fax: 301-846-7117; E-mail:
elkharroubia@mail.ncifcrf.gov.
§ Present address: Life Technologies Inc, Rockville, MD 20849.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M009392200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MEF(s), mouse embryonic fibroblasts; AzaC, 5-azacytidine; AzadC, 5-aza-2'-deoxycytidine; TSA, trichostatin A; WT, wild-type; AG, androgenetic; PG, parthenogenetic; RT-PCR, reverse transcriptase-polymerase chain reaction; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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