From the Laboratory of Mammalian Genes and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, August 19, 2002, and in revised form, September 12, 2002
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ABSTRACT |
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Understanding the molecular basis of
monoallelic expression as observed at imprinted loci is helpful in
understanding the mechanisms underlying epigenetic regulation. Genomic
imprinting begins during gametogenesis with the establishment of
epigenetic marks on the chromosomes such that paternal and maternal
chromosomes are rendered distinct. During embryonic development, the
primary imprint can lead to generation of secondary epigenetic
modifications (secondary imprints) of the chromosomes. Eventually,
either the primary imprints or the secondary imprints interfere with
transcription, leading to parent-of-origin-dependent
silencing of one of the two alleles. Here we investigated several
aspects pertaining to the generation and functional necessity of
secondary methylation imprints at the Igf2/H19
locus. At the H19 locus, these secondary imprints are, in
fact, the signals mediating paternal chromosome-specific silencing of that gene. We first demonstrated that the H19
secondary methylation imprints are entirely stable through multiple
cell divisions, even in the absence of the primary imprint. Second, we
generated mouse mutations to determine which DNA sequences are
important in mediating establishment and maintenance of the silent
state of the paternal H19 allele. Finally, we
analyzed the dependence of the methylation of
Igf2DMR1 region on the primary methylation imprint
about 90 kilobases away.
Mammals inherit two complete sets of chromosomes, one from the
mother and one from the father. Most autosomal genes are expressed equivalently from the maternal and the paternal alleles. Imprinted genes, however, are expressed preferentially from only one chromosome in a parent-of-origin-dependent manner (1). Because the
active and the inactive promoters of an imprinted gene are present in a
single nucleus, the differences in their activity cannot be explained
by differences in transcription factor abundance. Rather, the
transcription of imprinted genes represents a clear situation in which
epigenetic mechanisms restrict gene expression and therefore offers a
model for understanding the role of heritable DNA modifications such as
cytosine methylation in maintaining appropriate patterns of expression.
The imprinted Igf2-H19 gene pair is part
of a large cluster of imprinted genes on the distal end of mouse
chromosome 7. H19 and Igf2 share enhancers
and therefore share developmentally complex patterns of gene expression
(2). However, they are reciprocally imprinted. H19 is
expressed exclusively from the maternal chromosome, whereas
Igf2 expression is almost entirely paternal in origin (3, 4). The syntenic region in humans on chromosome 11p15.5 is highly
conserved in genomic organization and in monoallelic gene expression
patterns (5, 6). Loss-of-imprinting mutations at chromosome 11p15.5 are
associated with Beckwith-Wiedemann syndrome and with several types of
cancer (7-9).
Maternal chromosome-specific expression of H19 and paternal
chromosome-specific expression of Igf2 are each
dependent upon a cis acting imprinting
control element
(ICE),1 a 2-kb
region spanning
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 kb to
4 kb upstream of the H19
promoter (10, 11) (Fig. 1a)
and located about 90 kb downstream of Igf2 (distance
based on GenBankTM sequence NW_000336). However, the
mechanisms by which monoallelic expression is maintained at the two
loci are distinct (12). Igf2 is regulated by a
methylation-sensitive insulator element that also maps with the
ICE. This insulator element must be continually present in
its unmethylated state to maintain maternal Igf2
silencing. Repression of paternal H19 is at least a two-step
process. A paternally inherited ICE is required in the
developing embryo. The primary paternal imprint at the ICE
somehow induces further epigenetic changes at the locus that silence
the paternal H19 promoter such that the presence of the
ICE is not subsequently required for maternally restricted
transcription in H19 expressing cells. Thus, at
H19, the primary imprint established during gametogenesis
leads to a secondary imprint responsible for silencing the paternal H19 (12).
View larger version (28K):
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Fig. 1.
a, schematic diagram of the
Igf2/H19 locus showing the relative positions of the
H19 and Igf2 genes
(rectangles). Transcription start sites are shown as
arrows with promoters for H19 (P) and
Igf2 (P0, P1, P2,
and P3) indicated. Exons are shown as blackened
areas. Gray rectangles represent differentially
methylated regions H19DMR (D),
Igf2DMR0 (D0), Igf2DMR1
(D1), and Igf2DMR2 (D2). The
H19DMR is coincident with the imprint control element,
ICE (gray oval), for H19 and
Igf2. Shared enhancers (black ovals) for
expression of H19 and Igf2 in endoderm
(E1) and skeletal muscle (E2) have been defined
and are all located downstream of H19 (2, 32). b,
strategy for the deletion of a part of H19 exon I by gene
targeting. (i), wild type allele; (ii), targeting
vector; (iii), targeted allele,
H19exIflox neo; (iv), targeted
allele after the excision of the neomycin gene,
H19exIflox, with loxP sites flanking
the first 710 bp of H19; (v), targeted allele,
H19
exI, after deletion of
loxP-flanked region in vivo using
EIIa-cre transgenic mice; (vi), correctly
targeted clones and cre recombinase-mediated excision of
neomycin gene were confirmed by Southern hybridization. Genomic DNA
from the manipulated embryonic stem cell clones was digested
with KpnI and hybridized to a 1.5-kb
KpnI-BglII fragment from the region upstream of
H19. Correctly targeted clones give a 5-kb band from the
targeted H19exIflox
neo allele and a 7.7-kb
band from the wild type allele (lane 1). Subsequent to
cre recombinase-mediated excision of neo-tk, the
targeted allele, H19exIflox, hybridizes only to
the 7.7-kb DNA fragment (lane 2) like the wild type
(lane 3); (vii), EcoRI-digested
genomic DNA hybridized to a 1-kb BglII-EcoRI
fragment downstream of H19. Correctly targeted clones show
two bands of 12.7 and 10.7 kb from
H19exIflox
neo and the wild type allele,
respectively (lane 1). Subsequent to the excision of
neo-tk, the H19exIflox and the wild
type alleles are indistinguishable (lane 2) and resemble the
wild type (lane 3). DTA, diphtheria toxin gene;
neo-tk, neomycin resistance and thymidine kinase genes;
solid arrowheads, loxP sites; thick
lines, regions for homologous recombination; white
rectangles, regions used as 5' and 3' probes for hybridization;
RI, EcoRI; B, BsmI;
Bg, BglII; D, DraIII;
K, KpnI; S, SalI.
Developmental regulation of cytosine methylation at the H19 locus is consistent with, and most likely explains, the mechanisms of this two part silencing process. The ICE is so far indistinguishable from the H19DMR (or H19 differentially methylated region). Cytosine residues within this 2-kb region are methylated in sperm but not in oocytes (10, 13). This differential methylation survives the global changes in DNA methylation that occur during early mammalian development (14, 15). Post-implantation, the domain of paternal chromosome-specific methylation spreads to include the H19 promoter and exonic sequences (13, 16, 17). The mechanistic significance of these changes in DNA methylation is evidenced by the correlation between loss of biallelism and gain-of-methylation in the normally developing embryo (18, 19) and also by the loss of H19 monoallelism in mice carrying a deletion of the DNA methyltransferase gene (20). Mechanisms for this methylation spread are of great interest because of analogies with changes in DNA structure and expression of a number of tumor suppressor genes, as seen in many types of tumor cells.
In this investigation, we examined the silencing of the paternal
H19 promoter and several aspects pertaining to the spread of
cytosine methylation. We show first that the cytosine methylation of
the H19 promoter and exonic sequences is stable in the
absence of the originating differentially methylated ICE
even through mitosis. Second, we have characterized the sequences
required to establish and hold this secondary imprint. Finally, we show that the spread of the methylation can occur over long distances, as
the paternally methylated ICE is responsible for secondary methylation changes that occur about 90 kilobases upstream at the
Igf2 promoter.
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EXPERIMENTAL PROCEDURES |
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Generation of hCD2-cre Transgenic Mice-- The plasmid phCD2-cre, placing cre recombinase under the control of hCD2 promoter (21), was microinjected into mouse oocytes to obtain the transgenic mice.
Generation of H19exI Mice--
We used
cre-loxP-based deletion strategy for generating
the mutants (22). As shown in Fig. 1b, the targeting vector
carried an 11.7-kb BglII fragment with H19
sequences from between
2 and +9.7 kb (all base pairs are described
relative to the H19 transcriptional start site). A
loxP-flanked cassette with neomycin resistance and thymidine
kinase genes (neo-tk) was inserted at DraIII (+3 bp), and an additional loxP site was inserted at
BsmI (+710 bp). The diphtheria toxin-A gene was inserted for
negative selection. Linearized vector was electroporated into mouse
RI embryonic stem cells. Correct clones were identified by
Southern hybridization. A correctly targeted clone was then
re-electroporated with pBS185 (Invitrogen) to direct excision of
neo-tk, and the excision was detected by Southern
hybridization (Fig. 1b). Correct clones were injected into
C57/BL6-J blastocysts to generate chimeric founder mice that were mated
with EIIa-cre transgenic females (23) to generate strains
deleted for exon I (H19
exI). The exon I
excision was detected by PCR using primers Madhu25 (5'-GAA TTC TGG GCG
GAG CCA C-3') and Madhu20 (5'-TGG GAT GTT GTG GCG GCT GG-3') upstream
and downstream of the deletion, respectively. The 180-bp PCR product
was confirmed by sequencing.
Isolation, Purification, and Induced Proliferation of T
Cells--
Cells were isolated from lymph nodes of
+/DMRflox and
+/DMRflox,hCD2-cre mice, suspended in
complete RPMI medium, and enumerated. Mature T cells were enriched by
depletion of non-T cells with magnetic beads essentially as described
(24). The purity of the resulting T cell populations was 90%. T
cells were then labeled with the membrane-permeable intracellular
covalent coupling fluorescent dye carboxyfluorescein diacetate
succinimidyl ester (CFSE) as described previously (24). For in
vivo experiments, labeled cells were resuspended in
phosphate-buffered saline (1 × 107/ml) and
adoptively transferred into irradiated (T-depleted) hosts by
tail vein injection. For in vitro experiments, labeled cells were incubated at 37 °C in 12-well plates that had previously been
coated with stimulating antibodies (anti-CD3 and anti-CD28). The
proliferation of viable cells was assessed by analyzing fluorescence at
various time points on a FACSCalibur flow cytometer (BD Pharmingen).
Bisulfite-based DNA Methylation Analysis--
Genomic DNA was
digested with restriction enzymes outside the sequence of interest.
More specifically, DNA from T cells of +/DMRflox,hCD2-cre (carrying the
deletion of H19DMR, DMRD) and
skeletal muscle of +/+ and +/H19exI mutants
was digested with BamHI for analysis of H19
promoter methylation. DNA from hearts of
+/DMR
G and DMR
G/+
mutants was digested with EcoRI for analysis of
Igf2DMR1 methylation. The DNA was then treated with
sodium bisulfite in agarose beads (25). Subsequently the H19
promoter region or the Igf2DMR1 region was amplified
using a nested PCR strategy such that the primers recognized the
bisulfite-converted DNA (Tables I and
II). To ensure that the methylation information is derived for several chromosomes from each sample, PCR products from at least three separate
PCR reactions were cloned and sequenced for each DNA sample. The parental origin of the
sequences obtained was assigned by taking advantage of the polymorphic
bases (+167 in the H19 relative to the transcription start
site and
3776 in the Igf2 relative to the first
nucleotide of exon I on the transcript originating from promoter P1)
between domesticus and castaneus parental
alleles.
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Nuclear Run-on Analysis--
Nuclei were isolated from the
livers of p8 neonates and prepared as described (26). Briefly, the
liver was homogenized in chilled buffer containing 2.1 M
sucrose, 10 mM HEPES, pH 7.6, 2 mM EDTA, 15 mM KCl, 10% glycerol, 150 mM spermine, 500 mM dithiothreitol, 500 mM phenylmethylsulfonyl
fluoride, and 7 µg/ml aprotinin. Nuclei were pelleted by
ultracentrifugation through the same buffer, rinsed, resuspended in
buffer containing 40% glycerol, 50 mM Tris-Cl, pH 8.3, 5 mM MgCl2, 0.1 mM and EDTA and kept
frozen at 70 °C. Typically, nuclei isolated from one liver were
resuspended in 450 µl of buffer. The run-on reaction was carried out
with 150 µl of nuclei mixed with a buffer containing 10 mM Tris-Cl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 1 mg/ml heparin, 1 mM ATP, 1 mM GTP, 1 mM CTP, and 300 µCi of UTP (800 Ci/mmol) at 30 °C for 30 min with shaking. After DNase and
proteinase K treatment, radiolabeled RNA was isolated using Trizol
(Invitrogen). Separately, 5 µg of linearized plasmid DNA
containing the target probes was immobilized on nitrocellulose.
RNA equivalent to 2.5 × 107 cpm was hybridized to the
DNA blot for 40-48 h at 43 °C. The probes for the run-on assay were
parts of the H19 (1.9-kb PstI-SalI fragment of H19), Igf2 (first 640 nucleotides of the transcript initiated at P1), and actin
(region encompassing nucleotides 91-709 of the actin
cDNA) cloned in pBluescript.
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RESULTS |
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Stability of the ICE-mediated Epigenetic Modifications-- Earlier analyses have shown that during embryogenesis, the primary imprint at H19DMR directs secondary epigenetic modifications that silence the paternal H19 promoter (12). We wanted to test whether the acquired epigenetic changes are developmental in nature and stable through mitosis or are lost with cell division and need to be re-established during each cycle of mitosis. Therefore, we deleted the H19DMR element in proliferating cells and assayed for the stability of the secondary methylation imprint of the H19 promoter.
The stability of the secondary imprint was analyzed in T cells, because they retain their proliferative ability even after terminal differentiation. Although T cells do not normally express H19, when they are fused with embryonal carcinoma cells, the maternal H19 locus of the T cells is activated, whereas the paternal H19 remains silent (27). Also, the H19DMR and the H19 promoter regions are hypermethylated on the paternal chromosome as shown by Southern blot analysis (27). Thus using these two criteria, we concluded that the H19 locus in the T cells is imprinted.
We generated a transgenic mouse in which the expression of cre recombinase was under the control of the human CD2 (hCD2) promoter so that cre recombinase was expressed in T cells at an early stage of development (i.e. cells that are double negative for cell surface markers CD4 and CD8). Females hemizygous for the hCD2-cre transgene were mated with males homozygous for the DMRflox allele. At the DMRflox allele, loxP sites flank the H19DMR and hence H19DMR can be deleted dependent upon cre expression (12). Thus, the paternal H19DMR was expected to be deleted in the T cells of the +/DMRflox,hCD2-cre progeny.
Mature T cells were isolated from the lymph nodes of
+/DMRflox,hCD2-cre mice and their
+/DMRflox nontransgenic littermates. To assay
the mitotic stability of the secondary imprint at the H19
promoter, we induced the isolated T cells to proliferate in
vitro. We assayed proliferation after 6 days in culture both by
counting viable cells and by monitoring changes in CFSE-mediated
fluorescence during the in vitro culture. CFSE is a
nonspecific cell stain. Once a cell is labeled with CFSE, the
intensity of fluorescence will depend upon dilution of the dye due to
cell division (28). This is clearly evident in adoptive transfer
experiments (Fig. 2a).
CFSE-labeled cells, when transferred to T-depleted hosts, proliferate
and have a reduction in fluorescence with each successive division,
whereas similar cells transferred to T replete hosts, which do not
allow proliferation, exhibit unaltered CFSE fluorescence even after 30 days. By monitoring CFSE fluorescence during in vitro
culture (Fig. 2b) and by counting cells, we projected that
the vast majority of T cells had divided at least six times.
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As expected, in +/DMRflox, hCD2-cre
transgenic mice, loxP-flanked H19DMR (Fig.
3a) on the paternal chromosome
was excised efficiently in the T cells as evidenced by the appearance
of a 340-bp deletion-specific product concomitant with a decrease in
the DMRflox-specific 580-bp product (Fig.
3b) in the DNA isolated from T cells.
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Methylation of the H19 promoter region was analyzed in the T cells of +/DMRflox,hCD2-cre mice before and after in vitro proliferation (Fig. 3c). We used bisulfite sequencing to determine the methylation status of individual cytosine residues of the DNA in the region. Primers were designed to ensure that the information was acquired only from the chromosomes where the ICE had been deleted. Like the wild type paternal H19 promoter (10), the paternal H19 promoter carrying the ICE deletion was hypermethylated. These cells had undergone at least some proliferation in vivo as they differentiated into mature single positive T cells (CD4+ or CD8+). Thus, H19 secondary imprint survived mitosis in vivo despite the absence of ICE. The cells that were induced to proliferate extensively in vitro also exhibited hypermethylation of the H19 promoter region comparable with cells before proliferation. This demonstrated conclusively that the absence of ICE did not lead to a loss of methylation during mitosis.
Because, subsequent to the ICE deletion, there is no loss in methylation despite extensive proliferation in vivo or in vitro, we concluded that the secondary imprint at the H19 promoter is stable through mitosis and does not require continuous input from the primary imprint at the ICE.
Requirement of the H19 Structural Gene for the ICE Mediated Epigenetic Silencing-- We next wished to determine which regions of the H19 gene are essential for acquiring and/or maintaining the secondary imprint that actually represses paternal H19 transcription. On the wild type paternal chromosome, both the H19 promoter region and the H19 RNA coding sequences are hypermethylated (10). In fact, the first exon of H19 is the most consistently hypermethylated region outside the H19DMR. Previous transgenic and knock-in experiments have provided contradictory data regarding the relative importance of these two regions. Deletion of exon I from transgenic constructs results in the loss of transgene imprinting (29). Likewise, replacement of the whole coding region with firefly luciferase also results in biallelic expression of the unmethylated transgene (29). However, replacement of the whole H19 coding region at the endogenous locus with luciferase results in only sporadic activation upon paternal inheritance (30). Interpreting the luciferase constructions is complicated, and it is not clear whether the presence of luciferase or the absence of the test sequence is the cause for the loss of imprinting. We decided, therefore, to test directly the requirement of H19 exon I for silencing paternal H19 promoter. The endogenous H19 locus was manipulated by gene targeting methods to flank most of exon I with loxP sites (Fig. 1b). This region was deleted in the germ line using an EIIa-cre transgenic line and passed through the maternal and paternal germ lines to analyze the effect of the deletion on methylation of the promoter and on H19 gene expression.
We analyzed the status of methylation of H19 promoter by
bisulfite sequencing (Fig. 4). The
paternal promoters of H19exI mutants were
consistently hypermethylated like the wild type paternal chromosomes at
all of the CpG dinucleotides. Our results clearly demonstrate that
although highly methylated itself, exon I of H19 does not
carry any information for the acquisition of methylation on the
paternal H19 promoter.
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We wanted to determine whether exon I and its methylation are required
to maintain repression of paternal H19 transcription. We
initially attempted to quantitate expression via Northern analysis (Fig. 5b). The expression of
H19 in +/H19exI mutants was
similar to the +/+ control littermates, initially suggesting that
paternal H19 was not appreciably activated because of the
deletion. However, maternal deletion mutants
(H19
exI/+) also did not exhibit any
H19 expression, suggesting that either the initiation of
H19 transcript or its stability is abolished as a result of
the deletion. Keeping this in view, it was clear that Northern analysis
was not informative for discerning the effect of the deletion on
H19 imprinting. We performed, therefore, nuclear run-on
assays to derive information about the H19 imprinting in the
H19
exI mutants, tentatively assuming that the
deletion affects the stability of the transcript rather than its
initiation.
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To directly test the role of exon I in initiation of transcription, we
attempted nuclear run-on experiments, first using nuclei isolated from
the livers of +/H1913 and
H19
exI/H19
13 mice.
(The H19
13 mutant removes the entire
H19 promoter and the coding region (31) and thus allowed us
to be certain of the parental origin of any transcripts that we
measured.) The run-on analyses (Fig. 5c, lanes 1 and 3) showed that the mutated
H19
exI allele does initiate significant,
although reduced, levels of transcription when maternally inherited.
Signal strength in a run-on analysis is affected both by alteration in
the rate of transcript initiation and by alteration in the stability of
the transcript. The reduced H19 signal in
H19
exI/H19
13
mutants could be the result of either one or both of these factors. However, given that transcription initiation was not entirely abrogated
due to exon I deletion and that significant levels of H19
RNA were observed, it was clear that the run-on analysis could be used
to determine the functional necessity of exon I for silencing of
paternal H19.
We next looked at transcription from the
H19exI allele when it was paternally
inherited (Fig. 5c, lane 4). Like a wild type
paternal allele, the H19
exI chromosome
remains silent when paternally inherited. In fact, the levels of
transcription could not be distinguished from those noted in nuclei
entirely lacking the H19 gene (Fig. 5c,
lane 2).
Thus the absence of exon I sequences does not interfere with the ICE-mediated silencing of the H19 promoter. Even though exon I acquires ICE-mediated methylation, it is required neither for ICE-mediated establishment of the secondary imprint at H19 promoter nor for its maintenance.
Because H1913 mutation removes the
ICE in addition to the H19 gene, use of these
mutants allowed us to investigate the effect of the ICE on
Igf2 transcription initiation. ICE
deletion on the maternal chromosome activates normally silent maternal
Igf2 significantly in liver although not to the same
levels as the paternal allele (31). However, paternal deletion
also reduces Igf2 expression to some degree (31, 32).
In complete accordance with this steady state mRNA data, we
observed significant initiation of Igf2 transcription
in H19
13/H19
exI
mutants (Fig. 5c, lane 4) where the maternal
Igf2 allele is expected to be active and the paternal
H19
exI allele to be fully active. The signals
are also intense for
H19
13/H19
13 (Fig.
5c, lane 2) where both alleles are likely to
express but at reduced levels. The initiation is least in
+/H19
13 and
H19
exI/H19
13
mutants (Fig. 5c, lanes 1 and 3) where
the paternal allele has a reduced expression and the maternal allele is
expected to be silent. In other words, the gain of maternal
Igf2 expression seen in maternal ICE
deletion mutants (like H19
13) is attributed
directly to gain of transcriptional initiation on this chromosome.
Role of the ICE in Methylation of DMR1 at the Igf2
Locus--
Methylation of Igf2DMR1 located 90 kb
upstream of the H19DMR has been postulated to play a role in
maintaining the expression of Igf2 (33). Deletion of
Igf2DMR1 indicates that its role is specific to
muscle, and it is especially important in maintaining appropriate
expression in cardiac muscle (34). We therefore investigated the effect
of H19DMR on methylation of Igf2DMR1 in cardiac tissue. We used bisulfite sequencing to derive information about methylation of the Igf2DMR1 region in DNA
isolated from the hearts of wild type +/+ mice and of mice with
a paternally inherited deletion of
H19DMR,+/DMRG (12). Examining the
12 CpG residues, we found that the mutant paternal chromosome (Fig.
6b) although still methylated
was less so than the wild type paternal chromosome (Fig.
6a). The extent of methylation, in fact, resembled the wild
type maternal chromosome (Fig. 6, d and e). This
clearly indicated that the ICE is required for the paternal
hypermethylation of Igf2DMR1 in cis.
Comparing the methylation status of Igf2DMR1 in wild
type chromosomes from +/DMR
G and
DMR
G/+ mutants, it appears that the
ICE deletion also has an effect on
Igf2DMR1 methylation in trans, although
the effect of paternal ICE deletion on
Igf2DMR1 in cis is most
pronounced.
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Given the enormous difference in the expression of
Igf2 from the wild type paternal and the maternal
chromosomes, we were surprised to note that the maternal
Igf2DMR1 was also heavily methylated. The methylation
was seen at 70 and 50% of the total CpG residues on the paternal and
maternal chromosomes, respectively, in the wild type mice (Fig. 6,
a and d). Additionally, no single residue could
be identified that was specifically methylated on the paternal
chromosome. Both the wild type paternal and wild type maternal
chromosome populations had individual chromosomes that were heavily
methylated and others that were practically devoid of any methylation.
Deletion of ICE on the maternal chromosome activates
Igf2 expression to high levels in the heart (32), and
if Igf2 expression requires
Igf2DMR1 hypermethylation, we would expect
hypermethylation of Igf2DMR1 on the mutant maternal chromosome. Hence, we analyzed the effect of ICE deletion on
maternal Igf2DMR1 methylation. However, the extent of
methylation on the expressing mutant maternal chromosomes (Fig.
6f) is low, comparable with wild type chromosomes (Fig. 6,
d and e). Thus, comparing maternal and paternal
wild type and mutant chromosomes, we see a correlation of methylation
of Igf2DMR1 with a methylated H19DMR but
not with Igf2 expression.
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DISCUSSION |
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Repression of the paternal H19 allele is a two-step
process. Molecular evidence strongly supports the idea that a paternal imprint at the ICE between 2 and
4 kb upstream of the
H19 promoter marks the parental origin of the chromosome
(10, 11). Molecular and genetic studies demonstrate that during
development this primary H19DMR imprint directs further
epigenetic changes that actually silence the paternal H19
promoter (12). These changes certainly involve DNA methylation. Here we
demonstrate that the secondary methylation imprint is developmental.
That is, it does not require continued signaling from the primary
imprint at the ICE but is stable even during multiple mitoses.
The ICE acts epigenetically to modify the H19 promoter region and also part of the structural gene. Both of these regions are hypermethylated post-fertilization, and the paternal H19 promoter is silenced. In fact, the exon I region is most completely and consistently methylated. However, our results here indicate that the region is not required at all for acquiring the secondary imprint. Thus, CpG methylation does not always connote function but in some cases may occur only coincidentally with other changes.
Our results concerning the role of exon I in maintaining H19 imprinting are not really consistent with previous transgenic experiments (29). Our personal observation is that transgenic constructs are particularly sensitive to disruptions in imprinting. For example, many transgenes show a copy number dependence in imprinted expression and DNA methylation that is obviously not applicable to the endogenous locus. These experiments perhaps suggest that the imprinting signals may include large DNA structures and elements in a way that is really not yet appreciated. Until recently, most transgenic constructs extended only 4 kb on the 5' flank and therefore included neither the entire H19DMR nor all of the CTCF binding sites. Recent experiments from the Bartolomei laboratory (35) indicate that additional flanking DNA may provide copy number and position independence and may provide a better substrate for further mutagenesis of transgene constructs.
Interestingly, the loxP sites left downstream to the promoter after our manipulation of the locus were also methylated (data not shown). Thus it appears that any CpG residue at that locus may be modified during the spread of methylation irrespective of its sequence context. This result is consistent with appropriate imprinting of the NeoR gene inserted at the H19 locus (36). Thus, even the sequence of the H19 promoter may not actually play an important role for methylation spreading but may simply be a CpG-rich element in the right place. We are currently investigating the ability of the ICE to modify promoters other than H19 when they are placed in adjacent position.
Our experiments have also demonstrated that the ICE controls the imprinting of Igf2 at the transcript initiation level despite being 90 kb downstream of the promoter. This is in complete accordance with the presence of a transcriptional insulator at the ICE (32, 37, 38). Insulator elements, when present between the enhancer and promoter, prevent the expression of genes. The mechanistic details of the process seem to be diverse and not well understood (39). Our nuclear run-on analysis suggests that the insulator in the ICE truly prevents promoter activation and transcript initiation by the enhancer.
Finally, we investigated the role of H19DMR in methylation of the Igf2DMR1 element. Igf2DMR1 has been identified as methylated in a parent-of-origin manner (17, 33, 40). Subsequently, its crucial role in maintaining appropriate expression patterns of Igf2 in mesodermal tissues was demonstrated in vivo using embryonic stem cell-generated mutational analyses (34). Further, an Igf2DMR1-specific binding protein, GCF2, has been identified in which affinity is dependent upon the levels of CpG methylation (41). Our bisulfite sequencing results are puzzling in this regard because although we see a correlation between Igf2DMR1 methylation and the presence of a methylated H19DMR in cis, we do not note a correlation between Igf2 expression and the methylation of the 12 CpG dyads we examined. Further, investigations are clearly warranted.
Although our experiments do not support a crucial functional role for
Igf2DMR1 methylation, they do confirm and extend
previous DNA sequencing experiments indicating that the degree of
Igf2DMR2 methylation was altered in trans
upon deletion of the H19DMR (via the
H1913 mutation) (42). These results are
intriguing in that they suggest a communication between the maternal
and paternal chromosomes sometime after implantation, when the
methylation of the Igf2DMR1 is first established. The
idea that imprinted alleles interact and that such interactions are
crucial to maintaining monoallelic expression has always been an
attractive hypothesis but one with limited experimental support.
Paternal and maternal human 15q11-q13 domains interact specifically
during S phase (43) although no functional role for the interaction has
been subsequently supported. To date no such physical association of
the chromosomes has been noted at the
Igf2/H19 locus. We have never found any
evidence for transvection (i.e. communication between
enhancer and promoter elements in trans at the
H19 locus) despite specific attempts to record such
interactions (44). Finally, H19 transgenes do not require a
partner to exhibit imprinting. Rather single copy hemizygous transgenes
exhibit monoallelic expression even in an H19
13 genetic background (32). Nonetheless,
it is interesting to note that the ICE can alter methylation
patterns not only 90 kb away but also across other chromosomes.
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ACKNOWLEDGEMENTS |
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We thank Shirley Tilghman for the
H1913 mice and Heiner Westphal for the
EIIa-cre mice.
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FOOTNOTES |
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* 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: National Institute of
Immunology, Aruna Asaf Ali Rd., New Delhi 110067, India. Tel.:
91-11-6162281 or 91-11-6167623 (ext. 482); Fax: 91-11-6162125 or
91-11-6177626; E-mail: madhus@nii.res.in.
Published, JBC Papers in Press, September 20, 2002, DOI 10.1074/jbc.M208437200
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ABBREVIATIONS |
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The abbreviations used are: ICE, imprinting control element; DMR, differentially methylated region; CFSE, carboxyfluorescein diacetate succinimidyl ester.
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