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INTRODUCTION |
Genomic imprinting is defined as an epigenetic change leading to
differential expression of the two parental alleles in somatic cells
(1-3). Igf2 is one of the known imprinted genes for
which only the paternal allele is expressed, the maternal allele being silent. In cancers, the Igf2 imprinting is frequently
relaxed so that the silent maternal allele becomes active, resulting in biallelic expression (4, 5). Such loss of imprinting
(LOI)1 also occurs in normal
tissues adjacent to some cancers with LOI (6, 7) and has been
implicated in the Beckwith-Wiedemann syndrome (BWS), a congenital
overgrowth disorder that predisposes to embryonal tumors (8, 9).
Imprinting of the Igf2 gene is thought to be
controlled by sequences located at the H19,
Igf2, and other loci (10, 11). Such cis elements
usually contain the region that is differentially methylated on the
parental alleles (DMR) (12-16), containing CpG-rich repeats, which are
postulated to facilitate heterochromatination and gene silencing at
imprinted loci (17). In addition, antisense RNA has been shown to be
transcribed from regions including the DMRs at
Igf2 (18) and Kvlqt1 (KvDMR)
(15, 16), which has been proposed to serve a regulatory role in
silencing the sense orientation transcript (1, 2).
H19DMR is located upstream of the H19 promoter
and is capable of binding CTCF, a highly conserved zinc finger
DNA-binding protein with multiple roles in gene regulation. One of the
important functions of CTCF is as a chromatin insulator acting in a
methylation-dependent manner (19-22). H19 is
expressed from the maternal allele, whereas the paternal allele is
silent, indicating that the H19/Igf2 genes are reciprocally imprinted. Competition of these two genes for the use
of sets of common enhancers located downstream of H19 is now
a well documented model that explains this opposite allele specific
expression (19-22). On the paternal allele, CTCF cannot bind to the
hypermethylated H19DMR, and the common enhancer may be
utilized by the Igf2 promoters. Such hypermethylation
on the paternal H19DMR is established during male
gametogenesis and is maintained throughout development (23). On the
other hand, on the maternal allele where the H19DMR is
unmethylated, CTCF binds to the H19DMR to insulate
Igf2, and then the common enhancer may be utilized
only by the H19 promoter.
The Igf2 gene contains three DMRs, two of which
(Igf2DMR0 and DMR1) are located far
upstream of the Igf2 coding region, and the other
(Igf2DMR2) existing within the gene body (12-14,
18). Igf2DMR0 is methylated on the inactive maternal
allele (18), whereas Igf2DMR1 and DMR2 are
methylated on the active paternal allele (12-14). Deletion of the
region including maternal Igf2DMR1 has been reported
to result in Igf2 LOI (24), and it has been proposed
that a putative repressor may bind to the maternal unmethylated Igf2DMR1 to silence the maternal allele (12-14, 24).
Furthermore, there seems to be a functional link between the
H19DMR and Igf2DMRs, because deletion of
the maternal H19 gene results in decreased methylation at
the maternal Igf2DMR0 (18) and paternal
Igf2DMR1 and DMR2 (25). Moreover, deletion
of a large segment of Igf2 upstream, either from the
maternal or paternal alleles, is reported to lead to LOI not only of
Igf2 but also H19 (26).
KvDMR is located within an intron of the Kvlqt1
gene, where the maternal allele is hypermethylated, whereas the
paternal allele is unmethylated (15, 16). Although the
Kvlqt1 gene is transcribed from the maternal allele,
antisense transcripts, referred to as LIT1, are expressed from the
unmethylated paternal allele in both humans and mice (15, 16).
Demethylation of the maternal KvDMR associated with
biallelic expression of LIT1 has been detected in the majority of BWS
cases (15, 16), Smilinich et al. (15) reporting
IGF2 LOI to occur independently of changes in methylation or
expression of H19. Thus, KvDMR appears to be an
additional control center for the Igf2 imprinting,
independent of Igf2/H19 loci. However,
this remains controversial, because Lee et al. (16) reported
similar results but found that KvDMR demethylation was not
correlated with the IGF2 LOI, although it was consistently associated with LIT1 LOI.
The molecular events involved in the Igf2 LOI in
tumors have not been determined. In a subset of Wilms' tumors (27, 28) and intestinal tumors (7), it has been shown to be accompanied by
methylation at the maternal H19DMR together with silencing of the maternal H19 allele (27, 28). Although this pattern of Igf2/H19 expression is consistent with
the Igf2/H19 chromatin insulation model,
there is no reciprocal pattern of Igf2/H19
expression as well as no strict relationship between the
Igf2 LOI and methylation at the H19DMR in
various tumors (29-34). Furthermore, some cases of Wilms' tumor with
the Igf2 LOI show hypomethylation at the region
corresponding to mouse Igf2DMR0 (34), suggesting the possibility that altered methylation at Igf2DMRs may
be also involved.
In the present study, we therefore investigated whether
Igf2 LOI is associated with specific alterations of
methylation at H19DMR, Igf2DMR1, and
KvDMR. In an earlier report, we documented that cells of
hepatic tumors (HTs) chemically induced in C3H/HeJ × MSM
(Mus musculus molossinus Mishima,
Japanese wild mice) frequently show Igf2 LOI and
express Igf2 at high levels (35). Furthermore, consistent polymorphisms found between the parental C3H/HeJ and MSM
strains allow investigation of allele-specific methylation correlating
with allele-specific expression of Igf2/H19. We here report that HT cells with Igf2 LOI are composed of
diverse cell populations, with and without Igf2 LOI,
but even the monoclonal cells with the Igf2 LOI
retain the maternal H19 expression with normal
methylation patterns at H19DMR, Igf2DMR1,
and KvDMR.
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EXPERIMENTAL PROCEDURES |
Mouse HT Cell Lines--
Male F1 hybrid mice derived from
breeding female C3H/HeJ and male MSM mice were intraperitoneally
administered diethylnitrosamine (5 µg/g, body weight) at the age of 2 weeks. Cells were isolated by the collagenase perfusion method from HTs
that developed 12-14 months later. The HT cells were cultured at a low
density as described previously (36), and colonies that were present
4-5 days after the start of cultivation were individually isolated
using a cloning ring and expanded to establish cell lines. These were
then examined for Igf2 LOI, and when LOI (+) clones
were found, they were further cloned to determine how many subclones
showed the Igf2 LOI.
Reverse Transcription-PCR (RT-PCR)--
RNA was extracted from
HT cells and tissues and from normal liver tissues of male C3H/HeJ,
MSM, and C3H/HeJ×MSM F1 mice at various ages. Total RNAs were treated
with RNase-free DNase (GenHunter, Nashville, TN) to destroy
contaminating genomic DNA, and first strand cDNAs were
generated using Superscript II (Invitrogen). The specific primers and
conditions for each RT-PCR are listed in Table
I.
Igf2 LOI--
The Igf2 exon 6 containing the polymorphic CA repeat (35) was amplified by genomic or
RT-PCR using the 5'-6-carboxyfluorescein-labeled forward and unlabeled
reverse primers, and the products were analyzed by an Applied Biosystem
automatic sequencer (ABI Prism 377; Foster City, CA) with the GeneScan
software. RT negative controls were run in parallel and demonstrated to
be consistently negative.
H19 LOI--
To find the polymorphism in the H19
coding region, the H19 exons were amplified by PCR from the
genomic DNA of C3H/HeJ and MSM mice, and the products were sequenced
with the automatic sequencer. The region covering H19 exons
2-3, including a polymorphic base at nt 7144 (GenBankTM
accession number AF049091), G for C3H/HeJ and A for MSM, was then
amplified by RT-PCR. The products were also cloned using a TOPO TA
cloning kit (Invitrogen, Carlsbad, CA), and the clones were
individually sequenced for each sample.
Methylation-specific DNA Sequencing--
The polymorphic bases
at the H19DMR, Igf2DMR1, and
KvDMR in C3H/HeJ and MSM mice were determined by sequencing
the genomic DNA. Bisulfite treatment of the DNA was carried out using a
CpGenome DNA modification kit (Intergen, Purchase, NY). The regions
within the DMRs containing the polymorphic bases were then amplified from the bisulfite-treated DNA using the primers listed in Table I.
These PCR products were cloned using the TA cloning kit, and plasmid
clones were individually sequenced.
CTCF Mutations--
CTCF exons 2-7, including 11 zinc finger
domains, were separately amplified from cDNA using the primers
listed in Table I and sequenced as above.
Western Blotting--
Cells were isolated from the subconfluent
cultures and lysed using solution containing 50 mM Tris (pH
8.0), 120 mM NaCl, 0.5% Nonidet P-40, and 100 mM NaF. The cell lysates, each with 50 µg of denatured
protein, were run on 7% polyacrylamide gels containing 0.1% SDS and
then blotted onto nitrocellulose membranes. The filters were then
blocked with 5% nonfat dry milk in phosphate-buffered saline plus
0.2% Tween 20 and incubated with 1:100 diluted anti-CTCF antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After extensive
washing in phosphate-buffered saline plus 0.2% Tween 20, the filters
were reacted with the horseradish peroxidase-conjugated anti-goat
secondary antibody (Santa Cruz Biotechnology) and washed again.
Finally, bound antibodies were visualized using the ECLTM
system (Amersham Biosciences, Uppsala, Sweden).
Statistics--
The data were statistically evaluated with
Statview software (SAS Institute, Cary, NC). Differences were analyzed
using the
2 or Fisher's test, and significance was
concluded at p < 0.05.
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RESULTS |
Establishment of Monoclonal HT Cell Lines with Igf2
LOI--
Since the RT-PCR fragments from Igf2 exon 6 including the polymorphic CA repeat were four bases longer in the
paternal MSM allele than the maternal C3H/HeJ allele (35), it was
possible to distinguish the parental alleles from which
Igf2 was expressed (Fig.
1). Although Igf2 was
expressed in all 10 HT tissues and 54 of 62 cell lines, only 4 of 54 HT
cell lines were demonstrated to show the Igf2 LOI
(Table II). Then the four cell
lines with the Igf2 LOI were subcloned, and 11-33
sublines were produced from each. Analysis of each subclone revealed
mosaicism in terms of monoallelic/biallelic as well as
positive/negative Igf2 expression (Table II).
Therefore, three Igf2 LOI (+) clones from the first cell lines were further subcloned, and 9-12 sublines were produced from each. Analysis of these revealed 75-100% of sublines to show the
Igf2 LOI (Table II). Then one of the subclones
derived from the first Igf2 LOI (+) cell line and
three subclones derived from the second were employed together with
four Igf2 LOI (
) cell lines in the next
experiment.

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Fig. 1.
Allelic Igf2
expression assay by the gene scanning. A, the
sequence including the polymorphic CA repeats in the
Igf2 exon 6 was amplified from the genomic DNA from
C3H/HeJ, MSM, and C3H × MSM F1 mice and analyzed by gene
scanning. The three major peaks are observed for C3H/HeJ and MSM mice,
but those of MSM mice are 4 bases longer than those of C3H/HeJ mice.
The peaks of C3H × MSM F1 mice show the overlapping pattern of
the C3H/HeJ and MSM peaks. B, RT-PCR analysis of subclones
with (top) and without Igf2 LOI
(bottom). The pattern of the LOI (+) cells is similar to
that of C3H × MSM F1 mice, whereas that of the LOI ( ) cells is
similar to that of the paternal MSM mice.
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H19 Imprinting--
RT-PCR analysis directed to H19
exons 2-3 revealed that the normal adult mouse livers and all the cell
lines expressed H19 regardless to the Igf2
LOI (Fig. 2A). Sequencing of
the individual RT-PCR fragments revealed that they were all derived
from the maternal H19 allele in all the cell lines,
indicating that the H19 expression and imprinting are
retained (Table III and Fig. 2B).

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Fig. 2.
Expression and imprinting of
H19. A, RT-PCR of H19 exons
2-3. H19 is expressed in all HT cell lines, with or without
the Igf2 LOI, as well as the normal liver of a
9-week-old mouse. B, sequencing of the RT-PCR fragments
including a polymorphic base (arrows), G for maternal
C3H/HeJ and A for paternal MSM, demonstrates that H19
transcripts are derived from the H19 maternal allele
both in the Igf2 LOI (+) and ( ) cells.
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Methylation at the H19DMR--
Allele-specific methylation at the
H19DMR, including 13 CpGs and one of the four
CTCF sites (the fourth CTCF site at the 3'-end in
Refs. 19 and 20), was then analyzed (Fig.
3A). This region includes a
single polymorphic base at nt 3534 (GenBankTM accession
number AF049091), C for C3H/HeJ and A for MSM, allowing distinction of
the parental alleles. This region was found to be generally heavily
methylated on the paternal allele and hypomethylated on the maternal
allele in the normal liver in two mice as previously described (23),
but one sample showed less methylation on the paternal allele (Fig. 3,
A-1). Nonmethylated CpGs on the paternal allele were more
frequent on the 5' side, including the CTCF binding site.
When comparing cells with and without the Igf2 LOI
(Fig. 3, B and C), aberrant methylation in the
region including the CTCF site was found on the maternal
allele at low frequency. This change was detected in all of the four
Igf2 LOI (+) cells (Fig. 3C) but not in
the Igf2 LOI (
) cells (Fig. 3B)
(p < 0.05). Although the same region was less
methylated on the paternal allele in the Igf2 LOI
(
) cells (Fig. 3B) than the Igf2 LOI (+)
cells (Fig. 3C), this could be due to physiological
variation as observed in normal hepatic tissues, because the
Igf2 LOI (
) cell lines were derived from different
tumors that were present in different hepatic lobes in a single
mouse.

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Fig. 3.
Allele-specific methylation status in the
H19DMR. A, livers of 9-week-old
C3H × MSM F1 mice. Methylation-specific sequencing of the
bisulfite-treated DNA shows that the paternal allele is heavily
methylated, whereas the maternal allele is nonmethylated or
hypomethylated. Two CpGs enclosed by a
square are one of the four CTCF sites. In one
mouse (A-1), the paternal allele is less methylated in the
5' region than the other mice (A-2, 3).
B, Igf2 monoallelic cell lines. Degrees of
methylation on the maternal allele are not changed as compared with the
normal livers, but the paternal allele is less methylated, especially
at the CTCF site and its franking region. The nonmethylated
CpG is most frequent at the 5'-site. C,
Igf2 biallelic cell lines. Although aberrant
methylation at the CTCF site and its flanking region is seen
on the maternal allele in all four cell lines at low frequency (*), the
differential methylation pattern on the paternal and maternal alleles
is maintained in most plasmid clones.
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Status of CTCF--
RT-PCR revealed CTCF to be expressed in all of
the cell lines (Fig. 4A), and
direct sequencing of the CTCF cDNA detected no mutations
(data not shown). Western blotting analysis revealed that the
expression levels of CTCF protein were not different between the cell
lines with and without Igf2 LOI (Fig. 4B),
indicating that the CTCF may not be altered in any of
them.

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Fig. 4.
Expression of the CTCT mRNA and
protein. A, RT-PCR of CTCT exons 6-7. The
CTCF mRNA is expressed in all cell lines, with and without the
Igf2 LOI. B, Western blotting analysis of
CTCF. The CTCF protein is also expressed to comparable levels in both
Igf2 ( ) and (+) cell lines.
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Methylation at the Igf2DMR1--
Five CpGs in the
Igf2DMR1 were examined for allele-specific
methylation using the polymorphic base at nt 12826 (GenBankTM accession number MMU71085), A for C3H/HeJ and G
for MSM as a marker. Although degrees of methylation were variable in each normal hepatic tissue and cell line, some characteristic methylation patterns were noted (Fig. 5).
First, the CpGs at the first and fifth positions were more methylated
than the others, although this was not constant either in normal livers
or cell lines. Second, the CpG at the fifth position tended to be
differentially methylated on the paternal allele, but such a tendency
was not apparent for other CpGs, for which the results were variable in each sample. However, when cells with and without the
Igf2 LOI were compared, there was no clear cut
difference in the methylation patterns.

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Fig. 5.
Allele-specific methylation status for the
five CpGs at the Igf2DMR1. A,
livers of 9-week-old mice. B, Igf2 LOI
( ) cell lines. C, Igf2 LOI (+) cell
lines. Most samples tend to show hypermethylation at the first (nt
12,800-12,801) and fifth CpGs (nt 13,020-13,021) as compared with
other three. In addition, the fifth CpG tends to be more methylated on
the paternal than the maternal allele, although such differential
methylation is not distinct in one of the normal livers
(A-2) and one of the Igf2 LOI ( ) cell
lines (B-4). Comparison of Igf2 LOI ( )
and (+) cell lines reveals no apparent difference in the methylation
patterns.
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Methylation at the KvDMR--
Twelve CpGs in the KvDMR,
including a polymorphic base at nt 2494 (GenBankTM
accession number AF119385), with G for C3H/HeJ and C for MSM, were
generally hypermethylated on the maternal allele but
hypomethylated on the paternal allele in the normal livers (Fig.
6A), as previously described
(15). However, some PCR fragments derived from the paternal allele
showed hypermethylation, and a few maternal fragments showed
hypomethylation, indicating that the KvDMR creates a mosaic in terms of methylation at the individual DNA strand level in normal
hepatic tissues. On the other hand, such a mosaic pattern was less
prominent in the HT cell lines, presumably due to extensive selection
of the clones. However, there was no significant difference in
methylation patterns between the cells with and without
Igf2 LOI.

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Fig. 6.
Allele-specific methylation status for the 12 CpGs at the KvDMR. A, livers of
9-week-old mice. B, Igf2 LOI ( ) cell
lines. C, Igf2 LOI (+) cell lines. Most
CpGs on the paternal allele are hypomethylated, whereas those on the
maternal allele are hypermethylated. However, DNA strands with either
hyper- or hypomethylation are seen, respectively, in the paternal and
maternal alleles in the normal liver, although such aberrantly hypo- or
hypermethylated strands are less frequent in HT cell lines. There is no
significant difference in the methylation patterns between the
Igf2 LOI ( ) and (+) cell lines.
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DISCUSSION |
Although the examined mouse HT cell lines showed
Igf2 LOI at low frequency, all 10 HT tissues were
negative in this study. This suggests that the Igf2
LOI (+) cells may be derived from rare cells within the HT tissues or
generated de novo during establishment of the lines. Because
Igf2 LOI has been reported in cultured mouse and rat
fibroblasts (37, 38) and human T lymphocytes stimulated to proliferate
by phytohemagglutinin in vitro (39), culture conditions may
be responsible. Furthermore, Ungaro et al. (38) reported
that the Igf2 LOI that occurred in rat fibroblasts
held in the confluent state persisted over cell generations, when the cell confluence was released by trypsinization and dilution, suggesting that the Igf2 LOI may be irreversible. On the
other hand, we demonstrated that even the established HT cell lines
were composed of heterogenous cell populations with/without the
Igf2 LOI, as well as with/without Igf2 expression. Heterogeneity of cell populations in
solid tumors was reported according to Igf2 LOI in
Wilms' tumors (40) and to E-cadherin gene methylation and
expression in breast cancers (41).
Previous studies have demonstrated Igf2
LOI to be associated with loss of H19 expression, together
with aberrant methylation on the maternal H19 gene, in a
subset of Wilms' and other tumors (7, 27, 28), which lends support to
the Igf2/H19 chromatin insulation model
(19-22). However, in the present study, all the Igf2
LOI (+) cells retained monoallelic H19 expression from the maternal allele. Such a pattern of
Igf2/H19 expression may be due to mixed
populations of cells, with and without Igf2 LOI
(i.e. if one population had biallelic Igf2
expression without the H19 expression and another maintained
the normal Igf2/H19 imprinting, the
outcome would be biallelic Igf2 expression with
maternally monoallelic H19 expression). We therefore cloned
the Igf2 LOI (+) cells and isolated four subclones of
cells with biallelic Igf2 expression.
Allele-specific expression analysis, however, revealed the biallelic
Igf2 expression with maternally monoallelic H19 expression to still be evident in all cases.
Although methylation-specific sequencing detected aberrant methylation
at the maternal H19DMR at low frequency in the
Igf2 LOI (+) cells, the normal differential
methylation pattern was retained in individual DNA strands in both
Igf2 LOI (+) and (
) cells. This is in contrast to
the reported cases of Wilms' and other tumors (7, 27, 28), cells of
individuals affected by Beckwith-Wiedemann syndrome (8, 9), and human T
lymphocytes stimulated to proliferate in vitro (39), in
which IGF2 LOI occurs in association with aberrant
methylation at the maternal H19DMR. Thus,
Igf2 LOI definitely occurs independently of altered
methylation at H19DMR, and aberrant methylation of the
maternal H19DMR may not, to a major extent, contribute to
the Igf2 LOI in our cell lines.
It was noted that some samples showed less methylation at the paternal
H19DMR, especially at one of the four CTCF sites
(19, 20) and its flanking regions (Fig. 3, A-1 and
B-1-4), indicating variation in the degree of methylation
at the paternal H19DMR in individual mice. Such
demethylation might allow the binding of CTCF to paternal
H19DMR, resulting in the insulation of the paternal
Igf2. However, the fact that the paternal
Igf2 and maternal H19 expressions were
maintained in such cells indicates that such partial demethylation on
the paternal H19DMR does not affect the Igf2/H19 imprinting.
The observed low frequency of aberrant methylation of the maternal
H19DMR raises the possibility that the
Igf2 LOI may be caused by alterations of other
components in the Igf2/H19 insulation machinery. We therefore investigated alterations in CTCF,
because chromosomal loss at human Ch16q, where CTCF is
localized, has been frequently detected, in many types of tumor
including HTs (42, 43). Mutational changes in the CTCF gene
have been found in some human tumors (44), and overexpression of CTCF
in tumor cells leads to growth arrest and apoptosis (45). Furthermore, CTCF can act as a silencer for c-myc (44), which is
frequently overexpressed in various tumors including HTs. However,
because no CTCF mutations were detected, and also because
its protein levels were not different between Igf2
LOI (+) and (
) cells, the CTCF function may be intact in these cells,
although further experiments are required to confirm this.
The Igf2DMR1 is suggested to be able to bind to a
putative silencer in a tissue-specific manner (12, 13, 24). Deletion of
the 5-kb region corresponding to the mouse Igf2DMR1
results in activation of the silent maternal Igf2 in
mesenchymal tissues, while not affecting the maternal H19
expression (24). Such tissue specificity is speculated to be due to the
possibility that, although the access of the endoderm-specific common
H19/Igf2 enhancer to the
Igf2 promoter is efficiently blocked by the CTCF
insulator on the maternal chromosome, the mesoderm-specific common
enhancer may further need the Igf2 silencer to
suppress maternal Igf2 expression. Methylation
analysis revealed that the patterns of differential methylation
at the Igf2DMR1 were not apparent as compared with the H19DMR and KvDMR in the normal hepatic
tissue. Especially, although differential methylation was observed at
the fifth position of the five CpGs analyzed in the some samples, it
was not apparent for other CpGs. The fact that there was no significant
difference in the methylation pattern between the
Igf2 LOI (+) and (
) cells suggests that this region
may not be responsible for the Igf2 LOI in these cell lines.
In addition to the H19DMR and Igf2DMRs,
the elements so far substantiated according to the
Igf2 LOI are the KvDMR (15) and some newly
found enhancers upstream and downstream of H19 (46-48). Aberrant demethylation at the maternal KvDMR, associated
with biallelic LIT1 expression, is thought to be a major cause of BWS (8, 9, 15, 16). Because such BWS cases do not show any abnormality
at the H19DMR and loss of H19 expression, the
KvDMR is thought to be the locus for BWS independent of
H19DMR. LIT1 expression from the paternal KvDMR
is indicated to be related to regulation of various maternally
expressed neighboring genes at human 11p15.5 and mouse distal Chr7,
with a special importance for p57Kip2 (2). However,
because such demethylation at the maternal KvDMR has not
been detected in Wilms' tumors (49, 50) and also because the frequency
of embryonic tumors in BWS cases with the maternal KvDMR
demethylation and LIT LOI is much lower than in the cases with maternal
H19DMR methylation and loss of H19 expression
(51, 52), KvDMR may be less important than
H19DMR, or not relevant to pathogenesis of Wilms' tumors.
The present observation of no abnormality at KvDMR in our
cell lines may support the notion that the KvDMR may not
have a major role in maintenance of Igf2 imprinting
(16).
Further potential candidates are newly found tissue-specific enhancers
located upstream and downstream of H19, which are conserved between mouse and humans (46-48). These novel sequences may not only
interact with H19DMR and Igf2DMR1 but also
affect the methylation status of H19DMR, and deletion of
these sequences may lead to the Igf2 LOI.
Furthermore, in brain, where Igf2 expression is biallelic, the relevant enhancers are located upstream of the H19DMR (53). Therefore, the possibility that alteration(s)
of these elements may result in Igf2 LOI in tumors
remains to be investigated.
In conclusion, the present study demonstrated that
Igf2 LOI can occur independently of changes in
H19DMR, Igf2DMR1, and KvDMR in
mouse HT cell lines.