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J Biol Chem, Vol. 274, Issue 41, 29331-29340, October 8, 1999
From the Differential DNA methylation of the parental
alleles has been implicated in the establishment and maintenance of the
monoallelic expression of imprinted genes. H19 and
IGF2 are oppositely imprinted with only the maternal and
the paternal alleles expressed, respectively. In Wilms tumor, a
childhood renal neoplasm, loss of the H19/IGF2 imprinted expression pattern results in silencing of H19
and biallelic expression of IGF2. This was shown to be
associated with biallelic methylation of the H19 promoter
in the tumor and the adjacent kidney tissue suggesting that epigenetic
H19 silencing is an early event in Wilms tumorigenesis. An
imprinting mark region characterized by paternal allele-specific
methylation has been suggested to reside in a GC-rich region of
400-base pair direct repeats starting at Genomic imprinting describes the phenomenon of heritable
parent-of-origin-specific expression of genes. The molecular mechanisms that determine the monoallelic expression of imprinted genes are to
date not fully understood. However, it is a widely accepted concept
that parental allele-specific DNA methylation plays an important role
in the process.
The insulin-like growth factor 2 (IGF2) and H19
genes are located in a cluster of imprinted genes on human chromosome
11p15.5 that is syntenic with the mouse distal chromosome 7. The
maternally imprinted IGF2 gene is transcribed only from the
paternal allele in most normal human tissues except for adult liver,
choroid plexus, and leptomeninges (1, 2). In contrast, the
H19 gene is paternally imprinted hence maternally
transcribed (3-5). This reciprocal expression pattern is frequently
lost in Wilms tumor, a childhood renal neoplasm, and in the overgrowth
syndrome, Beckwith-Wiedemann syndrome, that predisposes to Wilms tumor
either by maternal loss of heterozygosity at chromosome 11p15 or by
loss of imprinting (LOI)1
(6). LOI of IGF2 in Wilms tumor was first described by
Rainier et al. (7)and Ogawa et al. (8). Since
then several groups have shown that LOI of IGF2 in Wilms
tumor and Beckwith-Wiedemann syndrome is associated with
transcriptional repression and hypermethylation of the maternal
H19 allele (9-14). These findings led to the hypothesis that early in embryonal development the maternal allele of
H19 becomes silenced by DNA methylation which then relaxes
maternal IGF2 silencing in cis thereby conferring
a growth advantage to the affected cell. Strong evidence supporting
this idea came from mouse studies in which biallelic
Igf2 expression was observed following maternal
transmission of deletions at the H19 locus (15, 16). More
recently Cui et al. (17) have reported that loss of
H19 expression occurs frequently in Wilms tumors and their precursor lesions irrespective of whether or not IGF2 is
mono- or biallelically expressed. At present, it is therefore not clear whether loss of H19 and gain of IGF2 expression
from the maternal allele in Wilms tumors are independent processes or not.
Differential DNA methylation of particular sites or regions is thought
to provide an epigenetic or imprinting mark that distinguishes the
parental alleles of imprinted genes (18). DNA methylation analyses of
the mouse H19 gene have identified a region of paternal allele-specific methylation that is maintained throughout murine development and may therefore function as an imprinting mark (19, 20).
A genomic deletion of 1.6 kb within this region has been shown to
abrogate allele-specific H19 and Igf2
expression highlighting the importance of this region in
H19/Igf2 imprinting control (21). The
human H19 gene and its 5'-flanking region are also
methylated on the silent paternal allele, whereas the maternal allele
is unmethylated (22, 23). A candidate region for a paternal methylation imprint of the human H19 gene has been proposed that
contains several unique 400-bp direct repeat sequences starting at Previous investigations of H19 methylation in Wilms tumor
examined only the promoter, gene body, and 3' flank of H19,
thereby solely relying on the usage of methylation-sensitive
restriction enzymes. The potential imprinting mark region has not yet
been investigated in Wilms tumors. In the present study we have
therefore analyzed H19 methylation in Wilms tumors at
several new upstream locations including the imprinting mark and the
promoter region using Southern blotting and bisulfite genomic
sequencing. Our analysis delineated the upstream boundary of the
imprinting mark to approximately Sequence Data--
The upstream H19 sequence used in
this study is available from GenBankTM accession number
AF125183.
Wilms Tumors IGF2 Allelic Analysis--
The Wilms tumors used in
this study were previously typed in our laboratory for 11p15 LOH using
polymorphic markers in the H19/IGF2 region. In
non-LOH cases mono- or biallelic expression of IGF2 was
determined as described (8).
Southern Blot Analysis--
Digests were performed with
RsaI (8 units/µg), MspI (10 units/µg), and
HpaII (13 units/µg). Southern blots contained 6 µg of
DNA per lane. Hybridization was at 65 °C for 16 h. Probes were obtained from plasmid clones containing H19 5' sequence by
restriction digest and purification of the appropriate size fragment.
To control for incomplete HpaII digestion, the Southern
blots were hybridized with a probe for mitochondrial DNA. All samples
showed a single HpaII fragment indicating complete digestion
(data not shown).
Bisulfite Treatment--
Prior to bisulfite treatment 4 µg of
genomic DNA were digested for 16 h at 37 °C with
PstI (40 units) or with PstI (40 units) and
HpaII (40 units) in combination. The digested DNA was
ethanol-precipitated and resuspended in 40 µl of H2O. The
bisulfite treatment was carried out as described by Clark et
al. (25) with the following alterations. The bisulfite reaction
under mineral oil was performed at 60 °C for 16 h in 525 µl
of total volume containing 2.4 M sodium bisulfite (Sigma)
and 123 mM hydroquinone (Sigma). Reactions were desalted using the QIAEX II gel extraction kit (Qiagen). The DNA was eluted in
50 µl of H2O, incubated with 5 µl of 3 M
NaOH for 15 min at 37 °C, neutralized with ammonium acetate (final
concentration of 3 M), and ethanol-precipitated. The
bisulfite-treated DNA was then resuspended in 50 µl of
H2O and stored at PCR--
To obtain the PCR products bis1 and bis2, nested PCRs
were performed using 5 µl of bisulfite-treated DNA in the first
amplification (25 µl total volume) and 5 µl of this PCR product as
template in the second amplification (50 µl total volume). All
reactions contained 0.6 µM primers, 0.2 mM
dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and
1.25 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer). PWO DNA
polymerase (1 units) was added during the second amplification to
obtain blunt-ended product for DNA cloning. The following primer pairs
were used under the given conditions. For bis1, outer primers 1133 (5'-TGATGGTGGTAGGAAGGGGTTTTTTGTGTT) and 1134 (5'-CTCCTCCAACACCCCATCTTCCCCTAATTA), 0.75 mM
MgCl2, 57 °C annealing temperature (AT); inner primers 1143 (5'-GGTATGGTGTTTTTTGAGGGGAGAT) and 1144 (5'-CATCCCACCCCCTCCCTCACCCTA), 1 mM MgCl2,
53 °C AT. For bis2, outer primers 1093 (5'-GAGATGAGAGGAGATATTTGGGGGATAGTGAAG) and 1099 (5'-CCAAACATTATAAAAAAAACTAACACAAAATCC); inner primers 1096 (5'-GTAGGGTTTTTGGTAGGTATAGAG) and 1091 (5'-CACTAAAAAAACAATTATCAATTC), both pairs at 2 mM MgCl2 and 50 °C AT.
The PCRs were performed on a Peltier Thermal Cycler-200 (MJ Research)
using the following programs: first round of amplification: 1 time at
95 °C for 13 min, 5 times at 94 °C for 1 min, AT for 2 min,
72 °C for 3 min, and 25 times at 94 °C for 30 s, AT for 2 min, 72 °C for 1.5 min, and 1 time 72 °C for 6 min; second round
of amplification: 1 time at 95 °C for 13 min, 5 times at 94 °C
for 1 min, AT for 2 min, 72 °C for 3 min, 18 times at 94 °C for
30 s, AT for 2 min, 72 °C for 1.5 min, hold on 4 °C to add 1 unit of PWO DNA Polymerase to every reaction, 10 times at 95 °C for
30 s, AT for 2 min, 72 °C for 1.5 min, and 1 times 72 °C for
6 min.
Cloning of PCR Products--
The PCR products bis1 and bis2
obtained from the second round of amplification were gel-purified using
the QIAquick gel extraction kit (Qiagen). Purified fragments were
cloned into the EcoRV site of the pBluescript
SK DNA Sequencing--
Plasmid clones were sequenced with the T3
primer using the ABI sequencing system (Perkin-Elmer Applied Biosystems).
Analysis of H19 Upstream Sequence--
The previous analysis of
3.4 kb of H19 upstream sequence by Jinno et al.
(23) identified a 400-bp direct repeat that is reiterated 3.5 times.
These repeats were suggested to harbor the paternal methylation
imprinting mark of the human H19 gene. To determine the
extent of this repeat motif, we obtained an additional 6.6 kb of
sequence 5' of H19 (Fig. 1,
GenBankTM accession number AF125183). The subsequent
analysis of the resulting 10 kb of 5' H19 sequence
identified two repeat units (1 and 2) that each consist of an
H19 proximal 450-bp direct repeat (A1 and A2) followed by
several 400-bp repeats (B1-7), two of which are incomplete (B4 and
B7). Repeat Unit 2 extends to Methylation Analysis of H19 Upstream Regions in Wilms
Tumor--
If an epigenetic error was the cause of H19
silencing in Wilms tumor, then this error would most likely occur in
the potential imprinting mark region that controls the establishment of
imprinted H19 expression. The maternal hypermethylation
found at the H19 promoter and sequences further downstream
might then be a secondary event that occurs after the maternal allele
has acquired a paternal epigenotype at the imprinting mark. The first
objective of this study was to test this hypothesis by investigating
the allele-specific methylation in the respective H19
upstream regions. We examined pairs of tumor and corresponding normal
kidney tissue samples from Wilms tumor patients that have retention of
IGF2 imprinting (ROI), loss of IGF2 imprinting or
maternal loss of chromosome 11p15.5 (LOH) in the tumor tissue, and a
sperm DNA sample from a healthy donor. The bisulfite genomic sequencing
method (26) was used to analyze a 327-bp fragment (bis1) in the
H19 promoter region and a second fragment (bis2) of 504 bp
located within the potential imprinting mark region (Fig. 1).
Polymorphisms were identified within both fragments, which allowed us
to distinguish between maternal and paternal alleles in heterozygous samples.
A second objective of this study was to find the upstream boundary of
differential H19 methylation. To do this Southern blots of
RsaI/HpaII double-digested Wilms tumor,
corresponding kidney, and sperm DNA were hybridized to the four 5'
H19 probes indicated in Fig. 1. Regions of differential
methylation were identified in this analysis by comparing the intensity
ratio of a larger RsaI fragment (derived from methylated
paternal alleles) to a smaller HpaII fragment (derived from
unmethylated maternal alleles) in kidney tissue and ROI Wilms tumor
samples. Wilms tumors with maternal 11p15 LOH showed only the paternal
methylation pattern, whereas LOI Wilms tumors with hypermethylation of
the 5' H19 region displayed a shift in signal intensity
toward the larger RsaI fragment that was proportional to the
number of cells in the sample that are methylated at the addressed
HpaII sites on the maternal alleles. The methylation data
obtained in the Southern blotting analysis have been summarized in
Table I.
Analysis of the Promoter Region by Southern Blotting (Probe
1)--
Southern blots were hybridized with probe 1 to determine the
methylation status of HpaII sites 43 and 44 (H43
and H44 in Fig. 1) in the promoter region (blots
in Fig. 2 and the legend in Table I). The
RsaI/HpaII-digested samples showed three
prominent fragments (indicated R7-R8, R7-H46, and
H43-H44 in Fig. 2, a and b). Fragments R7-R8 and R7-H46 corresponding in size to the respective restriction digest fragments (Fig. 1) were of paternal origin since they appeared in all tumor and kidney samples including the LOH cases. This indicated
that HpaII site 46 (H46) was paternally unmethylated in a
high proportion of cells in Wilms tumors and in kidneys regardless of
the IGF2/H19 imprinting status. We can therefore
conclude that H46 does not show strict paternal-specific
methylation.
The five ROI Wilms tumors and the corresponding normal kidney tissues
showed the expected 1:1 ratio of the maternal fragment H43-H44 (Fig.
2a) compared with the paternal fragments R7-R8 and R7-H46.
In contrast, four of six LOH Wilms tumors lacked the HpaII fragment indicating that the paternal alleles were methylated at sites
H43 and H44 (Fig. 2, a and b) in agreement with
previous reports (9, 14). Surprisingly, LOH Wilms tumors 47 and 55 displayed weak HpaII fragments (Fig. 2b)
indicating that the paternal alleles were unmethylated at H43 and H44
in a minor proportion of cells in these tumors. This was unlikely to be
caused by contaminating normal tissue considering the high purity of
the tumors.
The LOI Wilms tumors were hypermethylated to different extents in the
promoter region (Fig. 2b). HpaII fragments were
absent in LOI cases 30, 31, and 65, hence both parental alleles were fully methylated (except H46 as described above) (Fig. 2b).
The corresponding kidneys also contained a high proportion of
hypermethylated maternal alleles implied by the stronger intensity of
fragments R7-R8 and R7-H46 compared with H43-H44 (Fig. 2b).
Hypermethylation in the adjacent kidney tissue of LOI Wilms tumors has
previously been shown and has led to the conclusion that epigenetic
changes at the H19 locus are an early event in Wilms
tumorigenesis (14). The LOI Wilms tumors 43 and 49, however, were
hypermethylated to a lesser extent at H43 and H44 indicated by the
presence of HpaII fragments of different intensities (Fig.
2b). Sperm DNA (Fig. 2a) was unmethylated at both
sites consistent with previous reports (9).
Analysis of the Promoter Region by Bisulfite Sequencing
(bis1)--
The bisulfite genomic sequencing analysis of the 12 CpGs
closest to the H19 transcription start (bis1,
Fig. 1) gave detailed methylation patterns from single clones of
paternal and maternal alleles (Fig. 3).
Sperm DNA was unmethylated at nearly all 12 CpGs concordant with the
Southern blotting results for H43 and H44.
The predominant observation for all Wilms tumors and kidneys examined
was the variability of methylation patterns in the H19 promoter. Although paternal alleles were significantly more methylated than maternal alleles, none of the 12 CpGs examined was found to be
consistently methylated on all paternal or unmethylated on all maternal
alleles. The extent to which the paternal alleles were methylated
varied from 1/12 to 12/12 CpGs. Equally surprising were the maternal
methylation patterns in that the presumably normal maternal alleles of
normal kidneys and ROI Wilms tumors that were expected to be fully
unmethylated also harbored methylation. This was particularly obvious
for heterozygous ROI Wilms tumor and kidney 110 that contained up to 7 methylated CpGs on a single maternal allele.
LOI Wilms tumors 30, 31, and 43 showed hypermethylation of the maternal
alleles consistent with previous methylation analyses at
HpaII sites. The corresponding kidneys also contained
hypermethylated maternal alleles consistent with a previous study from
our laboratory (14). However, the extent and distribution of
methylation on the paternal and maternal alleles was strikingly
variable within the same LOI case, as well as between the three LOI
cases. The fact that the methylation patterns in the H19
promoter region were highly variable may argue against a causative role
of methylation in this region in the establishment or maintenance of
H19 silencing.
Analysis of the Upstream Repeat Region by Southern Blotting (Probe
2)--
The Southern blotting analysis with probe 2 (Fig.
4 a and b) gave a
complex fragment pattern indicating the methylation status of
HpaII sites 25, 34, and 35 (H25, H34, and H35) since this
probe hybridized simultaneously to two locations, repeat A1 and A2
(Fig. 1). These HpaII sites are located within the upstream
repeat region that was suggested to function as the human
H19 imprinting mark (23).
The methylation status of H34 and H35 was examined using probe 2 that
hybridized to repeat A1. The respective Southern blots showed the
RsaI fragment R6-R7 from the paternal alleles and the HpaII/RsaI fragments H34-R7 and H35-R7 from the
maternal alleles (Fig. 4, a and b). This
interpretation of the parental origin of the RsaI and
HpaII fragments is supported by the previously reported
paternal allele-specific methylation for this region (23) and has been
verified by our own bisulfite genomic sequencing results (Fig.
5). Analysis of the kidney and tumor
tissues of ROI cases showed two maternally derived fragments H34-R7 and
H35-R7, and the paternal fragment R6-R7 with approximately equal
intensity (Fig. 4, a and b). This band pattern
indicated that paternal alleles were methylated at H34 and H35, and the
maternal alleles were unmethylated at H34 but methylated to
approximately 50% at H35. This meant that only site H34 conformed to
the previously reported paternal-specific methylation, whereas H35 did
not (23). This contradiction can be explained by the PCR-based assay
used by Jinno et al. (23) that examined the methylation at
HpaII sites 33, 34, and 35 in one amplicon. The methylation
status of HpaII site 25 (H25) was also examined using probe
2 that cross-hybridizes to repeat A2 (Fig. 1). The ROI Wilms tumors and
corresponding kidneys exhibited a 1:1 ratio of methylated to
unmethylated alleles (compare intensities of fragment R4-R5 with H25-R5
in Fig. 4a) which indicated the region of paternal-specific
methylation; hence, the candidate region to harbor the paternal
methylation imprint extends from
The LOH Wilms tumors showed different fragment patterns for the
potential imprinting mark region. HpaII fragments H34-R7 and H35-R7 were absent in LOH tumors 86 and 88, but both cases displayed the fragment H32-R7 (Fig. 4a, lanes 86T and
88T). This indicated paternal methylation at H34 and H35 and
a considerable cell population being unmethylated at H32. LOH Wilms
tumors 100, 47, and 55 displayed weak signals for H34 and H35 (Fig.
4a, lane 100T, and Fig. 4b, lanes 47T and 55T) indicating that these LOH
tumors were paternally unmethylated at these sites in a minor
proportion of cells. LOH case 84 showed the fragment H35-R7 of similar
intensity to the RsaI fragment R6-R7 indicating that H35 was
unmethylated on approximately 50% of paternal alleles in this tumor
(Fig. 4b, lane 84T). For HpaII site 25 tumors 86 and 84 showed fully methylated paternal alleles (Fig.
4a, lane 86T, and Fig. 4b, lane
84T), whereas the other LOH cases displayed weak signals for H25
(Fig. 4a, lanes 88T and 100T, and Fig.
4b, lanes 47T and 55T). These data
suggested that, although the paternal alleles were highly methylated,
not all CpGs on the paternal alleles were methylated in every cell of
the LOH Wilms tumors.
The LOI Wilms tumors exhibited different degrees of hypermethylation in
the upstream repeat region (Fig. 4b). LOI cases 31 and 65 were fully methylated on both alleles over the entire upstream repeat
region indicated by the lack of RsaI/HpaII
fragments (Fig. 4b, lanes 31T and 65T). Kidney 65 displayed RsaI/HpaII fragments of very low
intensity (Fig. 4b, lane 65K) indicating the
presence of a large population of non-tumorigenic cells being
hypermethylated in the H19 upstream repeat region. Kidney 31 was hypermethylated to a lesser extent indicated by stronger
RsaI/HpaII fragment intensities. LOI Wilms tumors
30, 43, and 49 and the adjacent kidneys displayed different fragment
patterns. Tumor 30 showed strong hypermethylation for H34 and H35 but
no detectable hypermethylation for H25 (Fig. 4b, lane
30T). The corresponding kidney 30 showed a low degree of
hypermethylation at H34 and H35 (Fig. 4b, lane
30K). LOI Wilms tumors 43 and 49 showed hypermethylation for H25,
H34, and H35 compared with the corresponding kidneys, however of much
lesser extent than the other LOI cases (Fig. 4b, lanes
43T, K and 49T, K). In summary, the LOI Wilms tumors
were hypermethylated in the upstream repeat regions, although highly
variable fragment patterns and different fragment intensities for the
addressed HpaII sites suggested that maternal methylation is
not complete in these tumors and seems to be of different distribution
comparing the LOI cases with each other. It is interesting to note that
patients 31 and 65 whose LOI Wilms tumors showed the highest degree
(nearly 100%) of maternal hypermethylation in the potential imprinting
mark region were patients with systemic overgrowth phenotype (case 31 had hemihypertrophy and multiple WT; case 65 corresponds to case 1 in
Morison et al. (27)).
Analysis of the Upstream Repeat Region by Bisulfite Sequencing
(bis2)--
Using the bisulfite genomic sequencing method we analyzed
27 CpGs within Repeat Unit 1 (bis2, Fig. 1). The detailed
methylation patterns from single clones of paternal and maternal
alleles are shown in Fig. 5. Sperm DNA was highly methylated with the
number of methylated sites varying between 21 and 26 of the 27 CpGs. In
samples with normal IGF2/H19 imprinting (106, 110, 134, and kidney 55) the CpGs 1-20 of bis2 that map to the 400-bp
direct repeat B1 showed very consistent paternal-specific methylation. These findings confirm that the 400-bp direct repeats are likely to
harbor the paternal methylation imprint of H19. In contrast, some of the CpGs (numbers 21-27) that are located within the 450-bp direct repeat A1 showed biallelic methylation in the kidney and tumor
samples (except ROI-WT 110, Fig. 5). This variable biallelic methylation at CpGs 21-27 indicated that the A1 repeat sequence itself
is not part of the methylation imprinting mark. On the contrary, the A1
repeat may constitute the 3' boundary of the imprint that therefore may
reside solely within the 400-bp direct repeats (B1-B7).
For the LOI Wilms tumors 30, 31, and 43 that showed hypermethylation to
different extents in the upstream repeats on Southern blots (as
described above, Fig. 2 and Table I), we obtained more detailed
methylation patterns of both parental alleles for the bis2 fragment
(Fig. 5). LOI tumors 30 and 31 showed nearly identical patterns of
hypermethylation. In LOI tumors 30 and 31, 98% of the CpGs within bis2
were methylated on both parental alleles. Hence, for these two tumors
the methylation patterns of the maternal and paternal alleles were
practically identical in this region. LOI tumor 43, however, contained
normal unmethylated (numbers 13-27) as well as methylated (numbers
1-12) maternal alleles. The number of methylated CpGs ranged from 6 to
26 of 27 sites, and methylation was distributed randomly over the 27 CpGs. Consequently, the methylation patterns of hypermethylated
maternal alleles were significantly different from those of the
paternal alleles that displayed the expected high density of
methylation. The adjacent kidney tissues of the LOI Wilms tumors 30 and
31 also contained methylated maternal alleles and were therefore mosaic
for H19 methylation in the upstream repeat region as was
shown for the promoter region. The kidney of LOI case 43 gave only one
partly hypermethylated maternal clone and thus may not be mosaic.
However, considering the much lesser extent and variable distribution
of methylation on the maternal alleles in the tumor tissue of case 43, it was possible that some kidney cells contained maternal alleles
methylated at a limited number of CpGs that escaped our analysis.
Bisulfite sequencing of fragment bis2 from LOH Wilms tumor 55 verified
our previous Southern blotting results (Fig. 4b, lane 55T) which indicated that this LOH tumor was unmethylated to some extent in the imprinting mark region. LOH Wilms tumor 55 gave rise to a
number of methylated (alleles 1-9, Fig. 5) as well as partly or fully unmethylated alleles (alleles 10-15, Fig.
5) which clearly demonstrated that this tumor with maternal LOH
contained paternal alleles that were unmethylated in the potential
H19 imprinting mark region. This finding contradicted our
expectation that LOH Wilms tumors contain only fully methylated
paternal alleles. However, more cases will have to be investigated in
order to determine whether paternal demethylation in the imprinting
mark region frequently occurs in LOH Wilms tumors.
Comparison of Methylation Patterns in the Promoter and Upstream
Repeat Region--
The methylation patterns obtained by bisulfite
genomic sequencing for the H19 promoter (bis1) and the
upstream repeat region (bis2) were concordant with the results obtained
by Southern blotting using probes 1 and 2, respectively. The extent and
distribution of methylation at the 20 CpGs in repeat B1 was the same
for almost all paternal H19 alleles amplified from the
different Wilms tumor and kidney samples, whereas the paternal
methylation patterns in the H19 promoter region appeared to
vary considerably. This observation also applied to the pathological
methylation on the maternal alleles of LOI cases 30 and 31 that
presented remarkably uniform hypermethylation of repeat B1 but highly
variable methylation of both alleles in the H19 promoter
region. In conclusion, our data are concordant with a role for the
400-bp direct repeats 5' of H19 as the H19
methylation imprint and suggest that pathological methylation of the
maternal allele in this region rather than the promoter region may be
the cause of H19 silencing in Wilms tumors.
Methylation Analysis of Regions 5' to the 400-bp Repeats--
To
address the question whether the paternal-specific methylation extends
5' of Repeat Unit 2, we analyzed the methylation status of
HpaII sites 22 and 23 (H22 and H23) and 15-18 (H15-H18) by
Southern blotting using probes 3 and 4, respectively (Fig. 1). The
methylation data have been summarized in Table I (Southern blots are
not shown). Sites H22, H23, H17, and H18 that are located within Alu
elements (Alu-Sx and Alu-Y, Fig. 1) did not
exhibit differential methylation. Instead, all Wilms tumor and kidney samples were highly methylated at these sites, except ROI Wilms tumor
110 that was fully unmethylated within the Alu sequences. The
methylated state of these HpaII sites in most samples
corresponded well with reports of Alu repeats being highly methylated
in somatic tissues (28, 29). The hypomethylation observed for Wilms
tumor 110 might indicate that this tumor has lost the ability to
methylate Alu elements. HpaII sites 15 and 16 located
approximately 7.8 kb upstream of H19 (Fig. 1) were less than
50% methylated in kidney samples and are therefore unlikely to be
differentially methylated. However, all Wilms tumors including the ROI
cases (except tumor 110) showed hypermethylation to different extents
at H15 and H16 compared with the corresponding kidneys. In summary,
hybridization probes 3 and 4 revealed methylation patterns further 5'
to the 400-bp direct repeats upstream of H19 that were
inconsistent with paternal-specific methylation.
In conclusion, this DNA methylation analysis suggests that the region
of paternal allele-specific methylation 5' of H19 that is
also methylated in sperm might be confined to the 400-bp direct repeats
that extend from This investigation is the first to exploit the bisulfite genomic
sequencing method to investigate H19 methylation in humans. Combining this method with results from
methylation-dependent Southern blotting, we describe a
detailed investigation of methylation patterns of the H19
promoter and regions upstream in Wilms tumor. Abnormal methylation of
the H19 locus is important in the pathogenesis of Wilms
tumor and the fetal overgrowth syndrome, Beckwith-Wiedemann syndrome
(BWS). An H19/Igf2 imprinting control
region that harbors a paternal methylation imprint as determined by
bisulfite genomic sequencing has been identified in the mouse, which
upon genomic deletion led to H19 silencing and biallelic
Igf2 expression (20, 21). For humans an equivalent
methylation imprint was suggested to be located at a similar
H19 upstream position (23). However, H19
methylation within this upstream region that seems to be critical for
H19 and IGF2 imprinting had not been studied in
Wilms tumor prior to this investigation.
Previous studies have shown that pathological methylation of maternal
alleles in the H19 promoter is linked to loss of
IGF2 imprinting in Wilms tumor and cases of BWS based on the
analysis of five HpaII sites in this region (designated
42-46 in this study) (9-11, 14, 27). Our investigation initially
confirmed by Southern blotting that HpaII sites 43 and 44 in
the promoter region are hypermethylated in LOI Wilms tumors. However,
our results demonstrated that the extent of hypermethylation at these
sites differed between the LOI tumors. For HpaII site 46 in
the H19 promoter, we found a lack of differential
methylation in all Wilms tumors and kidney samples regardless of their
imprinting status. Bisulfite sequencing analysis subsequently showed
that the pathological H19 promoter methylation of maternal
alleles in LOI Wilms tumors is highly variable in extent and
distribution. Furthermore, in tumors and kidneys with normal
IGF2 imprinting the 12 CpGs in the promoter displayed only a
low degree of differential methylation. Although paternal alleles were
generally more methylated than maternal alleles, the number of CpGs
methylated on any paternal allele was found to be highly variable, and
the distribution of methylation appeared to be completely random. In
addition, some maternal alleles from tumors and kidneys with normal
imprinting showed a considerable number of methylated CpGs.
These results are of particular interest in view of other studies that
have used HpaII site 46 to examine changes in parental allele-specific methylation at the H19 locus in BWS patients
(11, 13, 30). These studies may potentially underestimate the
proportion of BWS cases that harbor hypermethylation of H19
by examining only this one site. In conclusion, the heterogeneity of
methylation at the H19 promoter indicated to us that this
region is inappropriate for future investigations into the pathological
hypermethylation of the H19 gene in Wilms tumor and BWS.
In contrast to the promoter region, the CpGs within repeat B1 displayed
highly consistent paternal allele-specific methylation in samples with
normal imprinting supporting the view that the 400-bp upstream repeats
may harbor the H19 methylation imprint in humans (23). Our
methylation analysis also refined 3' and 5' boundaries of the potential
imprinting mark. The location of a 3' boundary was indicated by
biallelic methylation of the CpGs within repeat A1 at A comparison of the results obtained for human H19 in the
present investigation to those obtained for mouse upstream
H19 regions (20, 31) reveals remarkable similarities in the
distribution of methylation patterns. In both species the methylation
imprint is located at approximately the same distance 5' to the
H19 transcription start and is characterized by uniform
paternal allele-specific methylation. In contrast, the regions
downstream of the imprint toward the H19 promoter seem to
harbor non-clonal patterns of less pronounced differential methylation,
and the region upstream of the imprint harbors biallelic methylation in
both species. These analogies between mouse and human underline the
importance of DNA methylation in this region in H19 imprinting.
The hypothesis that an epigenetic error at the H19 locus
early in embryonal development may cause biallelic H19
silencing in Wilms tumor implies that the error should affect the
imprinting mark that distinguishes the parental alleles. Our analysis
has provided support for this hypothesis since we demonstrated that Wilms tumors with LOI are methylated on both parental alleles in the
potential H19 imprinting mark which coincides with the 400-bp direct repeats. Because this region was also hypermethylated in
the adjacent non-tumorigenic kidney tissues of the LOI cases, we can
speculate that the epimutation critical for biallelic H19 silencing in Wilms tumor may initiate within the repeat region 5' of
H19. In this scenario the hypermethylation of the
H19 promoter and gene body would occur secondarily.
Therefore, future studies into the relationship between H19
and IGF2 imprinting in Wilms tumor should aim to investigate
epigenetic changes of H19 within this candidate imprinting mark.
We thank Sue Clark for kindly sharing the
bisulfite technique with us.
*
This work was supported by the Cancer Society of New
Zealand, the New Zealand Lottery Grants Board, and the Health Research Council of New Zealand.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF125183.
§
To whom correspondence should be addressed: Lerner Research
Institute, Cleveland Clinic Foundation, Dept. of Cancer Biology, NB40,
9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-9653; Fax:
216-444-3164; E-mail: frevelm@ccf.org.
The abbreviations used are:
LOI, loss of
imprinting;
ROI, retention of imprinting;
LOH, loss of heterozygosity;
bp, base pair(s);
kb, kilobase pair(s);
CpG, cytosine-guanine
dinucleotide;
BWS, Beckwith- Wiedemann syndrome;
PCR, polymerase
chain reaction;
AT, annealing temperature;
bis, bisulfite.
Methylation Sequencing Analysis Refines the Region of
H19 Epimutation in Wilms Tumor*
§,
Cancer Genetics Laboratory,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 kilobase pairs (kb)
relative to the H19 transcription start and extending
upstream. The upstream boundary of the potential paternal methylation
imprint of the H19 gene has yet to be defined. We sought to
define this upstream imprint boundary and investigate whether Wilms
tumors with loss of imprinting are biallelically methylated in this
imprinting mark region. The analysis of 6.6 kb of new upstream
H19 sequence determined in this study identified a series
of the direct 400-base pair repeats that extends to approximately 5.3
kb relative to the transcription start. DNA methylation analyses indicated that the upstream boundary of the potential imprint may
coincide with the 5' end of the direct repeats. We found that Wilms
tumors with loss of imprinting are biallelically methylated in the
H19 upstream repeat region, and we suggest that
pathological methylation in this region is the epigenetic error that
initiates H19 silencing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
kb and extending further upstream (23). A number of HpaII
and HhaI sites in this region were shown to be methylated in
sperm and on the paternal alleles in somatic cells, and this
methylation was found to be preserved in pooled 8-32 cell stage
embryos. An evolutionary conserved sequence within these potential
imprinting mark regions of mouse and human which may be involved in the
control of H19 and IGF2 imprinting was recently
identified in our laboratory (24).
5.3 kb. The bisulfite genomic
sequencing analysis revealed highly variable methylation patterns in
the H19 promoter region in Wilms tumors and adjacent kidneys
regardless of their imprinting status. In contrast, the potential
imprinting mark region showed very consistent paternal specific
methylation. Wilms tumors with LOI were hypermethylated at the
imprinting mark region consistent with the hypothesis that an
epigenetic error in this region may initiate H19 silencing.
EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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20 °C.
vector. Ligations were for 12 h at 16 °C and
contained circular pBluescript:insert at a molar ratio of 12:1, 200 units of T4 DNA ligase (New England Biolabs), 20 units of
EcoRV, and ligation buffer (New England Biolabs) in 10 µl
total volume.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5.3 kb upstream of the H19
transcription start and is separated from Repeat Unit 1 by 387 bp of
unique sequence. The 450-bp motifs A1 and A2 are 85.4% identical. The
400-bp motifs B1, B2, and B3 of Repeat Unit 1 as well as B5 and B6 of
Repeat Unit 2 show a remarkable 85-91% identity to each other.
Further upstream we found two complete Alu elements, one incomplete
retroviral long terminal repeat, four MLT1 elements, and a 29-bp
GC-rich repeat.
View larger version (12K):
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Fig. 1.
Model of 10 kb of upstream H19 sequence and bisulfite PCR fragments. a, on the
sequence line are depicted the H19 transcription start site
(arrow), HpaII sites (short vertical
lines), and RsaI sites (longer vertical lines
numbered R1-R8). HpaII sites that were addressed in
the methylation analysis are boxed and numbered
(e.g. H46). Southern blotting probes 1-4 are
indicated above the sequence line. Note that probe 2 hybridizes at two locations due to high sequence homology of repeats A1
and A2. Repetitive sequence elements are indicated below the
sequence line. Repeat Units 1 and 2 consist of homologous 450-bp direct
repeats A1 and A2, and several 400-bp direct repeats B1-7. B4 and B7
are incomplete and contain only 130 and 250 bp of homologous sequence,
respectively. The locations of the PCR fragments bis1 and bis2
generated in the bisulfite genomic sequencing analysis are indicated
below the sequence line. b, fragments bis1 and
bis2 are shown enlarged (the 100-bp scale bar applies). The short
vertical lines on bis1 and bis2 depict all CpGs found in these
fragments. Every fifth CpG within each fragment is numbered. CpGs that
coincide with an HpaII site are marked below according to
the nomenclature used in a. The arrows below bis1
and bis2 indicate the location of single base pair polymorphisms (in 5'
to 3' order: bis2, C/T, C/A, G/A, C/G, and C/T; bis1, G/A).
Summary of H19 methylation analysis in Wilms tumor, adjacent kidney,
and sperm
(fully unmethylated), over +/ (differential or 50%
methylation), to +++ (hypermethylation or 100% methylation). WT, Wilms
tumor; K, kidney.
View larger version (93K):
[in a new window]
Fig. 2.
Southern blot analysis of
HpaII methylation in the H19 promoter
region. Genomic DNA from five Wilms tumors (T) with
retained IGF2/H19-imprinting (ROI)
(a), five tumors with loss of imprinting of IGF2
(LOI) (b), and six tumors with loss of
heterozygosity at 11p15 (LOH) (a and
b), as well as genomic DNA from the corresponding normal
kidneys (K) and from a sperm sample (Sp)
(a) were digested with RsaI and HpaII
(R+H). Genomic DNA from ROI tumor 109 (a) and
from LOI tumor 30 (b) were digested with RsaI
alone (R) or with RsaI and MspI
(+M). The digested DNAs were analyzed by Southern blotting
with an H19 promoter probe (probe 1 in Fig. 1).
Lanes are marked with patient numbers. The far left
lane contains a size marker (Sm) with the fragment
sizes indicated in bp in the left-hand box. Marked in the
right-hand box are the digest fragments that correspond to
the nomenclature used in the text and in Fig. 1.
View larger version (57K):
[in a new window]
Fig. 3.
Bisulfite genomic sequencing analysis of the
H19 promoter region. Genomic DNAs from Wilms
tumors (WT), adjacent kidneys, and sperm were
bisulfite-treated, PCR-amplified, and single clones sequenced to give
the methylation pattern of 12 CpGs located in the H19
promoter region (bis1 PCR depicted in Fig. 1; + for methylated, for
unmethylated). The tumors differed in the IGF2 imprinting
status. ROI-WTs 110 and 106 have retained IGF2 imprinting,
LOI-WTs 30, 31, and 43 express IGF2 biallelically, and
LOH-WT 55 has maternal 11p15.5 chromosome loss. Sperm DNA from a
healthy donor was also analyzed. Clones are numbered and marked with an
asterisk, if they were obtained upon digestion with
HpaII prior to bisulfite treatment (see "Experimental
Procedures"). This predigest was necessary to overcome a PCR bias
that favored the amplification of unmethylated alleles. For samples
heterozygous at a G/A-polymorphism (depicted in Fig. 1), the parental
origin of alleles was identified, and the clones were grouped into
paternal and maternal alleles.
View larger version (86K):
[in a new window]
Fig. 4.
Southern blot analysis of
HpaII methylation in the H19 upstream
repeat region. These Southern blots are the same as in Fig. 2 (see
legend Fig. 2). The blots were stripped and reprobed with an
H19 upstream probe (probe 2 in Fig. 1).
2 to at least 4.7 kb relative to
the H19 transcription start site (including HpaII
sites 25-34, see Fig. 1).
View larger version (144K):
[in a new window]
Fig. 5.
Bisulfite genomic sequencing analysis of the
H19 upstream repeat region. Genomic DNAs from
Wilms tumors (WT), adjacent kidneys, and sperm were
bisulfite-treated, PCR-amplified, and single clones sequenced to give
the methylation pattern of 27 CpGs located in the H19
upstream repeats A1 and B1 (bis2 PCR depicted in Fig. 1; + for
methylated and for unmethylated). The tumors differed in the
IGF2 imprinting status. ROI-WTs 110, 106, and 134 have
retained IGF2 imprinting, and LOI-WTs 30, 31, and 43 express
IGF2 biallelically, and LOH-WT 55 has maternal 11p15.5
chromosome loss. Sperm DNA from a healthy donor was also analyzed.
Clones are numbered and marked with an asterisk, if they
were obtained upon digestion with HpaII prior to bisulfite
treatment (see "Experimental Procedures"). This predigest was
necessary to overcome a PCR bias that favored the amplification of
unmethylated alleles. For samples heterozygous at five polymorphic
sites within the bis2 fragment (depicted in Fig. 1; allele types TAGGT,
CCACC, or CCGCC), the parental origin of alleles was identified, and
the clones were grouped into paternal and maternal alleles. The
parental origin of alleles from homozygous individuals 106 and 55 was
assumed according to the methylation pattern.
2 to 5.3 kb relative to the H19
transcription start site. We have also shown that this region is
hypermethylated to different extents on maternal alleles in Wilms
tumors with loss of IGF2 imprinting and, furthermore, that
the pathological methylation is more pronounced in the upstream repeats
compared with the H19 promoter region.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2.0 kb relative
to the H19 transcription start. The 5' boundary of the
methylation imprint was refined to between 4.4 and 5.6 kb, with
HpaII site 25 being the most 5' methylation site that was
clearly differentially methylated. Since the 400-bp direct repeats
extend to 5.3 kb, it seems conceivable that the methylation imprint
might comprise the entire repeat region from 2.0 to 5.3 kb, but
this remains to be proven.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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