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© 2000 Oxford University Press
Human Molecular Genetics, 2000, Vol. 9, No. 20 3029–3035
Differential chromatin packaging of genomic imprinted
regions between expressed and non-expressed alleles
Takuya Watanabe1,2, Akira Yoshimura1, Yukio Mishima1, Yoshiro Endo1,
Toshihiko Shiroishi3, Tuyoshi Koide3, Hiroyuki Sasaki4, Hitoshi Asakura2 and
Ryo Kominami1,+
1First
Department of Biochemistry and 2Third Department of Internal Medicine, Niigata University School of Medicine,
Asahimachi 1-757, Niigata 951-8122, Japan and 3Mammalian Genetics and 4Human Genetics, National Institute of
Genetics, Yata 1111, Mishima 411-8540, Japan
Received 11 August 2000; Revised and Accepted 9 October 2000
Chromosomal regions subject to genomic imprinting
comprise a functional domain exhibiting parentalspecific expression of genes and hence may take a
unique chromatin structure. Here we have examined
the chromatin packaging state of allelic sites in the
Zfp127/Snrpn locus on mouse chromosome 7 and in
the Igf2r locus on mouse chromosome 17 with an
assay consisting of chromatin fractionation and
allele-specific detection. The results showed that
non-transcribed alleles of Igf2r are packaged more
compactly than transcribed alleles in F1 hybrid mice
of both types of cross between C57BL/6 and MSM
strains, whereas a non-imprinted gene, Sod-2, in the
vicinity of Igf2r does not show such a difference. This
indicates a close correlation between imprinting and
the differential packaging of chromatin. On the other
hand, the Zfp127/Snrpn locus showed such an allelespecific fractionation pattern only in F1 hybrid mice
of a cross but not in those of the reciprocal cross.
Analysis of the congenic mice produced for this
locus did not provide any difference. These results
suggest that chromatin of imprinted domains in
different compaction levels is affected by distinct
blueprints in homologous chromosomes that are
heritable through the germ line.
INTRODUCTION
Light microscopic studies of interphase nuclei of higher
eukaryotic cells distinguished two types of chromatin, a highly
condensed heterochromatin and a less condensed euchromatin
(1). The heterochromatin replicates late in S phase and is
enriched with unacetylated histone H4 (2–4). The euchromatin
can be further divided into at least two forms: ∼10% is in the
form of active chromatin, which is the least condensed, and the
rest is inactive euchromatin, which is more condensed than
active chromatin but less condensed than heterochromatin.
Rough separation of active chromatin from the remainder of
the chromatin is possible with several methods, such as differ+To
ential centrifugation of sonicated chromatin fragments, and
accordingly their biochemical differences have been studied
(5). At the DNA level, the chromatin organization is investigated by DNase I sensitivity assay and by digestion with
MNase that permits the determination of the positioning of
nucleosomes (6–8).
Genomic imprinting refers to the differential marking of
parentally inherited specific genes or chromosomal regions
during gametogenesis (9,10). Those imprinted genes are
expressed on only the maternal or paternal allele and are not
expressed on the opposite allele. Such genes include Zfp127
and Snrpn on mouse chromosome 7, which is syntenic to the
Prader–Willi syndrome (PWS)/Angelman syndrome (AG)
imprinted region on human chromosome 15 (11,12) and Igf2r
on mouse chromosome 17 (13). Several mechanisms have
been clarified by which imprinting can be achieved (14–16)
and one is the CpG methylation. Almost all imprinted genes
have sequence elements that are methylated on only one of the
two parental alleles. The differential methylation is a signal
that leads to an inactive state of chromatin probably through
binding to methyl-CpG-binding proteins, such as MeCP2,
which recruit histone deacetylase and corepressor complexes
(17–19).
The chromatin structure of imprinted gene regions have been
studied by DNase I sensitivity assay and by MNase digestion
(20,21). The transcribed allele is more accessible to DNase I
than the other non-transcribed allele and constitutes a nonnucleosomal organization. However, the chromatin was not
examined at the chromatin-compaction level. We previously
developed a novel method that is based on the chromatin fractionation by centrifugation, combined with allele-specific
detection (22). This is applicable to cells and tissues of heterozygous mice and allows quantitative measurement of the relative amounts of allelic DNA fragments in the heterochromatin
(H) and euchromatin (E) fractions. The H–E assay detects a
difference in packaging state of the Xist gene between active
and inactive X chromosomes in normal tissues and tumor cell
lines of inter-subspecific F1 mice (23). With this method, we
have compared chromatin compaction between expressed and
non-expressed alleles of the imprinted Zfp127/Snrpn region on
chromosome 7 and of the Igf2r region on chromosome 17. This
whom correspondence should be addressed. Tel: +81 25 227 2077; Fax: +81 25 227 0757; Email: [email protected]
3030 Human Molecular Genetics, 2000, Vol. 9, No. 20
Figure 1. Map and transcripts of the Zfp127/Snrpn locus on mouse chromosome 7 (top) and of the Igf2r locus on mouse chromosome 17 (bottom). Black
boxes and an oval indicate the position of genes and a Mit marker, respectively,
and arrows show transcription (12,13). The maps are not drawn to scale.
paper demonstrates an allelic difference in chromatin compaction and that the difference varies in the two imprinted loci.
RESULTS
Allelic difference in chromatin packaging was studied with H–E
assay in liver of F1 heterozygous B6 × MSM mice. Two loci
were examined (Fig. 1), one containing five genes, Zfp127,
Ndn, Snrpn, Ube3a and Myo-d1, on mouse chromosome 7. The
other is in the vicinity of Igf2r gene on chromosome 17. Cell
nuclei isolated from liver were subjected to sonication
followed by centrifugation at low speed, which gave an H fraction and further centrifugation of the supernatant at high speed
yielded the E fraction. The H fraction is enriched with compact
chromatin and the E fraction is enriched with chromatin of
open structure (22). DNA in the two fractions were used for
PCR amplification with primers on the five genes which were
able to distinguish B6 and MSM alleles. The primer sequences
were designed according to sequences that had been obtained
by analysis of B6 and MSM genomic DNA (data not shown).
Figure 2 shows gel electrophoretic patterns of PCR products of
three independent experiments for (B6 females × MSM males)
F1 mice. B6 DNA gave a band of Zfp127 more migrated than
MSM DNA, which was ascribed to a 1 bp substitution between
them (data not shown). The B6:MSM band ratio of F1 mice
was 0.65 (a mean value of four experiments) though it should
be theoretically 1 because the band signal should reflect the
amount of each DNA fragment. The H fractions showed an
average B6:MSM ratio of 1.1, more than the 0.65 of F1 mice,
indicating that the H fractions contained less MSM DNA than
B6 DNA (the MSM < B6 pattern). On the other hand, the E
fractions showed an average B6:MSM ratio of 0.44, i.e. an
inverse MSM > B6 pattern. The result suggests that the
paternal MSM allele is less compact than the maternal B6
allele. This is consistent with expression of only the paternal
MSM allele (Fig. 3A). The observed difference was clear but
much less prominent than those detected in genes on the
X chromosome (22,23). Ndn and Snrpn subject to imprinting
also provided similar results of fractionation, though the difference in Ndn was smaller than those in the other two (average
B6:MSM ratios shown in the legend to Fig. 2). The lower two
panels show gel patterns of Ube3a and Myo-d1 are transcribed
from both alleles in the liver (11,12). Either gene did not
exhibit such allelic differences. Analysis of the D7Mit200
Figure 2. Relative compaction of chromatin packaging in allelic sites on the
Zfp127/Snrpn region: (left) for livers of heterozygous F1 mice by (B6 males ×
MSM females) crosses and (right) for those by the reciprocal crosses. Three
independent experiments are shown. The loci examined are shown at the right
side of each panel (Fig. 1). B, M and F1 indicate B6, MSM and their F1 offspring, respectively; H and E denote H and E fractions obtained from F1 mice,
respectively. Average B6:MSM ratios in F1, H and E fractions of the (B6 ×
MSM) cross are as follows: Ndn, 0.99, 1.34 and 0.71, respectively; Snrpn, 2.2,
3.5 and 0.97, respectively; Ube3a, 0.24, 0.30 and 0.34, respectively; Myo-d1,
1.0, 1.3 and 1.3, respectively.
distal to the Zfp127 locus also showed no allelic difference
(data not shown).
Figure 2 shows the results of the reciprocal crosses, (MSM
females × B6 males) F1 mice. None of the five loci showed
allelic difference. This was our unexpected result. One
possibility to account for the result is that other genetic or
epigenetic factors exist which affect allele-specific chromatin
compaction. The B6 and MSM strains used belong to different
mouse subspecies and hence their genomes differ more than do
those of conventional laboratory strains. This genetic difference might affect epigenetic modifications during gametogenesis and possibly during development which lead to distinction
in chromatin compaction of paternal and maternal alleles. To
examine this possibility, we generated a congenic strain for the
imprinted region by backcrossing MSM to B6 mice. The
congenic mice possessed MSM-derived genomic sequence
only in a region covering Zfp127 on chromosome 7. Figure 4
shows H–E assays for hybrid mice between B6 and the
congenic strain. The results were similar to those of F1 mice
between B6 and MSM strains.
Allelic difference in DNase I sensitivity was investigated
using a primer set on a promoter region of Zfp127. The region
amplified by this primer set contained a polymorphism at the
AluI recognition site between B6 and MSM and a site showing
DNase I hypersensitivity (Fig. 5A). Nuclei prepared from liver
of the two types of cross between B6 and MSM were digested
with DNase I at various concentrations and then DNA was
isolated. Figure 5B shows gel electrophoretic patterns of AluI
digests of the PCR products. The band signal ratio of B6:MSM
markedly increased with amounts of DNase I added in
(B6 females × MSM males) F1 mice, whereas it decreased in
(MSM females × B6 males) F1 mice. The results indicated that
transcribed paternal alleles were more sensitive to DNase I
digestion than non-transcribed maternal alleles in both of the
Human Molecular Genetics, 2000, Vol. 9, No. 20 3031
(MSM:B6 ratios shown in the legend to Fig. 6). This contrasts
with the result of the imprinted genes on mouse chromosome 7.
We also generated the strain congenic for this Igf2r region of
B6 background (see Materials and Methods) and examined
differential chromatin packaging using hybrid mice between
B6 and the congenic strain. H and E fractions gave different
B6:MSM band ratios and the difference was essentially the
same as that of F1 mice between B6 and MSM (Fig. 7).
DISCUSSION
Figure 3. Expression (A) and methylation (B) of the Zfp127 gene. PCR products for Zfp127 and Ube3a are shown. B, M and F1 indicate liver cDNA of B6,
MSM and their F1 offspring, respectively, and BXM and MXB denotes cDNA
from liver of the reciprocal crosses. (B) Gel electrophoresis of AluI digests of
PCR products. DNAs were obtained from mice indicated above the panels,
digested with HpaII and then subjected to PCR amplification. Average
B6:MSM ratios are as follows: 2.5, ∼0, 5.8 and ∼0 in the HpaII digests of
(B6 × MSM)F1, (MSM × B6)F1, and (B6 × MSM) and (MSM × B6) of the
congenic mouse, respectively. An average B6:MSM ratio of undigested DNA
of (B6 × MSM) is 0.72.
reciprocal crosses. A similar analysis was carried out for
crosses between B6 and the congenic strain for a Zfp127 region
(Fig. 5C). Allele-specific DNase I sensitivity was also found in
transcribed alleles. In addition to the DNase I sensitivity we
examined expression and methylation of Zfp127. F1 mice of
the reciprocal crosses both showed expression of paternal
alleles of the Zfp127 gene but not of the Ube3a gene (Fig. 3A).
HpaII sensitivity also showed allelic difference (Fig. 3B).
However, B6 allele was fully demethylated when paternally
inherited but demethylation of the paternal MSM allele was
partial. These results are consistent with a previous analysis
(24) and with the idea of parental imprinting.
Another imprinted region on mouse chromosome 17 was
examined. Three primer sets were used to detect allelic difference in chromatin compaction: a promoter and a 3′-untranslated region (UTR) of the Igf2r gene subject to imprinting and
a flanking Sod-2 gene not subject to imprinting (Fig. 1).
Figure 6 shows the results of an H–E assay of the F1 generation
of the reciprocal crosses. As for (B6 females × MSM males) F1,
the B6:MSM band ratio of the 3′-UTR region was 1.1, 0.18 and
1.4 in F1, H and E fractions, respectively, and that of the
promoter was 1.1, 0.64 and 1.9 in F1, H and E fractions, respectively (Fig. 6, left half). On the other hand, Sod-2 did not show
such differences: the B6:MSM band ratio was 1.5, 1.5 and 1.6
in F1, H and E fractions, respectively. The differences between
H and E fractions in the Igf2r region indicated that H fractions
contained non-transcribed paternal alleles more than transcribed maternal alleles, consistent with the result of the
imprinted genes on chromosome 7. Of importance, however, is
that (MSM females × B6 males) F1 mice also showed allelic
difference (Fig. 6, right half); both of the 3′-UTR and promoter
primers provided patterns of non-transcribed paternal alleles
being recovered more in H fractions than in E fractions
Chromosomal regions subject to genomic imprinting are
known to comprise a functional domain that is conferred by a
gamete-determined group of epigenetic modifications. The
modification signals are stably transmitted to cells of the
developing organism and result in changes in gene expression,
cytosine methylation, chromatin structure and replication
timing within the imprinted domain (9–12,14,17,18,25–27). In
this study we show that the H–E assay, based on chromatin
fractionation and allele-specific detection, can be used as a tool
to study the organization of imprinted chromatin. Also we
provide the results of H–E assay for two imprinted loci. Nontranscribed paternal alleles of Igf2r on chromosome 17 are
packaged more compactly than transcribed maternal alleles,
and a non-imprinted gene, Sod-2, in the vicinity of Igf2r does
not show such a difference (Figs 6 and 7). This indicates a
close correlation between imprinting of Igf2r and its differential chromatin compaction between parental alleles.
As for the Zfp127/Snrpn locus on chromosome 7, however,
two reciprocal crosses of F1 mice showed different results.
DNA of (B6 females × MSM males) F1 mice gave H–E
patterns showing a correlation between imprinting of individual genes and their differential chromatin packaging, but
DNA of the reciprocally crossed (MSM females × B6 males)
F1 mice showed no difference (Fig. 2). This may reflect genetic
differences between B6 and MSM genomes, because there is a
precedence for genetic variation in imprinting. The genome of
Mus musculus molossinus, another mouse subspecies different
from M. m. domesticus, carries an allele called Imprintor-1
(Imp-1) that affects imprinting at the T-associated maternal
effect (Tme) locus on chromosome 17 (28). The Imp-1 gene is
unlinked to Igf2r, the probable gene responsible for Tme (29)
and has two alleles: Imp-1d causes imprinting at the Tme locus
in M. m. domesticus, and Imp-1m does not inactivate the
paternal copy of Tme and has been found so far in M. m.
musculus. Thus, to know whether or not an allele(s) in the
genome of M. m. molossinus affects imprinting signals, we
have produced congenic lines for the Zfp127 locus and for the
Igf2r locus by introducing MSM loci into the B6 genome.
Analysis of F1 mice between B6 and the congenic lines did not
provide any difference at either region. Therefore, no evidence
was obtained for supporting the idea that there is an allele(s)
that modifies the differential compaction of chromatin packaging detected by the H–E assay.
On the other hand, the results described above revealed that
variation in the packaging status of allelic sites in the imprinted
Zfp127/Snrpn region were inherited unchanged through generations of establishing the congenic lines (Fig. 4). This suggests
the presence of a variant blueprint for the regulation of chromatin compaction that is heritable through the germ line. Such
an idea for the blueprint controlling epigenetic modification is
3032 Human Molecular Genetics, 2000, Vol. 9, No. 20
Figure 4. Relative compaction of chromatin packaging in livers of heterozygous mice between B6 and congenic mice for the Zfp127/Snrpn region.
(Left) (B6 males × females of congenic mice) crosses; (right) the reciprocal
crosses. The results of three independent experiments are shown.
reported for allele-specific DNA methylation in several human
and mouse loci including the c-Ha-Ras-1 gene (30–32). Variation in the methylation of allelic sites is tissue specific and
reproducible after transmission through the germ line. A
putative cis-acting element(s) must be close to the Zfp127
locus to explain the complete cosegregation observed in seven
generations. If it is the case, the presence of the cis element
may explain why the two imprinted loci examined here differ
in differential chromatin packaging of paternal and maternal
alleles. We observed allelic difference in methylation of the
Zfp127 promoter: the MSM allele was fully demethylated
when paternally inherited but the B6 allele was only partially
demethylated (Fig. 3B). Although the partial demethylation
was found in the expressed B6 allele of (MSM females × B6
males) F1 mice, this might be a variant blueprint that explains
the difference in chromatin structure depending on the crosses.
The difference between two reciprocal crosses at the Zfp127
locus may be simply accounted for by the inability of the H–E
assay to detect compaction of chromatin packaging. However,
we think this possibility is less likely for the following reasons
(22,23). Firstly, we previously showed that the H–E assay
enriches the H fraction with the mouse satellite and the p53
pseudogene that comprise heterochromatin. Secondly, it was
successfully applied to detection of the differential chromatin
packaging of genes on the X chromosome such as Xist and
Pgk-1 between active and inactive X chromosomes. Thirdly,
we found a unique tumor cell line that showed biallelic expression of the Pgk-1 gene, which reflected impairment of
transcriptional repression of the Pgk-1 allele on inactive
X chromosome. Interestingly, this line exhibited little difference in the chromatin packaging of Pgk-1 between active and
inactive X chromosomes, suggesting that the H–E assay is able
to monitor the chromatin change in those tumor cells.
The Zfp127/Snrpn region on mouse chromosome 7 is
syntenic to the PWS/AG imprinted region on human chromosome 15q11–q13. Much effort has focussed on understanding
how parental identity is established for this chromosomal
region. Localized deletions upstream of the SNRPN gene
appear to disrupt the epigenetic program that regulates
Figure 5. Accessibility of the promoter region of the Zfp127 gene to DNase I.
(A) Map of the Zfp127 locus. AluI and HpaII sites are 258 bp and 170 bp 5′ to
the transcription start site and DNase I-sensitive site is between these two sites.
The 5′-end position of the three primers (F9, R9 and R10) is also shown. Polyacrylamide gel electrophoresis of AluI digests of the PCR products provided
bands of 98 and 25 bp for B6 and a band of 123 bp for MSM. (B and C) Nuclei
were treated with various concentrations of DNase I (from 1/16 to 2 U/µl indicated above the lanes) and analyzed with PCR primers detecting an AluI site
polymorphism between B6 and MSM.
Figure 6. Relative compaction of chromatin packaging in allelic sites on the
Igf2r region: left half for livers of heterozygous F1 mice by (B6 males × MSM
females) crosses and right half for those by the reciprocal crosses. The loci
examined are shown at the right side of each panel. Average MSM:B6 ratios in
the (MSM × B6) F1 cross are as follows: 3′-UTR, 1.1, 0.38 and 1.3 in F1, H and
E fractions, respectively; promoter, 0.98, 0.63 and 1.1 in F1, H and E fractions,
respectively; Sod-2, 0.74, 0.68 and 0.74 in F1, H and E fractions, respectively.
imprinted gene expression, defining a putative cis-acting
imprinting control center (IC) (12). Allele-specific methylation
and hypersensitivity to nucleases are observed at the promoter
and first exon of the SNRPN gene within the IC and therefore
this region is likely to control the parental-specific epigenotype
Human Molecular Genetics, 2000, Vol. 9, No. 20 3033
Figure 7. Relative compaction of chromatin packaging in livers of heterozygous mice between B6 and congenic mice for the Igf2r region. (Left) B6
males × females of congenic mice crosses; (right) the reciprocal crosses. The
results of two independent experiments are shown.
(12,33,34). Consistently, the IC is composed of an unusually
high density of certain DNA sequences as matrix attachment
regions, the maternal allele of which strongly associates with
nuclear matrix and is more condensed than the paternal allele
(35). Likewise, a region of an imprinting signal that maintains
expression of the maternal Igf2r allele is determined in the
second intron of the gene on chromosome 17 (36). A CpG
island within the second intron is subject to parental-specific
methylation. Additionally, paternal or maternal delay in
replication timing or sister chromatid segregation extending
over several mega base pairs has been reported for the Snrpn
and Igf2r loci (25,27).
In addition to those epigenetic properties described above, the
parental-specific chromatin compaction detected by the H–E
assay can be another characteristic of imprinted genes, since
our data show a correlation between imprinting of individual
genes and their differential packaging of chromatin. However,
functional relationship between this new parameter and the
others is not yet clear. As discussed above, the Zfp127 region
does not show any difference in the compaction between transcribed and non-transcribed alleles for F 1 mice of a cross. This
suggests that the observed chromatin compaction is different
from that of the cytologically and genetically defined heterochromatin, which represses transcription not only in the DNA region
itself but also in regions of chromatin adjacent to the heterochromatin domain. Consistently, our previous study showed that an
actively transcribed Xist allele on inactive X chromosome is
packaged into chromatin more compactly than an untranscribed
Xist allele on an active X chromosome (23). Therefore, the
allele-specific differential packaging of chromatin detected
here may simply represent a physical state of chromatin that
could be a secondary regional by-product of imprinting at a
specific gene locus. If it is the case, however, data on the state
of chromatin provide a clue to relation between chromatin
compaction in the cell nucleus and genomic imprinting and
possibly other epigenetic modifications.
MATERIALS AND METHODS
Mice
F1 heterozygous mice were obtained by mating C57BL/6(B6)
females with MSM males and by reciprocal crosses. MSM is
an inbred strain derived from the Japanese wild mouse, M. m.
molossinus. Mice congenic for either the Zfp127 region on
chromosome 7 or the Igf2 region on chromosome 7 were
obtained by backcrossing MSM mice to B6, using a markerassisted selection protocol (37). In this process Mit markers
were used to examine genomic composition (38). The
congenic mice for the Zfp127 region comprised an MSMderived region between D7Mit21 and D7Mit362 and those for
the Igf2 region contained an MSM-derived region between
D17Mit246 and D17Mit123. Backcross mice of generation 7
were mated with B6 mice to generate hybrid mice used for
experiments.
H–E assay for the packaging state of chromatin
The H–E assay was carried out as previously described (22). In
brief, the suspension of washed nuclear pellet in cation-free
0.25 M sucrose was sonicated at 200 W for 5–10 min into
smaller chromatin particles. After removing chromatin aggregates, the supernatant was centrifuged at 1700 g for 10 min.
The pellet was used as the H fraction. The supernatant fraction
was washed by centrifuging at 4500 g for 30 min. The resultant
supernatant was again centrifuged at 100 000 g for 60 min to
give the pellet, E fraction. DNA was extracted from those
pellets and subjected to PCR. To prove that the enrichment of
H and E fractions had been achieved, we examined every
sample using the p53 primer probe (22). Then, separate sets of
H and E fractions were used.
PCR analysis of DNA and RNA and single-strand
conformation analysis (SSCP)
PCR was carried out for cellular DNA and cDNA from liver in
10–20 µl under standard conditions as previously described
(22,23). The reaction was processed through 30–35 cycles of
amplification consisting of 30 s at 95°C, 30 s at 55–58°C and
30 s at 72°C, with the last elongation step lengthened to
10 min. One primer was end-labeled with 32P and used for
amplification. Products were analyzed by polyacrylamide gel
electrophoresis and some were subjected to SSCP analysis, i.e.
the products were heat-denatured and separated by electrophoresis
in 6% polyacrylamide gel containing 5 or 10% glycerol.
PCR primers for the H–E assay
Five primer sets were used for the H–E assay to detect allelic difference of genes in the Zfp127 region on mouse chromosome 7. Primers
for the 3′-UTR of Zfp127 and Snrpn were 5′-TTGGCTCTTGCTTCAGTACC-3′ (F1) and 5′-TCACAAGTCATCAGTTGACAG-3′
(R1), and 5′-CTTCCTTAGTTTTCTCCTTGCC-3′ (F2) and 5′TGGGGGATGAAAAGTAGACAC-3′ (R2), respectively. Polymorphisms were detected by SSCP analysis of their PCR products.
Primers for the 3′-UTR of Ube3a were 5′-CCTGGGTCTGGCTATTTACAATAA-3′ (F4) and 5′-AGAGTCTCCCAAGTCACG-3′ (R4), and a polymorphism was detected by MfeI digestion
of PCR products. Primers for Ndn and Myo-d1 were 5′-TAACCACTGAACCAAGTCTC-3′ (F3) and 5′-CCTTCGGATCAGAGCAGGAC-3′ (R3), and 5′-CTTCCACTCCCCTCACAGAG-3′ (F5)
and 5′-ATCTTTTGGGCGTGAAGAACC-3′ (R5), respectively.
Their polymorphisms were detected simply by polyacrylamide gel
electrophoresis.
Primers for the Igf2r promoter were 5′-CATCCTGTATATCAGCCCAG-3′ (F6) and 5′-TAAACACGTGCACAGCACAC-3′
3034 Human Molecular Genetics, 2000, Vol. 9, No. 20
(R6) and those for the 3′-UTR of Igf2r were 5′-AGGTCTCATCTCTTCAGGGTC-3′ (F7) and 5′-GGACACTGCCCTAGCACAG-3′ (R7). Their polymorphisms were detected by polyacrylamide gel electrophoresis of PCR products. Primers for Sod-2 were
5′-AGGGTTGAGAGTGCCCCAGCT-3′ (F8) and 5′-TCACAACTCGGACTGCAGTC-3′ (R8), and the polymorphism was detected by PvuII digestion of PCR products.
DNase I digestion of nuclei
Isolation of nuclei and DNase I digestion were carried out
essentially as described by Sasaki et al. (21). Nuclei equivalent
to 10 µg of DNA were resuspended in 90 µg of 0.3 M sucrose,
5% glycerol, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM
EGTA, 15 mM Tris–HCl pH 7.6 and 0.5 mM dithiothreitol.
The samples were mixed with 10 µg of the same solution
containing 20, 10, 5, 2.5, 1.25, 0.625 and 0 U of DNase I
(Takara, Kyoto, Japan) and incubated for 5 min at 25°C. The
reactions were terminated by adding an equal volume of
20 mM EDTA pH 8.0, 1% SDS containing 0.2 mg/ml of
proteinase K. After overnight incubation, DNA was purified
and subjected to PCR using F9 and R9 primers.
PCR primers for expression, DNase I sensitivity and
methylation
F1–R1 and F4–R4 primer sets were used for the detection of
allele-specific Zfp127 and Ube3a expression, respectively.
Their polymorphisms were detected by SSCP analysis and by
MfeI digestion of PCR products, repectively. Primers used for
DNase I sensitivity assay were 5′-CACAAATAAACTGCAATGTATACAGC-3′ (F9) and 5′-GCTTCTGCCGGCTTTCTAAG-3′ (R9). AluI digests of the PCR products
provided bands of 98 and 25 bp for B6 and a band of 123 bp for
MSM, since there is a polymorphism (T in B6 and C in MSM
3′ to the F9 sequence) in the promoter region of Zfp127 (Fig.
5A). F9 and 5′-TTGAGACACTGGGATGGGC-3′ (R10),
which was 3′ to R9, were used for methylation sensitivity
assay. Genomic DNA was digested with HpaII and then used
as a template for PCR amplification. Polymorphism was
detected using AluI digestion as described above.
ACKNOWLEDGEMENTS
This work was supported by a grant-in-aid from the Ministry of
Education, Science, Sports and Culture of Japan.
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