 |
INTRODUCTION |
In various natural and experimental scenarios, mammalian genomes
become the targets for foreign DNA insertions. Many DNA- and
RNA-containing viruses are capable of integrating their genomes into
the genomes of their host cells. Numerous experiments designed for the
artificial transfer of genes into mammalian cells aim at the permanent
fixation of these genes in established eukaryotic genomes. In
transgenic organisms that have been propagated from successfully
transformed embryonal cells, all cells carry integrated foreign DNA. We
have started to investigate the structural and functional consequences
of the insertion of foreign DNA into established mammalian genomes. The
de novo methylation of the integrated DNA and alterations in
the patterns of DNA methylation in the recipient genomes at the site of
insertion and remote from it have been of particular interest (for
review, see Ref. 1). By using different techniques including the
bisulfite protocol of the genomic sequencing technique (2, 3), we have
documented extensive changes in the patterns of DNA methylation at
several cellular sites remote from the loci of insertion of the DNA of
adenovirus type 12 (Ad12)1
and lesser changes in cells transgenic for the DNA of bacteriophage
(4, 5). Because
DNA is not transcribed in transgenic mammalian
cells, alterations of methylation patterns subsequent to foreign DNA
insertion are not dependent on foreign gene transcription. It has been
shown earlier that cellular DNA sequences immediately abutting the
foreign DNA integrates also exhibit changes in DNA methylation (6, 7).
It is presently unknown by what mechanisms the insertion of foreign DNA
affects the organization and function of the recipient genome. Does the
site of foreign gene integration determine where the remote effects
occur, and is a critical size of integrated DNA required? We
surmise that the acquisition of many kilobases or even a megabase of
inserted DNA alters the chromatin topology and can thus influence the
function of specific parts of the genome. At present, these
interdependencies are essentially unknown. We have therefore started a
step-by-step analysis of these alterations by studying changes in
transcription and DNA methylation patterns.
In this report, we have utilized a differential hybridization method
with cDNAs prepared from
DNA-transgenic cells as compared with
nontransgenic cells. We have used the method of methylation-sensitive representational difference analysis (MS-RDA) and the novel method of
methylation-sensitive amplicon subtraction (MS-AS) to detect cellular
DNA segments with altered methylation patterns in transgenic cells.
These genome-wide scanning methods have led to the isolation of several
cellular genes and DNA segments with changes in DNA methylation or
transcription. Differentially methylated DNA segments obtained with the
MS-RDA method usually are highly repetitive and show no homology to
known sequences. The newly developed MS-AS method on the other hand has
facilitated the cloning of DNA segments with high C+G contents, which
have been characterized by their nucleotide sequences; and their
functions have been derived from GenBankTM data collections.
 |
EXPERIMENTAL PROCEDURES |
Transgenic Mice--
The origin of mouse lines transgenic for a
construct carrying the chloramphenicol acetyltransferase gene under the
control of the E2A late promoter of adenovirus type 2 (pAd2E2AL-CAT)
has been described (8, 9). Microinjection of the M-HpaII
(5'-CCGG-3') premethylated construct generated the founder animal of
mouse line 8-1, and microinjection of unmethylated DNA generated that of line 7-1.
Cell Lines--
The Ad12-transformed hamster cell line
T637 (with multiple copies of integrated Ad12 DNA (10)) was described
elsewhere. The clonal BHK21 cell lines transgenic for the DNA of
bacteriophage
and plasmid pSV2neo (11) were presented earlier (5).
After selection, these cell lines were cultivated without the addition of G418. For control experiments, nontransgenic BHK21 cells were recloned three to four times, and the thus established clonal lines
were used. All cell lines were cultivated in Dulbecco's medium with
10% fetal calf serum.
Standard Techniques of Molecular Biology--
DNA extraction,
including RNase treatment, Southern transfer hybridization (12, 13),
restriction analyses with the methylation-sensitive endonucleases
HpaII, HhaI, or the control enzyme
MspI, and electrophoresis in 0.8-1.0% agarose gels were
all detailed earlier (e.g. Ref. 14). Autoradiograms were
evaluated quantitatively by using a Fuji X-BAS 1000 phosphorimager.
Identification and Isolation of Differentially Methylated DNA
Segments by MS-RDA (15)--
After HpaII cleavage of tester
or driver DNAs and ligation of the fragments to Rhpa adaptors,
amplicons were prepared by 25-35 PCR cycles using the Rhpa24
oligonucleotides as primers under the conditions described elsewhere
(16). In these experiments, DNA from the cell line T637 or from the
DNA-transgenic BHK21 cell lines was the "driver" and the DNA from
the nontransgenic BHK21 cells the "tester" DNA. Amplification
products were cut with MspI and purified by gel
filtration. Only the tester amplicon preparation (1 µg) was
subsequently ligated to 500 pmol of the Jhpa adaptor
(designations and sequences of adaptors available on request). All
adaptors carried a CG overlap, which was ligated to the GC overlap of
the HpaII and MspI cleavage products. When selective hybridization was performed, only self-reannealed tester molecules with adaptors at both ends became amplified efficiently in
the following PCR. Adaptor-ligated tester molecules only
self-reannealed in the absence of homologous partners in the driver
amplicon pool. Amounts of 40-100 ng of the ligation product were mixed
with 30-40 µg of driver amplicon DNA. The mixture was
phenol-extracted, ethanol-precipitated, and dissolved in 3 mM EDTA, 3 mM HEPES, pH 8.0. The
subtractive hybridization was performed by denaturing the tester-driver
DNA mixture at 96 °C for 10 min followed by reannealing at 67 °C
for 18 h in the presence of 1 M NaCl. After adding 45 µl of a prewarmed dilution buffer (1 M NaCl, 8 mM Tris-HCl, pH 7.4, 0.8 mM EDTA, pH 8.0),
one-tenth of the product was amplified with the Jhpa24 primer for
13-15 cycles. The PCR solution was then heated to 72 °C for 10 min,
and the linearly amplified fragments were cleaved with 100 units of
mung bean nuclease in the presence of 1 mM
ZnSO4. Double-stranded DNA was further amplified by PCR for
20-35 cycles using the Jhpa24 primer.
In a second cycle of competitive hybridization, 10-40 ng of the PCR
product was ligated to the Nhpa adaptor and again mixed with 40 µg of
driver amplicon DNA. The product was cloned into the pGEM-T vector
(Promega) and transfected into XL1BlueMRF' bacteria. The insert from
positive clones was PCR-amplified using the SP6 and T7 primers and
restricted with MspI. To test for the effectiveness of the
MS-RDA protocol, linearized and in vitro
HpaII-premethylated pGL2 control vector (Promega) or the pN3
plasmid containing human proto-RET cDNA p51 (17) was added in 1 or
2 genome equivalents to the driver DNA prior to HpaII
cleavage. The same amount of unmethylated plasmid was added to the
tester DNA. Southern blot analyses of amplicons, first- and
second-round difference products with the pGL2-control or the pN3
probe, revealed the levels of enrichment of the exogenously added DNAs
in the course of the MS-RDA procedure. The nucleotide sequences of all
primers and adaptors referred to in this section as well as the
annealing temperatures chosen in individual experiments were not
reproduced here but will be available on request.
Screening and Analysis of Second-round Difference
Products--
PCR-amplified plasmid inserts were denatured and gridded
identically on two GeneScreen Plus membranes. These membranes were then
hybridized against 32P-labeled amplicon DNA from either the
tester or the driver. Putative difference products as determined by
their differences in signal intensities between tester and driver were
selected and used for Southern blot analyses with cellular DNA. The
nucleotide sequences of differentially methylated clones were
determined with an Applied Biosystems 377 DNA sequencer by standard
methods (18), and a homology search was performed at a
GenBankTM Web site.
Isolation of Differentially Expressed Genes: Subtraction of
cDNA Libraries--
Differences in gene expression between
transformed or transgenic cells versus nontransgenic BHK21
cells were determined by the cDNA subtraction method (19).
Poly(A+) mRNA was isolated using an mRNA isolation
kit (Roche Molecular Biochemicals). From both the tester and the
driver cell lines, cDNA libraries were constructed by oligo(dT)
priming with 2 µg of poly(A+) mRNA. The protocol
specified by CLONTECH was followed for the subtraction of the cDNA libraries. For PCR reactions, the Advantage 2 polymerase mix (CLONTECH) was used. Difference
products were cloned into the pGEM-T vector (Promega) and transfected
into competent XL1BlueMRF' cells. Insert-positive colonies were
cultivated in 96-well plates, 3 µl of each culture was used for PCR
with the SP6 and T7 primers to amplify the plasmid inserts. The PCR
products were denaturated in 0.8 M NaOH, 50 mM
EDTA and subsequently arrayed on GeneScreen Plus membranes. The two
identical membranes were hybridized against the 32P-labeled
cDNA libraries from either tester or driver. Positive clones were
selected and further analyzed by Northern blot experiments using either
cytoplasmatic RNA isolated as described (20) or poly(A+) mRNA.
MS-AS: A Novel Strategy for Identifying Differentially
Methylated Sequences in Complex Genomes--
This new protocol (21)
facilitates comparisons between the representations of DNA fragments
that are present in one amplicon sample but not in another. Under the
conditions chosen, only fragments of up to 2.5 kilobase pairs were
effectively PCR-amplified. Larger fragments derived from methylated and
therefore HpaII cleavage-resistant DNA segments were not or
were inefficiently amplified. Differential methylation in the tester
and driver DNAs will therefore result in different representations.
Conventional RDA required several rounds of hybridization, more
material, and favored the amplification of repetitive sequences. To
overcome vast differences in abundance, the newly developed subtraction
protocol included two separate initial subtractive hybridizations with
representations (amplicons) of genomic DNA in combination with
suppressive PCR (19, 22). Genomic tester or driver DNAs (5 µg) were
cleaved with excess amounts of HpaII. After ligating 200 ng
of the HpaII fragments to the Rhpa adaptor, amplicons were
prepared by PCR using the Rhpa24 primer as described (16). The PCR
products were cut with RsaI to create blunt ends. The tester
DNA was subdivided into two 500-ng portions, which were ligated to
either adaptor L or R. Two rounds of hybridization followed. In the
first, 2 µl of RsaI-cleaved driver amplicon (1-2 µg)
was added to 1 µl of tester DNA (50 ng) and to 1 µl of
hybridization buffer containing 200 mM HEPES, pH 8.3, 2 M NaCl, 0.08 mM EDTA, pH 8.0, 40% (w/v)
polyethylene glycol (PEG 8000). The solution was overlaid with mineral
oil, heat-denatured (98 °C, 2.5 min), and annealed for 8 h at
68 °C. For the second hybridization, the two primary hybridization
samples were mixed without denaturation. Denatured driver (500 ng in 1 µl) was added and hybridized for an additional 14 h at 68 °C
and then diluted with 200 µl of preheated dilution buffer (20 mM HEPES, pH 8.3, 50 mM NaCl, 0.2 mM EDTA). The hybridization products were subsequently
amplified by primary and nested PCRs. After ligating the PCR products
into the pGEM-T vector, difference products were screened and analyzed
as described above.
 |
RESULTS |
Differential Expression of Cellular Genes in BHK21 Hamster Cells
Transgenic for Ad12 or
DNA--
The cDNA subtraction method
was applied to reverse transcripts of mRNAs isolated from the
Ad12-transformed BHK21 hamster cell line T637 or from BHK21 cells
transgenic for the DNA of bacteriophage
, e.g. cell lines
L10 and L18. After one cycle of subtractive hybridization to a cDNA
preparation from nontransgenic BHK21 cells, the PCR products were
cloned into the pGEM-T vector and arrayed on GeneScreen Plus membranes.
The DNA was then hybridized to 32P-labeled cDNA from
nontransgenic BHK21 cells, from T637 cells (Fig. 1A), or from the
DNA-transgenic cell line L10 (Fig. 1C). The clones with
array numbers as indicated in Fig. 1, B and D, showed marked differences, and these cDNAs were used for RNA
transfer hybridization (Northern blot) experiments with RNAs from BHK21 cells (B), T637 cells (T), or the L10 cell line.
All differential cDNA clones were strongly expressed in T637 but
not in BHK21 cells.

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 1.
Differential transcription of cellular genes
in BHK21 hamster cells transgenic for Ad12 or DNA. Individual PCR-amplified difference products were
gridded on GeneScreen Plus membranes as dot blot arrays of difference
products after one round of cDNA subtraction. Membranes were
hybridized against 32P-labeled cDNA preparations from
nontransgenic BHK21 or T637 cells (A) or from the DNA-transgenic cell line 10 (L10) (C). Clones
with marked differences in signal intensities between BHK21
(lanes B) and T637 (lanes T) or L10 were
analyzed further in Northern blot experiments using either 30 µg of
cytoplasmic RNA (B) or 2 µg of poly(A+)
mRNA (D). Samples were analyzed by electrophoresis on a
1% agarose gel containing 2.2 M formaldehyde. Upon
transfer to GeneScreen Plus membranes, the RNA was hybridized to
32P-labeled difference clones with array numbers as
indicated in B and D or to -actin
(open arrows) as shown in the lower
panels of B and D. Results from
phosphorimager analyses of signals obtained in D are
described in the text.
|
|
For the two
DNA-transgenic cell lines L10 and L18, 198 cDNA clones each were analyzed by these methods. Phosphorimager
analyses of signal intensities on Northern blot experiments revealed
differences between different mRNA levels of up to 6-fold.
Transcription of the E11 clone was not detectable in the
DNA-transgenic cell line L10 (Fig. 1D). Several of the
differentially expressed clones from cell line L10 were identified by
their nucleotide sequences, and the most convincing data base matches
for the nucleotide sequences of some of these clones were included in
Table I. The differential cDNA arrays
presented in Fig. 2 revealed seven
cDNA segments, clones 80, 4, 10, 29, 31, 32, 58, which were
expressed in cell line L18 but not, or to a very limited extent, in
BHK21 cells. The numbers 80, 4, 10, 29, 31, 32, 35, 58 designated
individual cDNA clones that were selected for DNA array analyses.
The transcription of known genes, murine DNA methyltransferase
(Dnmt1), ADPRT,
-actin, IAPI, or pSV2neo, showed no differences between
the L18 and the BHK21 parental cell line (Fig. 2). At the time the L18
line was analyzed, G418 selection had long been discontinued, and hence pSV2neo expression was not required. In several
DNA-transgenic cell lines without pSV2neo expression,
differences in DNA methylation or transcription were observed. In
contrast, some of the cell lines still expressing the resistance marker
did not exhibit these effects. In the
DNA-transgenic cell lines,
DNA transcripts were never found. Moreover, we observed no
differences in methylation in the cDNA clones analyzed between the
transgenic and nontransgenic cells. Of course, this experimental
protocol based on cDNA comparisons precluded promoter analyses that
would have been necessary for functionally meaningful investigations of
correlations between promoter methylation and activity.
View this table:
[in this window]
[in a new window]
|
Table I
Database homologies of differentially expressed DNA-segments
Details of these analyses were described in the text.
|
|

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
Differential gene transcription in the
DNA-transgenic BHK21 cell line L18. Shown are
reverse Northern dot blot arrays of preselected difference products
after one round of cDNA subtraction with the DNA-transgenic
cell line L18 as tester and the nontransgenic BHK21 cell line as
driver. Difference clones were gridded on GeneScreen Plus membranes
together with known genes, murine DNA methyltransferase
(Dnmt1), ADPRT, -actin,
IAPI, or pSV2neo. The DNAs were hybridized to
32P-labeled cDNA libraries from L18 or BHK21
cells.
|
|
Differentially Methylated Cellular DNA Fragments in Hamster Cell
Lines Transgenic for Ad12 (T637) or Bacteriophage
DNA (L12,
L18)--
Methylation patterns in a wide selection of cellular DNA
sequences were compared between nontransgenic BHK21 cells and Ad12 DNA-
or
DNA-transgenic BHK21 cells. The methods of MS-AS or MS-RDA
compare representations (amplicons) of DNA fragments and have not been
applied previously in analyses with transgenic cell lines. Amplicons
were produced by cleavage of genomic DNA with HpaII followed
by PCR amplification of these fragments using a universal adaptor that
was ligated to the HpaII fragments as primer annealing site.
The conditions chosen for PCR favored the amplification of small
fragments derived from hypomethylated and therefore
HpaII-restricted genomic areas. In contrast, large
HpaII fragments resulting from highly methylated DNA
were not enriched by PCR. Differences in DNA methylation between the
two cell lines resulted in different fragment representations. The goal
of each subtractive hybridization method was to enrich and clone DNA
fragments present in only one amplicon. After amplicon preparation, the
universal adaptors were removed, and only the tester amplicon fragments
were ligated to new adaptors.
During the subtractive hybridization of adaptor-ligated tester
fragments to excess amounts of driver amplicon, only tester fragments
without a homologous partner in the driver pool self-reannealed and
hence carried adaptor sequences on both ends of the DNA molecule. Fragments common to tester and driver formed heterohybrids with adaptor
sequences on only one terminus. The PCR following the subtractive
hybridization step led to the exponential amplification of
self-reannealed tester fragments. MS-RDA required several cycles of
subtractive hybridization and PCR to enrich differentially methylated
DNA fragments from the tester cell lines, as the method did not take
into account the large differences in relative abundance of individual
DNA sequences.
MS-AS used a normalization step and a special form of PCR to adjust for
the sequence abundance in the tester amplicon. A detailed protocol for
the MS-AS method has been described elsewhere (21). All differentially
methylated DNA fragments derived from both low abundance and
repetitive genomic sequences were equally enriched by MS-AS. In
addition, the number of false-positive fragments of repetitive DNA
fragments was reduced by the internal normalization step of the MS-AS protocol.
Individual DNA clones, which were identified by DNA array hybridization
(data not shown), were isolated and used as 32P-labeled
hybridization probes with DNA from the nontransgenic BHK21 cells, the
Ad12-transformed BHK21 cell line T637, or the
DNA-transgenic BHK21
cell lines L12 or L18. These DNAs had all been cleaved with
MspI (M), HpaII (Hp), or
HhaI (Hh) prior to electrophoresis and Southern
transfer. The data presented in Fig. 3,
A (T637) and B (L12, L18), demonstrate the
differences in cleavage patterns for the methylation-sensitive
restriction endonucleases HpaII and HhaI between
the DNAs from the nontransgenic BHK21 cells and the Ad12 DNA (T637,
Fig. 3A)- or
DNA-transgenic cell lines (L12, L18, Fig.
3B). The probe numbers refer to individual clones isolated
by MS-AS (Fig. 3A, probes t3, t4, t7, R-1, R-3,
R-4) or MS-RDA (Fig. 3B, probes 18, 79, 80, 32, 44). The data base homologies and gene assignments identified by
the nucleotide sequences of the probes characterized in Fig.
3A are listed in Table II. Probe 44 used in the L18 experiment of Fig. 3B corresponds
to intracisternal A particle (IAP) DNA that had been
analyzed previously in a different set of
DNA-transgenic cell lines
using different methods (4, 5). All of the differentially methylated
clones identified by the MS-RDA technique and the IAP
sequences (24) selected by the MS-AS method represented repetitive
sequences in the hamster genome. Most of the repetitive sequences with
altered methylation patterns are most likely not linked to the
insertion site of the transgene, but are located in
trans on different chromosomes. When the DNAs from nontransgenic
single cell-cloned BHK21 isolates were analyzed using the same probes
and methods, no differences in HpaII or HhaI
cleavage patterns were observed (data not shown).

View larger version (117K):
[in this window]
[in a new window]
|
Fig. 3.
A, detection of de novo
methylated cellular genes and DNA segments in the Ad12-transgenic BHK21
cell line T637 by applying the method of MS-AS is shown. The DNA (5 µg) extracted from BHK21 or T637 cells was cleaved with
HpaII (Hp), HhaI (Hh), or
MspI (M), and the fragments were separated by
electrophoresis on a 1% agarose gel. The DNA was then transferred by
Southern blotting to a GeneScreen Plus membrane and hybridized to
difference products isolated after one round of MS-AS (for details, see
"Experimental Procedures"). Most of the six differentially
methylated clones (t3, t4, t7, R-1, R-3, R-4) were
identified by their nucleotide sequence homologies to known genes (see
Table II). B, an increase in DNA methylation of cellular DNA
segments in the DNA-transgenic BHK21 cell line L12 or L18 is shown.
By applying the methylation-sensitive representational analysis to
HpaII cleaved genomic DNA from the nontransgenic BHK21 cell
line compared with the DNA-transgenic BHK21 cell line L12 or L18,
several differentially methylated DNA segments were isolated.
Experimental procedures for the Southern blot experiments were similar
to those described in A. The open
arrowheads indicate differences in the HpaII cleavage
patterns between the DNAs from cell lines BHK21 and L12 when clone18
DNA was used as a hybridization probe. The closed arrowheads
show the differences in the HpaII cleavage patterns between
cell lines BHK21 and L18 using the hybridization probe 44.
|
|
We conclude that the selection methods used to identify
differentially methylated DNA sequences in Ad12 DNA- or
DNA-transgenic BHK21 cell lines yield a spectrum of DNA sequences in
which the methylation patterns have been altered as a consequence of
foreign DNA insertion into the hamster cell genome.
Alterations in Levels of DNA Methylation in the DNA from Transgenic
Mice--
The B6D2F1 mouse strains 7-1 and 8-1 carried a
transgene construct that contained the adenovirus type 2 E2A
late promoter controlling the chloramphenicol acetyl transerase gene
(pAd2E2AL-CAT) (8). Other mouse strains (a gift of Klaus Schughart,
Strasbourg, France) were transgenic for the DNA of bacteriophage
DNA. We investigated about 10 different known mouse genes as
32P-labeled hybridization probes with DNA from
nontransgenic or transgenic mice that had been cut with
HpaII or MspI. For IL-10 and
Igf2r (insulin-like growth factor 2 receptor, an
imprinted gene), differences in the cleavage patterns of
HpaII were observed (Fig. 4,
A-C).

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 4.
Changes in DNA methylation patterns in
transgenic mouse strains. A, loss of methylation in the
IL-10 and Igf2r genes in
pAd2E2AL-CAT-transgenic mouse line 8-1. Genomic liver DNA (20 µg)
extracted from male animals of the transgenic mouse line 8-1 or from
male nontransgenic B6D2F1 (hybrid C57BL/6 × DBA/2)
animals with the same genetic background was cleaved with
BamHI only ( ) or in combination with HpaII
BamHI/Hp) or MspI (BamHI/M).
Experimental procedures for the Southern blot experiments were carried
out as described in the legend to Fig. 3A. The open
arrowheads indicate increases in relative signal intensities in
the BamHI/HpaII cleavage patterns of
IL-10 and Igf2r in the DNA of the
transgenic mouse line 8-1 compared with the nontransgenic
B6D2F1 control animals. Increases in signal intensities of
smaller fragments are due to the loss of methylation at these loci in
transgenic animals. After transfer of the genomic DNA fragments, the
DNA was hybridized to 12 different known gene probes. The results of
three of these hybridization experiments with the same membrane were
shown as examples. B and C, loss of methylation
in the IL-10 (B) and Igf2r
(C) loci in transgenic animals of the pAd2E2AL-CAT
transgenic 7-1 mouse line. Liver DNA (20 µg) from male transgenic 7-1 mice and from nontransgenic control animals with the same genetic
background (B6D2F1) was cleaved with
BamHI and HpaII. Further experimental procedures
for the Southern blot experiments (12) were similar to those described
in A. Loss of methylation in the IL-10 and
Igf2r loci as evaluated by phosphorimager
analyses of the fragment patterns ranged from 12 to more than
25%. The observed changes in DNA methylation were tissue-specific for
liver DNA. D, hypermethylation in the
Igf2r locus of DNA-transgenic mice. DNA extracted
from heart muscle of DNA-transgenic CD-1 animals or from
nontransgenic CD-1 animals of the same litter was cleaved with
HpaII or MspI. As hybridization probe, a
3.0-kilobase pair fragment of the imprinted
Igf2r locus (23) or of several other cellular genes
(here the 5-HT1C gene probe was shown as an example) were
used. Both male founder animals (filled squares) showed
hypermethylation specifically in the Igf2r locus of
heart muscle DNA as compared with nontransgenic litter mates. No
differences were apparent with the 5-HT1C hybridization
probe. The arrowheads indicate differences in
HpaII cleavage patterns between transgenic and nontransgenic
litter mates.
|
|
The IL-10 and Igf2r loci mapping to
different chromosomes were hypomethylated in liver DNA from the
transgenic animals 8-1 (Fig. 4A) and 7-1 (Fig. 4,
B and C). For the transgenic mouse line 7-1, the
BamHI/HpaII fragment patterns in the
IL-10 (Fig. 4B) and Igf2r (Fig.
4C) genes were determined. Both genes exhibited partial
methylation phenotypes (methylation mosaicisms). The relative signal intensities in each lane reflected the degree of methylation at
a given site. The results of phosphorimager analyses of the decrease in
DNA methylation at the IL-10 and Igf2r
loci in the transgenic mouse line 7-1 documented the loss of
methylation to a range between 11 and 25% (Tables
III and IV). The photostimulated luminescence (PSL) of each fragment band
was measured (Fig. 4, B and C) and the percentage
relative to the sum of total intensity/lane (relative signal intensity,
%PSL) was calculated for the BamHI/HpaII cleavage patterns of IL-10 (Table III) and
Igf2r (Table IV). The marker in Fig. 4B indicates the positions of fragment
bands included in the phosphorimager analyses. The standard deviations
of signal intensities for BamHI/HpaII DNA
fragments from nontransgenic control mice were always below 1% (Tables
III, IV). Numerous control hybridization probes, e.g.
pBE1, a glycosylase gene (Fig. 4A), revealed no
changes in DNA methylation at these sites on the same membranes.
View this table:
[in this window]
[in a new window]
|
Table III
Phosphorimager analyses of the IL-10 BamHI/HpaII cleavage patterns
, measured PSL yielded background values; %PSL, percentage of signal
intensity of a specific fragment band as compared with the total signal
intensity in each lane; kb, kilobase pair.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Phosphorimager analyses of the Igf2r BamHI/Hpall cleavage
patterns
%PSL, percentage of signal intensity of a specific fragment band as
compared with the total signal intensity in each lane; kb, kilobase
pair.
|
|
When BamHI/HpaII-cut DNA from nontransgenic
control mice from the B6D2F1 hybrid mouse strain or from the parental
C57BL/6 or DBA/2 strain was probed with 32P-labeled
IL-10 or
Igf2r DNA, identical cleavage patterns were observed
(Fig. 5). These data demonstrate that the methylation patterns
in the IL-10 and Igf2r genes do not vary
when comparisons are made between different control animals or between
any of the three mouse strains. The significant changes in DNA
methylation at the IL-10 and Igf2r loci
seem to be specific for the transgenic animals of the 8-1 and 7-1 lines. Other transgenic mouse lines, e.g. lines 6-2 and 5-8, which carried the same transgene construct, albeit at different loci
(8), showed no alterations in the IL-10 and
Igf2r methylation patterns (data not shown). Because IL-10 and Igf2r are located on different
mouse chromosomes, we reason that at least one of the genes with
altered DNA methylation is located in trans to the transgene
insertion site.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 5.
Methylation status of the IL-10
and Igf2r loci in nontransgenic control
animals. Liver DNA (20 µg) from nontransgenic control animals of
the DBA/2, C57BL/6, or hybrid B6D2F1 mouse strains was
cleaved with BamHI and HpaII. All experimental
procedures for the Southern blot experiments and the abbreviations used
were similar to those described in the legend to Fig. 4.
|
|
In two additional mouse strains transgenic for the DNA of bacteriophage
, strains 17 and 20, hypermethylation of the Igf2r gene was observed in the DNA from heart muscle (Fig. 4D).
The founder animals (filled squares in Fig. 4D)
of the
DNA-transgenic mouse lines showed increased methylation at
the Igf2r site as compared with the same site in
their nontransgenic litter mates (open squares in Fig.
4D). Differences in HpaII cleavage patterns are
indicated by black arrows. Numerous other gene probes,
e.g. 5-HT1C, revealed no changes in DNA
methylation at these sites (Fig. 4D, lower panel). The
MS-RDA analysis of
DNA-transgenic founder animals compared with
nontransgenic animals did not lead to the isolation of aberrantly
methylated genomic DNA fragments. Instead, DNA segments with
restriction fragment length polymorphisms in HpaII
sites were isolated irrespective of the organs from which the DNA was prepared.
 |
DISCUSSION |
The integration of foreign DNA into established mammalian
genomes can be considered a frequent event. Virus infection,
microinjection, or transfection of foreign DNA can lead to the
permanent insertion of foreign DNA into the recipient genome. It is
unknown how frequently and by what exact mechanism this insertional
recombination proceeds. It is not unusual for multiple copies of the
foreign DNA to be inserted at a single site. Hence, arrays of foreign
DNA comprising a total sequence of up to 1 megabase pair and more can
be added to mammalian genomes. At present, information is lacking as to what extent this megabase acquisition of foreign genetic material can
lead to perturbations of the chromatin structure at or remote from the
sites of insertion. There is evidence that in the nucleus of the cell
individual chromosomes are neighboring one another in a unique spatial
relationship (25-27). Thus, structural alterations at one site on one
chromosome might be transmitted to and affect the structure and
function of loci on adjacent chromosomes, i.e. in
trans, hence over considerable genetic distances.
Can these structural and functional consequences of foreign DNA
insertion be assessed by current technology? In the present communication and in previous work from our laboratory (4, 5), we have
analyzed hamster cells transgenic for and transformed by Ad12 DNA or
transgenic for the DNA of bacteriophage
DNA as well as mice
transgenic for the latter DNA. By applying several independent
subtractive hybridization protocols previously not used in the analyses
of methylation and transcription patterns in transgenic cell lines or
animals, we were able to expand this analysis to a genome-wide survey
and demonstrate marked changes in transcription and methylation
patterns in cells that carried integrated Ad12 or
DNA. Several of
the clones with aberrant methylation patterns in the transgenic cell
lines were derived from repetitive DNA sequences in the genome
irrespective of the techniques applied, either MS-RDA or MS-AS. This
finding either reflects a bias of these PCR-based techniques for highly
abundant DNA templates or indicates that repetitive sequences are
particularly prone to undergoing changes in DNA methylation when
foreign DNA is inserted into an established genome. In several
independent experiments using either the MS-RDA or the MS-AS method, a
specific CpG-rich region from the endogenous IAP
genomes was cloned and found to be hypermethylated in transgenic cells.
Control experiments, in which patterns of methylation have been
assessed by the same set of methods in a large number of subcloned lines of nontransgenic BHK21 cells have never shown differences in
methylation or transcription patterns among individual BHK21 cell
clones for the probes tested (see also Ref. 5). Hence, we have not
pursued the possibility that the BHK21 cell population could be mosaic
with respect to methylation or transcription patterns. Similar
control experiments performed with the DNA from three different mouse
strains showed no differences in the HpaII cleavage patterns
with the IL-10 and Igf2r probes (Fig.
5).
As the integrated
DNA is not detectably transcribed in the
transgenic BHK21 cells, the possibility that products of the integrated
foreign DNA might be involved in the observed alterations is
unlikely. The pSV2neo gene, usually present as a
cointegrate with
DNA, has usually been silenced in the
DNA-transgenic cell lines, since G418 selection had been discontinued.
Several of the
DNA- and pSV2neo-transgenic cells, which
still expressed pSV2neo, did not show alterations in
transcription and methylation patterns. In contrast, in the
Ad12-transformed hamster cell line T637 and Ad12 genes are
transcribed, and their products may contribute to the induction of
changes in transcription and methylation patterns (4). However,
revertants of the T637 cell line devoid of Ad12 genomes,
e.g. TR3 or Ad12 DNA-transgenic cell lines (e.g.
H-Ad12neo2/5), which do not express the integrated Ad12 DNA, still
exhibit the marked alterations in methylation patterns (4). On the
other hand, analyses of a different Ad12-transformed hamster cell line, A2497-3, or of Ad12-infected BHK21 cells, both of which express parts
of the Ad12 genome, show no altered methylation (4). Thus, factors
other than transgene transcription and/or viral gene products must be
decisive in eliciting alterations in cellular DNA methylation and
transcription patterns.
It is conceivable that the selection of combinations of genes and DNA
segments with altered methylation and transcription patterns depends on
the sites of foreign DNA insertion and on the loci adjacent to these
sites. This selection may also be subject to possible structural and
functional effects. In this context, it will be important to
investigate whether the insertion of foreign DNA per
se can contribute to the mechanism of oncogenesis in
virus-induced and/or "naturally occurring malignancies."
Of course, the observation that the insertion of foreign DNA into
the mammalian (hamster, mouse) genome can have structural and
functional consequences for the target cell will be of interest for the
interpretation of experiments in which regimens have been applied that
lead to foreign DNA insertion, such as in gene transfer studies,
transgenic animals (knock-out, knock-in), and human (somatic) gene
therapy. The interpretation of the results adduced with these methods
may be complicated.