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Journal of Virology, September 2000, p. 8382-8389, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Relationship between Retroviral DNA Integration and
Gene Expression
Joanne Barnes
Weidhaas,
Elizabeth Lloyd
Angelichio,
Sabine
Fenner, and
John M.
Coffin*
Department of Molecular Biology and
Microbiology, Tufts University, Boston, Massachusetts 02111
Received 2 March 2000/Accepted 16 June 2000
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ABSTRACT |
Although retroviruses can integrate their DNA into a large number
of sites in the host genome, factors controlling the specificity of
integration remain controversial and poorly understood. To assess the
effects of transcriptional activity on integration in vivo, we created
quail cell clones containing a construct with a minigene
cassette, whose expression is controlled by the papilloma virus E2
protein. From these clones we derived transcriptionally active
subclones expressing the wild-type E2 protein and transcriptionally silent subclones expressing a mutant E2 protein that binds its target
DNA but is unable to activate transcription. By infecting both clones
and subclones with avian leukosis virus and using a PCR-based assay to
determine viral DNA integration patterns, we were able to assess the
effects of both protein binding and transcriptional activity on
retroviral DNA integration. Contrary to the hypothesis that
transcriptional activity enhances integration, we found an overall
decrease in integration into our gene cassette in subclones expressing
the wild-type E2 protein. We also found a decrease in integration into
our gene cassette in subclones expressing the mutant E2 protein, but
only into the protein binding region. Based on these findings, we
propose that transcriptionally active DNA is not a preferred target for
retroviral integration and that transcriptional activity may in fact be
correlated with a decrease in integration.
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INTRODUCTION |
Integration, or the insertion of a
double-stranded DNA copy of the viral genome into the hosts' genomic
DNA, is a central event in the retrovirus life cycle. While the DNA
breaking and joining reactions mediating integration are biochemically
well understood (5, 6, 7, 9, 10, 18), the determinants of
retroviral integration site selection have been difficult to elucidate.
In vitro integration systems have provided a powerful tool with which
to study the determinants of integration site preferences on the DNA
level. These assays have shown that hot spots for integration can be
created by changes in local DNA structure, such as by the methylation
of a run of alternating CpG dinucleotides (17) or by the
creation of nucleosome-associated regions of DNA in minichromosomal DNA
(26, 27). Favored integration sites in nucleosome-associated regions were shown to be due to DNA bending (24), with the
most distorted sites within the nucleosome core being the most
preferred for integration (25). Consistent with this idea,
several DNA binding proteins known to create sharp bends in their
target DNA, such as the Escherichia coli integration host
factor, also create hot spots for integration within their binding site
regions (3). By contrast, the binding of some other DNA
binding proteins, such as bacterial transcriptional repressors, have
been shown to suppress integration in the vicinity of their binding
sites (28). Despite the wealth of information from in vitro
systems, the effect of DNA binding proteins on integration into
chromosomal DNA has never been determined.
Attempts to study integration in vivo have been difficult due to the
scarcity of integration events in the large mammalian genome. Early in
vivo studies with murine leukemia virus and avian sarcoma-leukosis
virus found that integration was not sequence specific and that a large
number of sites in the host genome could serve as integration targets
(5, 39). Other in vivo studies have suggested a specificity
in target site selection for certain regions of the chromosome, such as
those that are transcriptionally active (31) or those
associated with other features, such as DNase I hypersensitivity
(11, 29, 30, 40). All of these early in vivo studies
suffered from potential biases such as small sample sizes, the
isolation of stably integrated proviruses, and the selection of cloned
proviruses. A system was designed in our laboratory that enabled study
of large numbers of integration events by using a virus with a
selectable marker and creating libraries of clones with provirus
together with host flanking sequences. Analysis of these libraries
found a small number of highly preferred sites for integration
(33). However, recent work by Carteau et al. studying
integration site libraries from human immunodeficiency virus-infected
cells found no evidence for highly preferred sites or for any increase
in the efficiency of integration near transcriptionally active DNA
(8).
Most recently, a PCR-based assay was developed in our laboratory that
enabled study of integration into newly infected cells and avoided any
possible biasing of observed results through cloning (42).
This assay was sensitive enough to detect a single integration event
within a population of 5 million cells, enabling the study of a large
pool of unselected integration events simultaneously. Initially, the
assay was used to study integration into 11 randomly chosen regions of
the avian genome. It was found that while all of the regions tested
were used for retroviral integration at a frequency not significantly
different from that expected for random, certain nucleotide positions
within these regions were used at up to 280-fold more than random
frequency. We hypothesized from these findings that while all or most
regions of the genome were accessible for integration, strong
integration site preferences could be determined at the local DNA
level. These initial studies were unable to determine what role, if
any, transcriptional activity of target DNA or protein binding had on
retroviral integration.
In this report, we describe a study in which the primary goal was to
determine and separate the roles of transcriptional activity and
protein binding on retroviral integration in vivo. Our strategy was to
establish cell lines carrying a minigene, the expression of which
could be regulated by the presence or absence of an appropriate transcriptional regulator, the bovine papillomavirus (BPV) E2 protein,
and to then monitor integration patterns into this minigene as a
function of the level of E2-stimulated transcriptional activity. In
addition, a mutant of E2 that could bind its target DNA without activating transcription allowed us to separate the effects of protein
binding from transcriptional activity on retroviral integration.
We found that E2-mediated activation of the transcription of our
minigene led to an overall decrease in integration events both
within the E2 binding region and within the actively transcribed gene.
In contrast, expression of the mutant E2 protein led to a decrease in
integration only into the regulator's binding region, with no change
in integration frequency within the untranscribed gene. In agreement
with earlier in vitro work, our findings show that protein binding in
vivo can suppress integration in the vicinity of protein binding sites.
However, contrary to earlier predictions, our findings also suggest
that transcriptional activity is not associated with increased
retroviral integration and in fact might be associated instead with a
decrease in integration frequency in vivo.
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MATERIALS AND METHODS |
Cells and virus.
The QT6 cell line used in this study was
originally derived from chemically induced tumors of Japanese quail
(22). The virus used, RAV-1, is a replication-competent
exogenous member of the avian sarcoma-leukosis virus genus which was
rescued from a molecular clone (32).
Plasmids.
pMJG1, a kind gift from M. Grossel, was derived
from pTKGH (Allegro Scientific) by the insertion of three E2 binding
sites between the NdeI and HindIII sites in
the multiple cloning site of pTKGH (1). Plasmids pCGE2 and
pCGE2 340-G (4) are derivatives of pBluescript (Stratagene)
and pSP65 (Promega). They both contain a cytomegalovirus (CMV)-driven
version of the entire 410-amino-acid E2 protein and are identical
except for a single C-to-G amino acid change at position 340. Plasmid
pCB60-95 (2), a kind gift from J. A. T. Young,
contains the neomycin gene cassette and was used for cotransfection and
selection with pMJG1. Plasmid pME18SHyg B contains the hygromycin gene
cassette and was used for cotransfection and selection with the pCGE2 plasmids.
Derivation of QT6 clones and subclones.
Clones and subclones
were derived from QT6 cells by transfection with the plasmids described
above using the Lipofectamine method (Gibco BRL). For each
transfection, 2.7 µg of the desired construct and 0.3 µg of the
selectable DNA were incubated in 300 µl of Lipofectamine for 45 min.
A 2.7-ml amount of serum-free Dulbecco's modified minimal essential
medium was then added, and this mixture was placed on the cells for
22 h before serum-containing medium was added. Clones were
selected with neomycin (300 to 500 µg/ml) and subclones were selected
with hygromycin B (200 to 400 µg/ml). Appropriate clones and
subclones were expanded and infected with RAV-1 as described below.
Southern analysis of clones and subclones.
Genomic DNA was
digested with restriction enzymes, transferred to nylon membranes, and
hybridized to a random-primer labeled probe derived from pMJG1
according to standard protocols (21). Three different sets
of enzymes were used to ensure that the entire plasmid was present and
to determine the copy number. The first digestion was with
XmnI and SacI to ensure that the upstream portion of the plasmid was present, the second was with EcoRI to
ensure that the downstream end was present, and the third was with
SacI alone to determine the copy number.
Detection of the E2 protein.
The E2 protein was detected by
Western blotting using an ECL (enhanced chemiluminescence) kit
(Amersham). The membrane (NEN) containing the samples of interest was
rocked for 1 h with the primary antibody, a monoclonal mouse
antibody to the E2 protein (B202; a kind gift from D. Breiding), which
was diluted 400-fold in 5 ml of Tris-buffered saline-0.2% Tween plus
2.5% milk. Next, the secondary antibody, a horseradish
peroxidase-labeled mouse antibody that binds to the primary antibody,
was added to the membrane at a 1,000-fold dilution in 5 ml of the same
solution. The membrane was rocked for 1 h at room temperature and
washed according to the manufacturer's instructions. The results were visualized by autoradiography.
Infection of cells.
Large amounts of infectious virus were
produced by first infecting a plate of QT6 cells with 1 ml of frozen
RAV-1 stock plus 1× Polybrene (15 µg/ml). The primary infected plate
was expanded, and supernatants were monitored for the level of reverse
transcriptase activity. When infected cells were efficiently producing
virus (two to three passages) and were almost confluent, the medium was
replaced with 8 to 9 ml of fresh medium. Supernatant from these
cultures was collected 16 to 18 h later, filtered through a
0.22-µm pore size filter, and immediately used to infect the test
cells. The cells to be infected were plated at a density of 2 × 106 per 100-mm-diameter culture dish in 11 ml of medium 16 to 18 h before infection. They were subjected to three rounds of
infection as follows. The medium was removed immediately before
infection, and 2 ml of RAV-1-containing supernatant with 1× Polybrene
was added to the cells for 45 min. At the end of this incubation, 8 ml
of regular uninfected growth medium was added for 45 min. The second
round of infection was identical to the first, and the third differed
in that no Polybrene was added to the viral supernatant before it was
added to the cells. To minimize selection for or against cells with
specific integration sites as well as minimize reduplication of
integration events by cell division, the genomic DNA for study was
collected 2 to 3 days after infection by standard procedures
(21).
Immunoassay.
Production of the human growth hormone (hGH)
protein by cells was first assayed using an immunological assay
(hGH-Transient Gene Expression System kit; Nichols Institute
Diagnostics), which used two antibodies to the hGH protein, one of
which was avidin labeled and one of which was labeled with
125I. Serum from the cells was first combined with the two
antibodies, and then a biotin-coated bead was added. This mixture was
rocked for 4 h at room temperature. The beads were washed and then
counted in a gamma counter, and the amount of hGH per sample was determined.
RNase protection assay.
Total cellular RNA from clones and
subclones was isolated using an RNeasy kit (Qiagen) according to the
manufacturer's recommendations. RNA was then eluted in water, treated
with DNase I, and repurified using the RNeasy kit prior to
A260/280 determination. RNA was divided into appropriate
amounts and frozen at 
70°C until use.
A [32P]UTP-labeled antisense riboprobe was generated from
pBluescript containing a 110-bp fragment from the fourth exon of the hGH gene by using a Riboprobe Systems kit (Promega) according to the
manufacturer's recommendations. Threefold dilutions of cellular RNA in
the range of 1.5 to 22.5 µg (brought to equal total RNA levels with
tRNA) were hybridized to the riboprobe (50,000 cpm/sample) for 16 h at 45°C. The samples were treated with RNases A (4 µg/ml) and
T1 (11 U/ml) for 45 min at 30°C and then with a sodium
dodecyl sulfate (SDS)-proteinase K solution for 30 min at 37°C.
Samples were then extracted with phenol-chloroform, ethanol precipitated, resuspended in 95% formamide loading buffer, preheated, and loaded onto a 5% polyacrylamide gel containing 8 M urea. Gels were
analyzed with a Storm PhosphorImager (Molecular Dynamics). Bands were
quantitated using Imagequant software (Molecular Dynamics).
PCR assay to detect in vivo integration.
PCRs were performed
using 20 µg of the infected cell genomic DNA, an amount equivalent to
approximately 107 cells. At our estimated multiplicity of
infection of two to three proviruses per cell, we predicted
approximately 2.5 × 107 integration events in every
20 µg of DNA analyzed per PCR. Since the haploid genome is
109 bp, and we were examining approximately 200-bp regions
in each experiment, we expected to see only two to three integration
events per copy of the target DNA in each region analyzed by PCR.
The PCRs for analysis of integration site distribution within a given
region were prepared as follows. Genomic DNA isolated
from infected or
uninfected QT6 clones and subclones was diluted
to 1 µg/µl, heated
at 100°C for 5 min, and then placed in a 80°C
heating block. Twenty
microliters (20 µg) of the DNA was added
to 50 µl of a reaction
mixture (10 mM Tris-HCl [pH 8.3], 3 mM
MgCl
2, 50 mM KCl,
0.01% gelatin, 411 µM each deoxynucleoside triphosphate,
0.6 µM
each primer [DNA specific and virus specific]) and 3.75
U of
Taq polymerase (AmpliTaq; Cetus-Perkin Elmer), overlaid with
50 µl of mineral oil, and prewarmed to 80°C for 5 min. The reaction
mixtures were transferred directly into a PCR machine preheated
to
80°C, heated to 94°C for 5 min, and then amplified for 29 cycles
at
94°C for 1 min, 69°C for 1.5 min, and 72°C for 2 min. For the
final step in the last cycle, the samples were heated to 72°C
for 3 min. The entire PCR mixture was then purified using a QIAquick
PCR
purification kit (Qiagen) according to the manufacturer's
directions
and elution in a final volume of 50 µl of 10 mM Tris-HCl
(pH 8.3).
PCR products were visualized by extension of an end-labeled primer. Ten
microliters of each purified PCR product was dried
and annealed with
approximately 0.2 pmol of an internally nested
-
32P-labeled primer (10
6 counts per
reaction) in 1× reaction buffer (40 mM Tris-HCl [pH
7.5], 20 mM
MgCl
2, 50 mM NaCl). Extension was carried out for
30 min at
42°C. Samples were analyzed on a prewarmed 6% polyacrylamide
denaturing gel under standard conditions, with sequencing ladders
derived from each region run in parallel to provide size standards.
These gels were then dried for 30 min and exposed to a
PhosphorImager
screen
overnight.
Analysis of integration events.
Analysis was performed using
the Imagequant software in conjunction with a Storm PhosphorImager,
both from Molecular Dynamics. Band intensity was determined by
densitometry, and the total number of integration events per region was
calculated. For each region, the same number of PCRs was analyzed for
clones and subclones to compare the number of integration events.
Oligonucleotides.
Oligonucleotides used in this study (Table
1) were selected using the PRIMER version
0.5 program (20), which selects primer pairs compatible with
specific reaction component concentrations and annealing temperatures.
Primers were synthesized and purified by M. Berne (Tufts University).
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RESULTS |
Experimental design.
We chose to use the BPV E2 protein as our
transcriptional regulator, since it requires only a simple promoter to
activate downstream transcription, and there are known E2 mutants that bind their target DNA without activating transcription (12). The E2 protein binds as a dimer to its target sequence in DNA and when
bound to multiple binding sites can enhance downstream transcription
(13, 23, 34, 35). E2-mediated transcriptional enhancement is
believed to require interaction with at least one additional cellular
factor, such as Sp1 (19, 38), which is thought to assist in
the recruitment of TFIID to the promoter site (14). The
mutant E2 protein (E2 340-G) was shown to be identical to the wild-type
(wt) E2 protein in that it is DNA binding competent, dimeric, and
localized to the nucleus but unable to support transcription
(12).
Clones of QT6 cells were created by transfection with plasmid pMJG1
(
12), consisting of a minigene cassette with the hGH
cDNA sequence under the control of an E2-dependent transcription
control element (Fig.
1). The hGH protein
is a useful reporter
because its mRNA is quite stable, it is secreted
by the transfected
cells into the medium, and it can be measured using
a simple immunological
assay.
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FIG. 1.
The E2-driven expression system. Plasmid pMJG1 was used
to create clones by transfection of QT6 cells. The plasmid contains
three E2 binding sites, followed by a TATA box, two Sp1 binding sites,
the herpes simplex virus TK gene promoter, and the hGH coding region,
as indicated. The arrow above the expanded region shows location of the
transcription start site. The TK gene promoter cap site and hGH
translation initiation site are depicted with arrows below the line.
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Subclones were derived from clones following transfection with plasmids
expressing either the wt or mutant E2 protein under
control of the CMV
early gene promoter (
4). Transcriptionally
active subclones
(wt subclones) with at least a threefold induction
of hGH as determined
by the immunoassay were successfully isolated
from two different clones
(clones 1 and 2 [Table
2]); a subclone
containing the mutant E2 protein (mutant subclone) was also isolated
from clone 2. The presence and copy number of pMJG1 were confirmed
to
be identical by Southern analysis between clones and subclones
used in
this study (data not shown), with three copies of the
plasmid in clone
1 and nine in clone 2. Some of the copies of
pMJG1 in clone 2 appeared
to be in tandem. The presence of the
E2 protein in subclones was
confirmed by Western analysis (Fig.
2).
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FIG. 2.
Expression of E2 protein in subclones. Extracts from
clones and subclones transfected with the wt or mutant E2 expression
construct were analyzed by SDS-polyacrylamide gel electrophoresis
followed by Western blotting and ECL detection of E2 protein using a
monoclonal antibody (B202) for the BPV E2 protein that binds to both wt
and mutant E2 protein (both 42 kDa).
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To confirm and better assess the level of transcriptional activity in
our subclones, in addition to the immunological assay
we used an RNase
protection assay to directly quantitate levels
of hGH mRNA. From
analysis of three separate experiments, we found
by densitometry that
both wt subclones had an approximate five-
to sevenfold induction of
hGH expression, and our mutant subclone
had approximately twice the
level of the hGH RNA expression as
did its parent clone (Fig.
3). These findings were similar to
the
results which we found using our immunologic assay and confirm
that
there is a direct increase in transcription of our hGH reporter
in the
presence of the wt E2 protein.
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FIG. 3.
Expression of hGH RNA in clones and subclones. Total RNA
from clone 1 (A) and clone 2 (B) and their E2-expressing subclones was
extracted and annealed with a 32P-labeled riboprobe. The
protected fragment after RNase digestion was analyzed by polyacrylamide
gel electrophoresis. Levels of cell RNA used were 2.5, 7.5, and 22.5 µg for clone 1 and its subclone and 1.5, 4.5, and 13.5 µg for clone
2 and its subclones. Gels were analyzed with a PhosphorImager. M, size
markers.
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Detection of integration events.
To determine patterns and
frequencies of integration into the hGH gene, we isolated DNA from the
clones and subclones 2 to 3 days after infection and subjected it to
the PCR assay previously described by Withers-Ward et al.
(42) (Fig. 4). This assay is exquisitely sensitive, enabling detection of a single molecule resulting from a specific integration event against a background of
millions of events at other sites. PCR was performed with a primer
complementary to one of five sites within pMJG1 and a primer complementary to the viral long terminal repeat. An example of integration patterns obtained using this assay (Fig.
5) shows the results of integration into
the JB-10 region of clone 2 (left) and into its wt subclone (right).
The patterns shown were derived from replicate PCR amplifications of
three independent infection experiments. Each band represents a single
PCR-amplified radiolabeled integration event from one infected cell,
with a darker intensity indicating multiple integration events at the
same location (i.e., from different cells in the pool). As observed
previously (42), the integration patterns are highly
nonrandom, with some sites used quite frequently and others not at all.
The use of many of the same sites for integration in multiple analyses
from separate infection experiments, as indicated by arrows, supports
the conclusion that these sites represent local hot spots for
integration, not fortuitous reduplication by cell division. As is
clearly visible in Fig. 5, while some hot spots remain unchanged
between the parent clone and its wt subclone, there were significantly
fewer integration events in the presence of the wt E2 protein than in
its absence. There were also distinct changes in the distribution of
integration sites when E2 was present, discussed in detail below.
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FIG. 4.
Detection of integration events in clones and subclones.
Clones and subclones were infected with RAV-1, the genomic DNA was
collected, and PCR was performed. Two oligonucleotide primers, one
derived from a sequence in pMJG1 (JB-n) and the other from a
sequence in the viral U3 region (U3-RAV), were used to amplify
integration events. The resulting PCR products were used as templates
to extend an end-labeled primer, and the samples were separated by
polyacrylamide gel electrophoresis.
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FIG. 5.
Pattern of integration into the upstream region of
pMJG1. Parent clone 2 and its wt subclone were infected with RAV-1, and
the integration site distribution was determined by PCR with primer
JB-10 (Fig. 4). Locations of the TATA box, Sp1 sites, and transcription
start site (arrow) are shown. DNA from three different infection
experiments (1, 2, and 3) was divided into two or three samples of 20 µg each (A, B, and C) and analyzed in independent PCRs. The
double-headed arrows indicate hot spots for integration conserved
between the clone and subclone, and single-headed arrows indicate hot
spots found in only the clone or the subclone.
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We analyzed a large segment of the minigene, beginning 5' of the E2
binding sites and extending 1 kb into the hGH coding region.
For each
region we analyzed a total of three to eight PCRs from
three separate
infections and pooled the results. Comparing overlapping
results from
contiguous regions, we found that the same events
were amplified with
different primers (data not shown). To better
visualize and analyze our
results, we used PhosphorImager analysis
to quantitate the position and
intensity of the bands. Below we
present separately the results
obtained for integration events
upstream and downstream of the
transcription start
site.
Integration upstream of the transcription start site.
The
integration patterns into the area upstream of the transcription start
sites in clones 1 and 2 and their respective subclones are shown in
Fig. 6. In contrast to previous
predictions, we found that the transcriptionally active wt subclones
showed no enhancement of integration but in fact showed a noticeable
decrease in integration events into this region. In general, the
differences in integration that we observed between each parent clone
and its wt subclone were common to the two sets and were as follows:
first, a decrease in integration upstream and within the E2 binding
sites; second, a decrease in integration into the areas upstream of the
TATA box; third, a loss of integration directly into the Sp1 sites; and
finally, a decrease in integration in the region of the transcription start site.
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FIG. 6.
Effects of the E2 protein on integration into the
upstream region. The integration patterns and frequencies into the
upstream region of pMJG1 of clone 1 (A) and clone 2 (B) and their
subclones as determined by densitometry are shown as the sum of the
products of all like reactions divided by the number of reactions
analyzed. The location along the pMJG1 construct is shown with the E2
binding sites, TATA box, and Sp1 sites marked. The location in base
pairs is shown on the abscissa, with 0 representing the transcription
start site. The right half of the pattern in panel B is derived from
the gel shown in Fig. 5. mut-Subclone 2, mutant subclone 2.
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In the mutant subclone 2, integration into the upstream region had
features resembling both the wt subclones and the parent
clones. First,
in the area containing the E2 binding sites as
well as in the area
upstream of the TATA box, there was a general
decrease in integration
similar to that seen in the wt subclones.
However, in the region
containing the two Sp1 sites as well as
in the area 5' of the
transcription start site, the mutant subclone,
similar to the parent
clones, showed a heavy use of this region
for
integration.
Integration into the hGH coding region.
We next examined the
frequency of integration downstream of the transcription start site, as
shown for clone 1 and its wt subclone in Fig.
7. Again, no increase in integration
targeting resulting from transcriptional activity could be observed. In fact, there was a decrease in the amount of integration into this area
compared to the parent clone. These results were confirmed by analysis
of clone 2 and its wt subclones. By contrast, levels of integration
into the untranscribed hGH gene in the mutant subclone 2 were not
significantly different from integration levels in parent clone 2 (data
not shown).
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FIG. 7.
Integration into the transcribed region of clone 1. Results from PCR analysis of integration into the 1,000 bp downstream
of the transcription start site in DNA from infected clone 1 (A) and
its wt subclone (B) are plotted as described for Fig. 6. The results
from different primers are again normalized for comparison.
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Integration into nonregulated sequences.
Although we knew from
the Southern analysis that the number and distribution of our hGH
minigene constructs were identical among the clones and their
subclones, we also wanted to ensure that the differences in integration
frequency observed were specific for transcriptional activity and not
due to some unknown effect of the E2 protein on infectability or on
integration in general. We therefore first compared the amounts of
integrated viral DNA between clones and subclones by Southern analysis
and found that the levels of integrated viral DNA were equivalent (data
not shown), implying the absence of significant differences in all
early steps of infection.
Next, to address the issue of possible nonspecific effects on
integration more directly, we compared patterns of integration
between
clones and subclones into the glyceraldehyde-3-phosphate
dehydrogenase
(GPDH) gene, a multicopy housekeeping gene (
41).
As can be
seen in Fig.
8, the number of integration
events into
the GPDH gene did not vary significantly with expression of
the
wt or mutant E2 protein, as it did in the hGH minigene in the
same experiment.
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FIG. 8.
Integration into a housekeeping gene in clones and
subclones. The pattern and frequency of integration of RAV-1 DNA into
the GPDH gene in clone 2 and its subclones are shown. The negative
control on the left is DNA from infected QT6 cells not transfected with
pMJG1. PCR was performed using the U3-RAV and GPDH-PCR primers (Table
1). The DNA used for analysis was one sample from each of the two or
three infections. Similar results were obtained with clone 1 and its
subclones (data not shown).
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To further protect against distortion of our analysis by any minor
differences among clones and subclones, we used the number
of
integration events into the GPDH gene to normalize the relative
frequency of integration into our minigene. With these normalized
values, we were able to calculate the relative amount of integration
between the subclones and their parent clones (Fig.
9). These
calculations revealed that
integration into the upstream regions
of both clone 1 and clone 2 was
reduced by approximately 60% in
the presence of the wt E2 protein and
by approximately half that
amount in the presence of the mutant E2
protein. In the transcribed
region, in the presence of the wt E2
protein (and increased transcriptional
activity) there was a smaller
but still significant decrease in
integration, whereas in the presence
of the mutant E2 protein
there was no effect on integration.
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FIG. 9.
Summary of integration into pMJG1. The integration
events were normalized for each type of infected clone by dividing the
number of events into the indicated regions of pMJG1 by the number of
integration events into GPDH. The standard errors were calculated based
on the differences in the relative frequency of integration among
regions amplified with separate PCR primers.
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DISCUSSION |
Transcriptionally active DNA has long been hypothesized to be
preferred for integration (30, 31, 40), based on studies of
small numbers of selected events and the logic that transcriptionally active DNA would provide a more suitable environment for expression of
the integrated DNA provirus. The experiments described here were
designed to test this hypothesis by directly assessing the effects of
transcriptional activity and protein binding on retroviral integration
into chromosomal DNA. In our model system, we found that enhancement of
transcriptional activity as well as protein binding without
transcriptional stimulation did not lead to enhancement of integration.
Rather, both were associated with an overall decrease in integration
events. While we did not study the difference in integration between
DNA in heterochromatin and euchromatin in this model, our results
clearly do not support the hypothesis of a positive link between
increased transcriptional activity and targeting of integration.
The minigene system.
The model that we chose for this
study was a simple minigene construct consisting of an hGH cDNA
reporter sequence downstream of the herpes simplex virus thymidine
kinase (TK) gene promoter (which provides the cap site, TATA sequence,
and two Sp1 sites). Expression of this gene is controlled by binding of
the BPV E2 protein to an array of three binding sites upstream of the
TK gene promoter. This artificial gene system was chosen over naturally inducible sequences (such as metallotheinin or heat shock genes) because of its compactness and simplicity and out of concern that inducing agents such as heavy metal or heat might affect integration directly. It also allowed us to use a mutant E2 protein to separate effects due to protein binding from those due to transcriptional activation. Finally, its presence in multiple copies in the cell lines
tested allowed us to collect more integration events than into a
single-copy gene. Also, the presence of multiple copies of the target
sequence reduced concern over possible effects due to specific
positions. The similarity of our results in two separate sets of clones
and subclones gives strong support that our results are correct and not
due to effects of location in the genome or copy number. Thus, although
the construct is artificial, and we cannot rule out that different
interactions with specific transcriptional control elements might occur
in some genes, we are confident that our results will apply in a
general way to most or all natural genes. Direct analyses of
integration into other types of cellular sequences are under way in our laboratory.
Decrease in integration in the factor binding region.
In the
region upstream of the transcription start site, the presence of the E2
protein was associated with an overall decrease in integration events
in both the wt subclones and the mutant subclone compared to their
parental clones, although the loss was greater in the wt subclones. We
hypothesize that the loss of integration into this area reflects
binding of the transcriptional initiation complex to the DNA, which
thereby blocks accessibility to the integration machinery. Indeed,
binding of transcriptional regulatory proteins to DNA has been shown to
interfere with integration of retroviral DNA in vitro (28).
In the wt subclones, the decrease in integration included the entire
area upstream of the TATA box extending through the Sp1 sites up to the
transcription start site. In the mutant subclone, by contrast, we did
not see a loss of integration into the Sp1 sites or the region 5' of
the transcription start site. We hypothesize that the difference
between the two may reflect the inability of the mutant E2 protein to
successfully recruit the Sp1 protein, and thereby the transcriptional
initiation complex, which would also explain its failure to activate transcription.
Integration within the coding region.
The region downstream of
the transcription start site also showed a decrease in integration in
the presence of the E2 protein, but only in our wt subclones. This
effect was not seen in the mutant subclone, even though there was a
twofold increase in the basal level of hGH expression in the presence
of the mutant protein. Although proteins must bind to the DNA to induce
transcription, the absence of a decrease in integration in the mutant
subclone suggests that the decrease in integration seen in the wt
subclones was due to their transcriptional activity. The decrease in
integration observed could be due to direct interference of integration
by the transcriptional apparatus itself or might reflect indirect effects, such as displacement of nucleosomes (36, 37) and loss of associated hot spots. It could also reflect additional changes
in the conformation of the DNA with transcriptional activity not yet appreciated.
Effects of DNA structure on integration.
Changes in DNA
structure, particularly bends due to the association with nucleosomes
(27) or introduced by DNA binding proteins (3),
have been shown to introduce hot spots for integration of retroviral
DNA in vitro. Similar hot spots have also been observed in phased
chromatin-associated DNA in cells (26). We did not observe
the creation of obvious hot sites as a function of E2 binding or
transcriptional activation, although E2 is known to introduce bends
into its DNA target (23). We would propose that in vivo such
effects of individual proteins are blurred by the binding of additional
proteins and protein complexes. Our observation of decreased
integration into transcriptionally active DNA supports in vitro work
showing that nucleosomal DNA is preferred for integration, since
transcriptionally active DNA has been shown to be dynamic and involves
shifting of nucleosomes resulting in out-of-phase nucleosomes (36,
37).
Transcriptional activity and integration.
Based on the results
of this study, we consider it improbable that there is any specific
interaction of the retroviral integration apparatus, either with a
component of the transcription machinery or with a
transcription-associated change in DNA structure. A well-established
example of the former interaction is found in the case of the
retrovirus-like Ty3 element of yeast, where a specific interaction of
the preintegration complex and a polymerase III-specific transcription
factor directs integration to the upstream region of tRNA genes
(16). The finding that a protein related to a generalized
yeast transcription factor can interact with human immunodeficiency
virus type 1 integrase (15) has been taken to suggest a
similar effect for retroviruses, but the relevance of this interaction
in cells remains to be established.
These studies have provided insight into the long-standing question of
the effects of transcriptional activity on retroviral
integration. We
found that transcriptionally active DNA is not
preferred for
integration over the same DNA when it is less active.
Rather,
increasing transcription led to a decrease in integration,
most likely
due to direct or indirect blocking of integration
by components of the
transcriptional machinery. This approach
may eventually provide a
useful tool for analysis of DNA chromosomal
structure in vivo and lead
to a better understanding of the changes
in DNA structure that occur
during
transcription.
| |
ACKNOWLEDGMENTS |
We thank Elliot Androphy, Marty Grossel, and David Breiding for
supplying components of the E2 system and the E2 antibody. We also
thank John Strasswimmer for assistance with Western blot analysis of
the E2 protein and John Aschoff for guidance and materials for the
RNase protection assay.
This work was supported by grant R35 CA from the National Cancer
Institute to J.M.C. J.M.C. is American Cancer Research Professor of Molecular Biology and Microbiology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Microbiology, Tufts University, 136 Harrison
Ave., Boston, MA 02111. Phone: (617) 636-6528. Fax: (617) 636-8086. E-mail: jcoffin_par{at}opal.tufts.edu.
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Top
Abstract
Introduction
