Next Article 
Journal of Virology, March 2000, p. 2489-2501, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Nuclear Matrix Attachment Regions of Human
Papillomavirus Type 16 Repress or Activate the E6 Promoter,
Depending on the Physical State of the Viral DNA
Walter
Stünkel,
Zhonghui
Huang,
Shyh-Han
Tan,
Mark J.
O'Connor,
and
Hans-Ulrich
Bernard*
Institute of Molecular and Cell Biology,
National University of Singapore, Singapore 117609, Republic of
Singapore
Received 1 October 1999/Accepted 15 December 1999
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ABSTRACT |
Two nuclear matrix attachment regions (MARs) bracket a 550-bp
segment of the long control region (LCR) containing the epithelial cell-specific enhancer and the E6 promoter of human papillomavirus type
16 (HPV-16). One of these MARs is located in the 5' third of the LCR
(5'-LCR-MAR); the other lies within the E6 gene (E6-MAR). To study
their function, we linked these MARs in various natural or artificial
permutations to a chimeric gene consisting of the HPV-16
enhancer-promoter segment and a reporter gene. In transient transfections of HeLa cells, the presence of either of these two MARs
strongly represses reporter gene expression. In contrast to this, but
similar to the published behavior of cellular MARs, reporter gene
expression is stimulated strongly by the E6-MAR and moderately by the
5'-LCR-MAR in stable transfectants of HeLa or C33A cells. To search for
binding sites of soluble nuclear proteins which may be responsible for
repression during transient transfections, we performed electrophoretic
mobility shift assays (EMSAs) of overlapping oligonucleotides that
represented all sequences of these two MARs. Both MARs contain multiple
sites for two strongly binding proteins and weak binding sites for
additional factors. The strongest complex, with at least five binding
sites in each MAR, is generated by the CCAAT displacement factor
(CDP)/Cut, as judged by biochemical purification, by EMSAs with
competing oligonucleotides and with anti-CDP/Cut oligonucleotides, and
by mutations. CDP/Cut, a repressor that is down-regulated during differentiation, apparently represses HPV-16 transcription in undifferentiated epithelials cells and in HeLa cells, which are rich in
CDP/Cut. In analogy to poorly understood mechanisms acting on cellular
MARs, activation after physical linkage to chromosomal DNA may result
from competition between the nuclear matrix and CDP/Cut. Our
observations show that cis-responsive elements that regulate the HPV-16 E6 promoter are tightly clustered over at least 1.3 kb and occur throughout the E6 gene. HPV-16 MARs are context dependent
transcriptional enhancers, and activated expression of HPV-16 oncogenes
dependent on chromosomal integration may positively select tumorigenic
cells during the multistep etiology of cervical cancer.
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INTRODUCTION |
Human papillomavirus type 16 (HPV-16) and several related HPV types cause cancer as a consequence of
persistent infection of epithelial cells of the transformation zone of
the uterine cervix. Three oncoproteins, products of the genes E5, E6,
and E7, deregulate the cell cycle and intracellular signaling, induce immortalization, and increase mutation rates by forming complexes with
cellular factors which include p53, E6-AP, E6-BP, Rb, and paxillin (for
reviews, see references 30, 53 and
70, for recent references; 40).
The expression of E6 and E7 is controlled by a promoter upstream of the
E6 gene, which is called P97 in the case of HPV-16. It is generally
believed that the strength of transcription from P97 will determine the
concentration of the oncoproteins E6 and E7 in situ and that increases
or decreases in the concentration of these proteins will favor or
disfavor the carcinogenic process.
The activity of P97 and of the homologous E6 promoters of other genital
HPV types is under the control of about a dozen different transcription
factors (for a review, see reference 46) and the chromatin structure surrounding P97 (61). These various
factors modulate P97 in correlation with the identity of the infected epithelial cell (13, 15, 26, 27), the differentiation state
of this cell (1, 22, 50), the physiology of the host (12, 51), and viral feedback loops (17, 63). An
example of how enhanced expression of E6 and E7 may favor cellular
transformation is the transcriptional stimulation of P97 by
progesterone (12), which leads to increased transformation
in cell culture (51) and may be the molecular mechanism
underlying the epidemiological observation that the number of
parturitions a women has undergone positively correlates with the
likelihood of developing cervical cancer (56). Another
example is that P97 is repressed by a negative feedback loop exerted by
the viral E2 protein. In tumor cells, the HPV genomes have frequently
recombined with cellular DNA downstream of the E7 gene. As such a
rearrangement leads to a shutoff of E2 expression, disruption of this
negative feedback is considered to be important for progression of
neoplasia (58). Here, we report that two nuclear matrix
attachment regions (MARs) also influence P97 activity in a manner
relevant for carcinogenesis.
The cellular nucleus contains, in addition to the chromatin, a variety
of substructures, which establish spacially defined compartments with
specialized functions (8, 36, 60). One of these structural
elements has been described as the nuclear matrix based on biochemical
fractionation (7, 8), and it is apparently identical to a
ribonucleoprotein containing network of fibrils and granules identified
by electron microscopy (45). One of the functions of the
nuclear matrix is to serve as an anchor for the attachment of chromatin
loops, and such attachments are thought to isolate a loop-internal gene
from surrounding genetic elements. DNA segments with a high affinity
for the nuclear matrix are called MARs. MARs have functions beyond the
anchoring of chromatin loops, as they often occur close to
transcriptional enhancers and promoters. Most enzymatic machineries
that handle DNA and RNA associate with insoluble nuclear structures
(see references 33 and 67 and
references therein), and MARs seem to bring together
cis-responsive elements, the nuclear matrix, and its attached enzymatic machineries, topological changes that eventually would result in transcriptional modulation.
HPV-16 P97 activity is modulated by transcriptional activators and
repressors that bind a DNA segment with a size of about 550 bp, roughly
between genomic positions 7450 and 97. This DNA segment is flanked by
two MARs, one in the 5' third of the LCR, approximately from positions
7150 to 7450. The second MAR overlaps with the coding sequence of the
E6 gene (62). Our research addresses the question of whether
these MARs affect the function of the enhancer-promoter segment. In
studies of the function of cellular MARs, it has been observed that
enhancers and promoters linked to MARs can be transcriptionally
repressed in transient transfections, where DNA mostly occurs in
episomal form, but stimulated in stable clones, where the same
constructs have become integrated into the chromosomal DNA of the
recipient cell (9, 10, 14, 34, 57). We report similar
regulatory phenomena for HPV-16 MARs, have begun to unravel the
underlying molecular mechanisms, and discuss the implications of this
regulation for cervical carcinogenesis.
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MATERIALS AND METHODS |
Plasmid constructs.
For all cloning procedures we used
Escherichia coli JM109 (recA). All HPV-16
subclones were derived from the HPV-16 reference genome, subcloned into
the BamHI site of pSP65 (Promega). Contiguous segments of
the HPV-16 genome containing either the epithelial cell-specific
enhancer plus promoter (positions 7450 to 100, EP segments) or the same
segment plus the MAR in the 5' part of the LCR (5'-LCR-MAR) (positions
7150 to 100, L-EP segments) were amplified by PCR, subcloned into the
SrfI site of pCR-ScriptT-SK(+) (Stratagene, San Diego,
Calif.), and recloned in the form of KpnI-SacI
fragments into the luciferase expression vector pGL3 basic (Promega).
Additional MARs of HPV-16 were cloned in the form of
BamHI-SalI fragments into the pGL3 basic
derivatives downstream of the luciferase gene. This second MAR was
either the E5 gene including the early late intergenic region (E5-MAR,
genomic positions 3536 to 4337), the E6 gene (E6-MAR, positions 105 to
560), or the 5'-LCR (long control region)-MAR (positions 7150 to 7450).
For generation of the construct E6EP-Luc-E6, the 5'-LCR-MAR was cut out
with EcoRI and replaced by the E6 gene in the form of a
PCR-generated EcoRI fragment. All constructs are
schematically represented in Fig. 1A.
Three deletion mutants of the E6-MAR (L-EP-Luc-delmut1 to 3) were
generated by PCR (Fig. 1B). Descriptions of CCAAT displacement protein
the CDP expression vector pMT2-CDP and the parental vector pMT2
(44) have been published elsewhere.
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FIG. 1.
Schematic structures of test vectors to examine
transcription regulatory functions of three different MARs of HPV-16
(A) and deletions of the E6-MAR (B). The recombinant reporter gene
(uppermost scheme) consisted of a contiguous genomic segment of the
HPV-16 LCR with the epithelial cell-specific enhancer (E) and the E6
promoter P97 (P) fused to the luciferase gene (Luc). This reporter gene
was linked in natural or artificial sequence to the MARs in the 5' part
of the LCR (L), in the E6 gene (E6), or overlapping with the E5 gene
and the early-late intergenic region (E5). Details of the constructs
are described in Materials and Methods.
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Generation of stable cell lines.
For analyses of MAR
functions, we transfected HeLa and C33A cells. Both cell lines are
derived from cervical cancers; HeLa contains chromosome internal copies
of HPV-18 genomes, while C33A is free of HPV genomes. All cells were
grown under standard conditions in Dulbecco's modified Eagle medium
with 10% fetal calf serum. Plasmid pXJ418, which confers resistance
against the neomycin homologue G418, served as a selection marker for
stably transfected cells.
Stable transfectants were generated by electroporation of HeLa or C33A
cells with 10 µg of
XmnI-linearized HPV-16-luciferase
test vectors mixed with 1 µg of pXJ418, linearized with
BamHI.
At 48 h after transfection, the cells were split
and selection
was started by adding G418 (500 µg/ml) to the medium.
The resistant
colonies were pooled after 2 weeks of selection and
subjected
to analyses of the copy number and expression level of the
reporter
gene. To investigate stably transfected clones, individual
G418-resistant
colonies were picked and expanded. The copy number of
the reporter
gene was determined by Southern blotting after preparation
of
genomic DNA according to standard protocols. Five micrograms of
genomic DNA was blotted onto a Hybond N membrane by slot blotting.
The
blot was dried at 80°C for 2 h and hybridized against a
32P-random-primed luciferase
gene.
Transient transfection studies and luciferase assays.
HeLa
cells were transfected using the Lipofectamine reagent (Gibco-BRL)
according to the instructions of the manufacturer. Cells (2 × 105) were seeded into six-well plates in a volume of 2 ml
of Dulbecco's modified Eagle medium DMEM; 5 to 10 µg of plasmid DNA
was mixed with 15 µl of Lipofectamine reagent and adjusted to a total
volume of 200 µl with serum-free medium. After complexes had formed
during a 30-min incubation, 800 µl of serum-free medium was added,
and the samples were added to the cells. Eight hours later, the mixture was removed; after a wash with phosphate-buffered saline (PBS), new
medium containing 10% fetal calf serum was added. The cells were
harvested after 48 h by removal of the medium, washing with PBS,
and lysis in 200 µl of 1× cell lysis buffer (Promega). The plates
were rocked for 20 min at room temperature, and the lysates were
transferred to 1.5-ml Eppendorf tubes. The activity of the firefly
luciferase was measured using a Turner TD-20/20 luminometer as
instructed by the manufacturer (Promega); 10 µl of cell lysate was
mixed with 50 µl of luciferase assay substrate (Promega), and
luciferase activity was recorded as the mean of three independent transfections. As an internal standard, the cells were cotransfected with a plasmid encoding 
-galactosidase, and luciferase activity was
standardized against the activity of this enzyme.
Oligonucleotides and EMSAs.
The sequences of all synthetic
oligonucleotides are presented in Table
1. For use in electrophoretic mobility
shift assay (EMSA), approximately 50 ng of each annealed
oligonucleotide of the 5'-LCR-MAR was labeled with
[
-32P]dATP and [-32P]dCTP with Klenow
polymerase (Boehringer), while E6-MAR oligonucleotides were labeled
with [
-32P]ATP and polynucleotide kinase; 20,000 cpm
of the labeled oligonucleotides was used in an EMSA in the presence of
1 µg of poly(dI-dC) (Boehringer) (49). Samples were loaded
onto 4% polyacrylamide gels and run in 0.25× Tris-borate-EDTA buffer
for 2 h at 150 V; the gels were transferred onto Whatman blotting
paper, dried under vacuum, and autoradiographed overnight at 
80°C.
For competitions, a 100-fold excess of either specific or unspecific
nonradioactive oligonucleotide was incubated with the protein fraction
prior to the addition of the labeled oligonucleotide.
Purification of proteins by column chromatography.
To
identify any factor(s) binding to oligonucleotide 10 (5'-AATACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTAT-3') of the E6
gene (E6-10), HeLa nuclear extracts were separated by chromatographic
methods, and activities were monitored by EMSA. Nuclear extracts were
either prepared from HeLa cells (21) or purchased from the
Computer Cell Culture Center (Brussels, Belgium). Ten milliliters of
nuclear extract (protein content of 150 mg) was applied to a
heparin-Sepharose column (Pharmacia), previously equilibrated with 5 volumes of buffer D (0.1 M KCl, 20 mM HEPES [pH 7.9], 20%
[vol/vol] glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride). Subsequently, the column was washed
with 2 volumes of buffer D. Column-bound proteins were eluted with
stepwise increases of KCl concentrations in buffer D. Band shift
activity was found in the 0.3 M fraction. After dialysis of active
fractions against buffer D, we further fractionated the preparation by
ammonium sulfate precipitation. Activity was found in the 40% ammonium sulfate precipitate, as determined after overnight dialysis against buffer D. Active fractions (200 µl) were subjected to gel filtration (Sephacryl S400; Pharmacia) in the presence of buffer D, where the
activity eluted near the void volume of the column with an apparent
molecular mass of 200 kDa.
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RESULTS |
Three MARs of HPV-16 suppress transcription from the HPV-16
enhancer-promoter in transient transfections.
Two short segments
of the HPV-16 genome have strong affinity to the nuclear matrix: the 5'
third of the LCR and the E5 gene together with the early-late
intergenic region downstream of E5. A third segment, with moderately
strong affinity, overlaps with the E6 gene. Computer-based sequence
analysis makes it likely that MARs in similar positions are conserved
in many or even all genital HPVs. This suggests that they play an
essential role during the life cycle of genital HPVs (62).
In this paper, we refer to these three nuclear MARs as 5'-LCR-MAR,
E6-MAR, and E5-MAR; in the names of recombinant plasmids, we further
abbreviate them as L, E6, and E5. Some other portions of the HPV-16
genome have low affinity to the nuclear matrix, and we have not
included them in this study. MARs are typically identified through
attachment to nuclear matrix preparations in vitro and in vivo, but
investigations going beyond binding studies have measured functional
correlates, e.g., influences on the transcription of linked genes.
Several studies have reported diverging effects on the transcription
during transient and stable transfections (9, 10, 14, 34,
57).
First, we addressed the question of whether the MARs of HPV-16 may
influence the transcription by the HPV-16 enhancer and
E6 promoter P97
in transient transfection studies. We compared
the expression of the
luciferase reporter gene directed by the
HPV-16 enhancer and promoter
with the expression from constructs
containing in addition to the
enhancer and promoter either one
or two HPV-16 MARs, positioned
upstream or downstream or on both
ends of the
enhancer-promoter-luciferase (EP-Luc) segment (e.g.,
we refer to a
construct with the 5'-LCR-MAR upstream and the E6-MAR
downstream of
this chimeric reporter as L-EP-Luc-E6). Some of
these constructs
reflect the natural relative position of these
MARs, while others are
artificially permutated. All constructs
are listed in Fig.
1A; deletion
mutants (see below) are depicted
in Fig.
1B.
Figure
2A shows luciferase expression
after transient transfection of six of these recombinant clones into
HeLa cells. With
L-EP-Luc, a vector that had the 5'-LCR-MAR positioned
upstream
of the enhancer, the natural organization in HPV-16, we
observed
expression 3.5-fold lower than that generated by the MAR-free
vector EP-Luc. Complementation of this clone with E6-MAR or E5-MAR
downstream of the luciferase gene further suppressed luciferase
expression. Suppression was also profound when, in an unnatural
alignment, two identical MARs were present upstream and downstream
of
the EP-Luc segment (Fig.
2B). We conclude that MARs act in
transient
transfections on the HPV-16 enhancer-promoter as
cis-responsive
repressors of transcription.
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FIG. 2.
MARs of HPV-16 in the 5' LCR, the E6 gene, and the E5
gene repress transcription from the HPV-16 enhancer-promoter in
transient transfections of HeLa cells. The presence of the MAR in the
5' part of the LCR in its natural position relative to the EP segments
(L-EP-Luc) represses luciferase reporter gene expression by 68%, while
various artificial constructs reduce enhancer-promoter activity by 60%
(L-EP-Luc-L) to 87% (L-EP-Luc-E5). Schematic structures of the test
vectors are shown in Fig. 1.
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Strong and moderate stimulation of transcription from the HPV-16
enhancer-promoter in stable transfectants by the E6-MAR and 5'-LCR-MAR,
respectively.
It is difficult to examine details of the molecular
organization of transiently transfected DNA. While during such a short experiment most of the transfected DNA will exist episomally rather than recombined with host chromosomal DNA, it can only be speculated whether, in the case of a virus, such an episomal state is mimicking the natural episomal maintenance and allows for the natural
transcription biology. On the other hand, in stably transfected clones,
the transfected DNA has become integrated into the host's chromosomal DNA, just as it occurs with HPV DNA in the majority of malignant cervical lesions. As MARs are known to modulate linked enhancers and
promoters after chromosomal recombination in a manner differing from
that during transient transfection, we studied HPV-16 MAR-EP-Luc recombinants in stably transfected HeLa and C33A cells.
C33A cells are derived from a cervical cancer but do not carry
endogenous HPV genomic copies, while HeLa cells carry endogenous
HPV-18
genomes. Both cells were investigated to exclude potential
influences
of integrated HPV genomes or its gene products on transfected
vectors.
To obtain cell lines harboring stably integrated copies
of the EP-Luc
constructs with and without MARs, we transfected
these cells by
electroporation with linearized DNA in the presence
of a G418
selectable vector. Linear DNA was chosen to ensure integration
through
the DNA termini without disruption of the fusion genes
during the
process of integration. G418-resistant colonies were
harvested, pooled,
and prepared for luciferase assays. Dot blots
confirmed that different
pools of transfectants contained similar
numbers of genomes (e.g., top
of Fig.
3). Because of this, and
because
of the randomness of the integration in pools of transfectants,
differences in luciferase expression must originate from the nature
of
the recombinant molecule rather than from a gene dosage effect.
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FIG. 3.
The E6-MAR strongly stimulates transcription from the
HPV-16 enhancer-promoter in stable transfectants. Luciferase values
were determined for pools of stably transfected HeLa cells (A and B)
and pools of C33 A cells (C and D). The reporter gene EP-Luc is only
weakly expressed in either cell line, and these expression levels are
augmented slightly (L-EP-Luc and L-EP-Luc-E5 in C33A cells) or not at
all by linkage to the MAR in the 5' part of the LCR or overlapping with
the E5 gene. In contrast, expression from a construct with the E6 MAR
in its natural position downstream of the EP segment, but separated
from it by the luciferase reporter gene (L-EP-Luc-E6), and from an
artificial construct having two E6 MARs upstream and downstream from
the reporter gene is stimulated 100-fold in HeLa cells and 3.5- to
7-fold in C33A cells. At the top is an example of dot blot experiments
that we performed for each of these pools of transfectants to ascertain
comparable HPV-16 genomic copy numbers.
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Compared with transient transfections, luciferase activity of the basic
construct was found low due to low copy numbers (EP-Luc
constructs in
Fig.
3A to D). Complementation of EP-Luc with the
5'-LCR-MAR or the
E5-MAR led to an approximately threefold stimulation
in C33A cells
(Fig.
3C) but had no strong effect in HeLa cells.
There was no
alteration by supplying two copies of the 5'-LCR-MAR
or the E5-MAR on
either side of the reporter construct. In contrast,
constructs having
the E6-MAR downstream or on either side of the
EP-Luc segment were
stimulated by about 2 orders of magnitude
in HeLa cells and by a factor
of 5 to 10 in C33A cells. This activity
does not require the complete
E6 gene but only sequence elements
3' of position 246, as a deletion
mutant of sequences between
positions 104 and 246 leads to a small
increase, but further deletion
to position 356 leads to a complete loss
of function (Fig.
1 and
3E). These observations identify the E6-MAR as
strong and the
5'-LCR-MAR and E5-MAR as weak
cis-responsive
activators whose
functions depend on the physical organization of the
DNA as part
of cellular chromosomes. This observation was intriguing,
as we
had not anticipated a regulatory element within an HPV gene, nor
did the moderate nuclear matrix affinity of the E6-MAR, in contrast
to
the strong affinity of the other two MARs, suggest the possibility
of
such a
function.
Stimulation of reporter gene expression depends on the E6-MAR but
not on the site of chromosomal integration.
The recombination of
transfected vectors normally occurs at random sites throughout various
chromosomes. In the previous experiments, we had used pools of stable
transfectants to average out the effect that the site of chromosomal
integration may have on expression of the reporter gene. To further
study the contributions of the constructs and of the site of insertion,
we picked individual G418-resistant clones from a similar transfection
of HeLa cells and determined the luciferase activity for each of these
clones separately (Fig. 4). Four clones
which contained EP-Luc showed barely detectable luciferase activity.
Four additional clones with the 5'-LCR-MAR in the natural position
(L-EP-Luc) had luciferase activities significantly above background.
Eight clones, having the 5'-LCR-MAR upstream and the E6-MAR downstream
of the E6 gene, had activities much greater than those of the EP-Luc
clones and even 10- to 20-fold above those of the L-EP-Luc clones,
although they differed by up to a factor 3 among one another. We
conclude that the increased activity of the individual clones as well
as of the pools of clones originates mostly from the MARs and is little
influenced by the chromosomal environment. These experiments further
identify the E6-MAR as strong and the 5'-LCR-MAR as weak physical
context-dependent transcriptional enhancers.
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FIG. 4.
Transcription of the luciferase reporter gene in
individual stable transfectants is slightly stimulated by the MAR in
the 5' LCR and strongly by the E6 MAR and does not significantly depend
on the site of chromosomal integration. Four individually isolated
stable transfectants of EP-Luc into HeLa cells showed nearly
undetectable levels of luciferase expression, which is significantly
stimulated by the presence of the MAR in the 5' LCR (L-EP-Luc) in four
individual clones. Fusion of these constructs with the E6 MAR leads to
a further 10- to 20-fold stimulation of luciferase expression in eight
independent stable transfectants (L-EP-Luc-E6).
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An EMSA screen of oligonucleotides representing the 5'-LCR-MAR and
the E6-MAR reveals numerous binding sites for soluble nuclear
proteins.
MARs are typically AT-rich regions with a high
propensity for stable base unpairing under superhelical strain. They
are bound by numerous proteins with little sequence specificity that
are intrinsic components of the nuclear matrix, such as topoisomerase II, nucleolin, lamins, SAF-A, and even cell-type-specific factors such
as SATB1 (19). These proteins are candidates for being responsible for the activation function of MARs. Unfortunately, it is
technically difficult to map the interactions between MARs and these
intrinsic matrix proteins.
There are only few reports about intrinsic matrix proteins with the
properties of typical transcription factors, such as the
B-cell-specific factor Bright (
66). On the other hand, it
has
been noted that many sequence-specific transcription factors (for
a
review, see reference
11) as well as components of
the general
transcription machinery, including the RNA polymerase II
holoenzyme
(
33), have high affinity to the nuclear matrix.
By binding to
their DNA target sequences, such proteins may generate
some affinity
of this DNA sequence to the nuclear matrix. Here, we
decided to
investigate whether the HPV-16 MARs can bind any of these
soluble
and sequence-specific
factors.
Toward this end, we dissected the 5'-LCR-MAR and the E6-MAR into short
segments, represented by overlapping oligonucleotides,
and used these
oligonucleotides in EMSAs with soluble proteins
from nuclear extracts
(
21). Table
1 shows oligonucleotides
with lengths of 31 bp
representing the complete 5'-LCR-MAR, from
the L1 gene on the 5' side
to the epithelial specific enhancer
on the 3' side (LCR-1 to LCR-21),
and oligonucleotides with lengths
of 40 bp representing the complete E6
gene (EP-1 to EP-15). Figure
5 shows the
outcome of an EMSA analysis of these oligonucleotides
with HeLa nuclear
extracts. As can be seen, there are at least
seven bands, labeled A to
F, which are apparently derived from
specific protein-DNA interactions,
as they appear only with some
but not with other oligonucleotides.
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FIG. 5.
EMSA screen of oligonucleotides derived from the
5'-LCR-MAR and the E6-MAR, showing strong binding sites for several
nuclear proteins, some of which are common to both MARs. (A) Band
shifts obtained with HeLa nuclear extracts and overlapping 31-mer
oligonucleotides representing the 5' third of the HPV-16 LCR from the
L1 gene to the E2 binding site most distal from the E6 promoter; (B)
band shifts with 40-mer oligonucleotides representing the complete E6
gene. For details, see the footnote to Table 1 and Materials and
Methods. The control (Co) is band shift of an oligonucleotide of the
CDP/Cut binding site of the gp91phox gene
promoter (59). The slowest-mobility complex was termed C1,
as it became clear later in this study that it is identical to the
complex forming on the PSM, which we termed C1 in a previous study
(48). The weak complex C2 that migrated even slower than C1
and is visible only in some slots may be a dimer of the protein giving
rise to the C1 complex but could also be a heteromer. It forms
efficiently on PSM, which has two flanking binding sites for C1, but
only weakly on nonrepeated binding sites. Band B represents binding of
YY1 to a previously described site (49), and band A
represents binding of the transcription factor USF (data not shown).
This study did not make an attempt to identify the proteins giving rise
to bands D to G. FP, free probe.
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We decided to study in detail complex C, as it occurs at least five
times with high affinity in each of the two MARs (LCR-2,
LCR-6 to
LCR-9, and LCR-12 to LCR-15; E6-3, -5, -7, -10, and -13),
and as these
complexes include some of the strongest signals of
this experiment. The
weak formation of complex C with some additional
oligonucleotides may
stem from the high AT content of the HPV-16
genome, which appears to
favor the formation of this complex (see
below). To identify complex C,
we used in this figure the terms
C1 and C2 (the latter term designating
the weak trailing band
visible in some slots), as it became clear later
during this research
that this complex is identical to one that we
detected binding
HPV-16 sequences elements elsewhere (
47,
48) (see below).
Two complexes, A and B, form on one
oligonucleotide each. In experiments
not shown here (reference
49 and data not shown), we identified
A and B as
being derived from the binding of the transcription
factors USF and
YY1, respectively. Yet four other, often weak
complexes, D to G, also
occur with oligonucleotides derived from
either MAR and multiple times
in each MAR. The nature of these
complexes is not known. Complex C also
formed with some oligonucleotides
representing the E5-MAR but not
strongly with oligonucleotides
representing the epithelial
cell-specific enhancer (data not
shown).
Biochemical purification, EMSA competition, and supershift analyses
identify a principal factor binding to both HPV-16 MARs as
CDP/Cut.
To identify the nature of the factor in complex C1, we
used the E6-10 as a reference for all 10 binding sites of the C1 factor in both MARs. HeLa nuclear extract was loaded onto a heparin-Sepharose column, and fractions with EMSA activity on E6-10 were eluted at a salt
concentration of 0.3 M (Fig. 6A). These
fractions were further processed by a fractionated precipitation with
ammonium sulfate. The band shift activity was retained in the 20 to
40% fraction (Fig. 6B). To estimate the molecular mass of the protein, the ammonium sulfate precipitate was dissolved and loaded onto a
Sephacryl S400 gel filtration column. The band shift activity eluted
shortly after the void volume with an estimated molecular mass of
around 200 kDa (Fig. 6C). A lower-molecular-mass protein that also
bound to E6-10 may be a degradation product of the C1 factor, as it was
not detectable after ammonium sulfate fractionation.
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FIG. 6.
Biochemical enrichment of a principal factor binding
numerous sites in the 5'LCR-MAR and the E6-MAR. The purification
profile of the factor binding E6-10, as monitored by EMSA, is similar
to that of the differentiation-specific factor CDP/Cut. (A)
Ion-exchange chromatography purification of HeLa nuclear extracts
(N.E.) on a heparin-Sepharose column demonstrates that PSM binding
protein activity is maximal in the 0.3 M KCl fraction. FT, flowthrough.
(B) Ammonium sulfate precipitation of E6-10 binding fractions of the
heparin-Sepharose chromatography defines the activity in the 20 to 40%
fraction. (C) A gel filtration experiment, with the 20 to 40% ammonium
sulfate fraction from the heparin-Sepharose column, indicates that the
protein binding E6-10 is a very large protein of approximately 180 kDa
(as defined by the -amylase molecular weight marker).
V0, void volume; FP, free probe. The purification profile
presented here is consistent with that described for the
differentiation-specific transcriptional repressor CDP/Cut
(44).
|
|
One of the few known transcription factors with such a high molecular
mass is CDP/Cut. CDP/Cut has a molecular mass of 180
kDa and,
reminiscent of the repressor function of these MARs in
transient
transfections, is one of the few well-characterized
mammalian
repressors of transcription (
44). Also, CDP/Cut has
been
found to bind the 5' part of the LCR of HPV-6 as well as
sites in early
genes of this virus (
1,
52). Against this
background, we
performed EMSA competitions and supershift analyses
to determine
whether CDP/Cut could be the C1 factor. Figure
7A
shows that the band shift generated
with E6-10 is abolished by
competition with two oligonucleotides
harboring known binding
sites for CDP/Cut, namely, one of the
gp91
phox gene (
59) (slots 6 and 7)
and one of the sea urchin sperm histone
H2B-1 gene (
6)
(slots 6 to 9). It is also eliminated by an
oligonucleotide
representing the HPV-16 papillomavirus silencing
motif (PSM) (slots 2 and 3), which we found to bind and functionally
depend on CDP/Cut
(
47). In contrast, competition with an E6-MAR
oligonucleotide which did not form complex C1 (slots 10 and 11)
or with
a mutant version of PSM (slots 4 and 5) did not affect
complex
formation. Complex C1 is also eliminated by antiserum
raised against
CDP/Cut but not by preimmune serum (Fig.
7B). We
conclude from this
that the transcriptional repressor CDP/Cut
is the factor that gives
rise to complex C1 with the E6-10.
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FIG. 7.
Band shift competition and supershift analyses identify
CDP/Cut as the factor that binds at least 10 sites in the 5'LCR-MAR and
the E6-MAR. (A) E6-10 generates a strong C1 band shift as well as the
weak trailing band C2 (lane 1). Formation of both complexes is
eliminated by competition with an oligonucleotide representing the
HPV-16 PSM (lanes 2 and 3) (47, 48) and the well-studied
CDP/Cut binding sites of the promoters of the
gp91phox gene (lane 6 and 7) (59) or
the histone H2B gene (lanes 8 to 9) (6) but not by a mutant
PSM oligonucleotide (lane 4 and 5). As outlined by O'Connor et al.
(48), we interpret complex C2 as reflection of a bound dimer
binding. FP, free probe. (B) An anti-CDP antibody (-CDP) abolishes
the formation of the C1 and C2 complexes on E6-10. This effect is
specific and is not seen upon the addition of a preimmune serum (PI) to
the EMSA reaction. (C) LCR-6 produces C1 and C2 complexes similar to
those seen with E6-10. The band shift is eliminated by the CDP/Cut
binding sites of the gp91phos promoter, the
HPV-16 PSM, and E6-10 but not by a mutated PSM oligonucleotide (PSM*).
This experiment is representative of numerous crosswise competitions
which identify at least five CDP/Cut binding sites in each of the two
HPV-16 MARs.
|
|
To investigate whether the slowly migrating complexes seen with other
oligonucleotides of the 5'-LCR-MAR and the E6-MAR that
comigrated with
E6-10 complex C1 are also caused by CDP/Cut, we
also analyzed the
behavior of these C1 complexes. Figure
7C is
an example of several
EMSAs, and shows competitions with LCR-6.
As can be seen, the
oligonucleotide derived from the gp91
phox
promoter and that from the HPV-16 PSM site are both able to compete
for
CDP/Cut binding, while a PSM mutant oligonucleotide has no
effect on
complex formation (compare lanes 2 to 5 with lanes 6
and 7). From this
observation and similar data for other oligonucleotides
(data not
shown), we conclude that all C1 complexes of the 5'-LCR-MAR
and the
E6-MAR are generated by the binding of CDP/Cut and that
CDP/Cut binds
strongly to at least five sites within each of the
two
MARs.
Mutations of an oligonucleotide derived from E6 gene sequences
identify an AT-rich segment as CDP/Cut binding site.
To identify a
binding motif for CDP/Cut, we performed EMSAs with the wild-type
version of E6-10 (Fig. 8B, slots 1 and 8)
and with six different mutated derivatives thereof (Fig. 8A slots 2 to
7). As can be seen, binding of the factor that generates C1 depends on
an AT-rich stretch in the 3' part of the oligonucleotide. CDP/Cut is
known not to have a long and highly conserved target sequence rather,
it bind to AT-rich sequences which normally include the sequence
5'-TAAT-3' (3, 28). This behavior is reflected in our EMSA
results. Alteration of the TAAT element in the 3' part of this
oligonucleotide either eliminates (V and VI) or reduces (II and III)
CDP/Cut binding, while alteration of two other AT-rich stretches (I and
IV) has no effect on the CDP/Cut band shift. We take this as additional
support for the identification of these 10 CDP/Cut binding sites.
Similarly, among the oligonucleotides used to scan the 5'-MAR and the
E6-MAR (Table 1; Fig. 5), all 11 oligonucleotides that gave
particularly strong CDP/Cut band shifts were particularly AT rich. Nine
of them contained the sequence 5'-TAAT-3' (LCR-2, -6, -7, -8, and -12;
E6-3, -7, -10, and -13), while the remaining two (LCR-15 and E5-5)
contained sequence elements deviating in one position from 5'-TAAT-3'.
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FIG. 8.
Mutational analysis of one of the CDP/Cut binding sites
within the E6 MAR of HPV-16. CDP/Cut does not bind to a particular
highly conserved sequence but rather binds to AT-rich sequences that
normally include the sequence 5'-TAAT-3' (3, 28). This
behavior is confirmed by an EMSA analysis of mutations of the CDP/Cut
binding site within E6-10. Alteration of the TAAT element in the 3'
part of this oligonucleotide either eliminates (V and VI) or reduces
(II and III) CDP/Cut binding, while alteration of two other AT-rich
stretches (I and IV) has no effect on the CDP/Cut band shift.
|
|
Effect of CDP/Cut overexpression on transient and stable
transfectants.
To confirm that CDP/Cut represses HPV-16
transcription during transient transfection, we cotransfected HeLa
cells with the construct E6-EP-Luc-E6 and two different concentrations
of the CDP/Cut expression vector pMT2-CDP (Fig.
9A, columns 2 and 3) and the parental
vector pMT2 (column 1); we observed more than 95% repression of
luciferase activity with the higher amount of the CDP/Cut expression
vector (Fig. 9C). This suggests that the high concentration of CDP/Cut
factor in HeLa cells does not saturate the corresponding binding sites
on HPV-16 and is in line with an earlier study where we failed to
detect CDP/Cut binding in footprint experiments (27). We
next examined whether the repression by CDP/Cut can still act on
chromosomally integrated copies although it is overridden by the
MAR-dependent stimulation. Under these conditions, CDP/Cut can still
function as a weak repressor by reducing reporter gene expression by
35% (Fig. 9B and C) which is reminiscent of the ability of SATB1 to
repress transcriptional stimulation by a chromosomally integrated MAR
(35).
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FIG. 9.
Effect of CDP/Cut overexpression on transient and stable
transfectants. (A) Transient transfection of HeLa cells with 1 µg of
E6-EP-Luc-E6 and 4 µg of pMT2 (column 1) and 4 and 12 µg of
pMT2-CDP (columns 2 and 3). (B) Pools of HeLa cells stably transfected
with E6-EP-Luc-E6 were transiently transfected with 4 µg of pMT2
(column 1) and 4 and 12 µg of pMT2-CDP (columns 2 and 3). (C)
Repression by CDP/Cut in stable and transient transfection, as
expressed by comparison of columns 1 and 3 in panels A and B. (D)
CDP/Cut binding site mutants of PSM (E*P-Luc) without or with the
E6-MAR tested in transient transfections of HeLa cells.
|
|
In both of these experiments, CDP/Cut may act through the five binding
sites in the E6-MAR as well as through the two binding
sites in the
PSM. To separate these effects, we constructed CDP/Cut
binding site
mutants of the PSM (E*P-Luc), and tested them in
transient
transfections of constructs that contained or lacked
the E6-MAR. Figure
9D shows that the mutant E*P-Luc with mutated
CDP/Cut binding sites of
the PSM and no MAR sequences is not affected
by CDP/Cut overexpression.
In contrast, CDP/Cut overexpression
represses 30% of the activity of
the mutant E*P-Luc-E6, which
is void of the two CDP/Cut binding sites
of PSM but contains the
CDP/Cut sites within the E6-MAR.
| |
DISCUSSION |
CDP/Cut, a transcriptional repressor that is down-regulated during
differentiation, binds conserved clusters of sites in most genital HPV
types.
Activity of the epithelial specific enhancer and the E6
promoter of genital HPV types depends on a dozen different
transcription factors, which induce epithelial specificity and couple
transcription to physiological signals from the host. In the model
systems HPV-6, -11, -16, -18, and -31 (for a review and references, see
reference 46), and probably in all genital HPVs,
most of the binding sites for these factors are positioned in a 550-bp
segment between the E2 binding site most distal from the E6 promoter,
at position 7450 in the case of HPV-16, and the transcription start
site. In spite of sequence divergence among these HPVs, this genomic region is similar in composition to the most conspicuous transcription factor binding sites. Outside this 550-bp region, only few
cis-responsive elements have been found upstream of the
promoter-distal E2 binding site (2, 29, 32, 65); until
recently, no cis-responsive element has been detected
downstream of the E6 promoter.
This traditional view has been completely altered by our findings and
by reports from Roman and colleagues (
1,
52), as
it emerges
that large genomic segments outside the classical EP
segment, including
protein-encoding sequences, are bound by numerous
nuclear proteins.
These segments are
cis-responsive elements and
exert
dramatic influences on HPV transcription. The most prominent
factor
among these nuclear proteins is, with about 10 binding
sites, the
factor CDP/Cut. Beyond this, our EMSA screens point
to several other,
not yet identified soluble factors, while interactions
with the nuclear
matrix suggest the binding of yet other little
soluble
proteins.
CDP/Cut is a repressor of transcription that acts by two alternative
mechanisms, through displacement of activators (
6,
42,
66,
69) and by binding the histone deacetylase HDAC1,
whose activity
changes nucleosomes such that it becomes difficult
for the
transcriptional machinery to access the DNA (
38). CDP/Cut
is
regulated during the differentiation of a variety of cell types,
having
high activity in stem cells that decreases during differentiation
to
the target cell type. Consequently, genes that are repressed
in stem
cells become derepressed during differentiation (
42,
44).
This behavior of CDP/Cut has been extended to epithelia
by studies of
Roman and colleagues (
1), who showed that CDP/Cut
is
abundant in undifferentiated and down-regulated in differentiated
epithelial cells. As a consequence, CDP/Cut represses in
undifferentiated
epithelial cells three HPV-6 promoters by binding to
promoter-flanking
regions, and this repression is released in
differentiating cells.
One of these promoters is the E6 promoter, whose
homologue we
studied in HPV-16, the second is the E7 promoter, which
does not
exist in HPV-16, and the third is the E1/E4 promoter
downstream
of the HPV-16 sequences that we investigated. HeLa cells
(
44)
and probably other cervical carcinoma-derived cell
lines (our
unpublished observations) have high CDP/Cut activity and in
this
respect resemble undifferentiated epithelial cells. This property
leads to low enhancer-promoter activity of transiently transfected
genital HPVs (
55).
Roman and colleagues used large genomic segments of HPV-6 for the study
of CDP/Cut binding, which did not allow exact localization
and
quantification of the binding sites. Despite this limitation,
comparison of their research on HPV-6 with ours on HPV-16, and
alignments of the LCRs and E6 genes of many other genital HPVs
(
43), suggest that these viruses contain a cluster of
CDP/Cut
binding sites in the 5' LCR, a directly repeated site between
the enhancer and the promoter overlapping with the replication
origin
(
47,
48), and another cluster of CDP/Cut binding sites
in
the E6 gene. These three elements appear to cooperate to repress
HPV,
probably through interaction of CDP/Cut with HDAC1 (
38),
which leads to deacetylation and structural changes of nucleosomes
resulting in decreased transcription factor access. This mechanism
appears to apply to HPV-16, as we recently observed release of
repression by the CDP/Cut-dependent silencer PSM in response to
the
HDAC1 inhibitor trichostatin A (
47), probably due to
alteration
of two specifically positioned nucleosomes that limit
transcription
factor access to the HPV-16 enhancer and promoter
(
61). This
finding can probably be extended to the
transcription of HPV-11
and the replication of all genital HPVs, as
there are reports
on the stimulation of HPV-11 transcription under the
influence
of trichostatin A (
68) and inhibition of
papillomavirus replication
by nucleosomes (
37a) and its
release by association of the E1
protein with components of the SWI-SNF
complex (
37). The upper
part of Fig.
10 summarizes the repression of HPV-16
by cooperation
of CDP/Cut, HDAC1, and nucleosomes in the form of a
model. The
lower part of Fig.
10 shows the binding of transcription
factors
in the absence of nucleosomes and proposes an additional
somewhat
more speculative activation mechanism by displacement of
CDP/Cut
through unknown components of the nuclear matrix.
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FIG. 10.
Model of a switch of the HPV E6 promoter between
repression by CDP/Cut, HDAC1, and two specifically positioned
nucleosomes (top) and activation by interaction between unknown
components of the nuclear matrix and cellular transcription factors
(bottom). The figure shows 12 CDP/Cut binding sites in two MARs
flanking the EP segment of HPV-16 and in a silencer motif that overlaps
with the binding site of the viral replication origin, as well as the
binding site of the replication protein E1 and of the most extensively
researched transcriptional regulators. Elements identified previously
are the MAR (62), the enhancer and its elements
(46), the silencer elements (47 and
48), the replication origin (46), and the
promoter elements (17 and 63).
Abbreviations for genes: L1, L1 gene; E6, E6 gene; E7, E7 gene.
Abbreviations for transcription factor binding sites: AP1, activator
protein 1; CDP, CCAAT displacement factor/Cut; GR, glucocorticoid
receptor; NFI, nuclear factor 1; TF1, transcription enhancer factor 1;
oct, octamer binding factor 1.
|
|
The MARs of HPV-16 are context-dependent transcriptional
enhancers.
The clusters of CDP/Cut binding sites in the 5' part of
the LCR and in E6 (but probably not the CDP/Cut binding sites of PSM) function in many genital HPVs as MARs, as judged by attachment assays,
clusters of topoisomerase II targets, and computerized sequence
analyses (62). The data in this paper do not answer the
questions of what kind of DNA-protein interactions (i) give rise to the
affinity of the HPV-16 MARs to the nuclear matrix and (ii) lead to
transcriptional activation after integration of HPV-16 into the
cellular DNA. It is unlikely that CDP/Cut itself is responsible for
these activities, as it is a soluble factor, not an intrinsic part of
the nuclear matrix, and as repression is its only known function.
CDP/Cut is sometimes referred to as a component of the nuclear matrix
(
5) based on the observation that it binds certain
MARs
(
5,
66). The assumption that MAR behavior and CDP/Cut
binding are expressions of the same underlying mechanism may require
further investigation, as two laboratories presented evidence
for an
alternative scenario, in which CDP/Cut is a soluble repressor
that
prevents MARs from interacting with the nuclear matrix, which
would
induce transcription. Wang et al. (
66) demonstrated that
the
activity of the immunoglobulin heavy-chain enhancer depends
on the
interaction between a MAR and the matrix-bound activator
Bright and
that this interaction is inhibited by CDP/Cut. In another
study, it was
found that SATB1 and CDP/Cut can both bind a MAR
at the promoter of a
mouse mammary tumor virus, thereby repressing
it (
39). SATB1
and CDP/Cut are able to bind to one another in
the absence of DNA
binding sites. The authors proposed that equimolar
concentrations of
SATB1 and CDP/Cut annihilate their DNA binding
ability, and
transcriptional induction may result from the interaction
of the vacant
SATB1 and CDP/Cut sites with the nuclear matrix.
Similar mechanisms
could apply to HPV-16, and CDP/Cut might not
only repress through the
HDAC1 pathway but also limit matrix
access.
There is presently no general explanation for the transcriptional
activation by MARs. MARs have been reported to alter nucleosomal
organization, to bring a gene into regions of the nucleus with
high
concentrations of transcription factors, RNA polymerases
and
topoisomerases, and to cause topological stress of flanking
regions due
to local unwinding (
9,
10,
24,
41,
66).
To achieve this, the
nuclear matrix and MARs interact through
DNA binding proteins other
than typical transcription factors,
such as SAF-A (
54),
SATB1 (
19), nucleolin (
20), or lamins
(
41). These proteins have neither nucleotide recognition
specificity
nor transcription activation domains, and targeting and
unwinding
of MARs are possibly a cause of transcriptional induction. It
is unclear why this mechanism is efficient when a MAR is part
of the
chromosomal DNA, and why transient transfection may not
allow the MAR
to exert this function. Against this general lack
of knowledge, we do
not know whether transcription from transiently
transfected HPV DNA
resembles the gene expression from a stable
episome. Alternatively, it
is quite possible that episomally replicating
HPV DNA is regulated by
the nuclear matrix in the same manner
as we observed only in stable
transfectants. These alternatives
require further research, as do the
molecular properties that
make the E6-MAR an strong enhancer and the
5'-LCR-MAR a weak one,
although the latter one binds the matrix more
strongly.
While the nuclear matrix consists of structural proteins with little
sequence specificity, sequence-specific transcription
factors such as
YY1, nuclear factor I, and steroid receptors (for
a review, see
reference
11) and the RNA polymerase II holoenzyme
(
33) can associate with the nuclear matrix. Should CDP/Cut
also
be able to associate with the nuclear matrix (
39), one
could
envisage a scenario where HPV MARs are alternatively bound by
structural matrix proteins or by matrix attached CDP/Cut, resulting
in
a surface-bound switch between an active and an inactive
state.
MARs, cis-responsive elements, and transcription factor
binding sites are located within HPV genes.
In cellular genes,
MARs, cis-responsive elements, and transcription factor
binding sites are normally in nontranscribed regions or in introns.
Papillomaviruses provide an increasingly impressive example of how the
lack of space on condensed viral genomes has led to deviations from
such paradigms. Papillomaviruses diverge from most cellular genes by
having no noncoding introns, by using some cistrons for more than one
protein, and by having polycistronic mRNAs with very short
nontranslated leaders. Published studies (1, 52, 62) and our
data reported here show that they have (i) MARs in the E6, E5 gene, and
other genes and (ii) transcription factor binding sites and
cis-responsive functions in the E6, E7, and E5 genes. The
combination of these observations raises the possibility that 50% or
even more of the viral genome may make contact with DNA binding nuclear
factors that modulate the HPV life cycle. These factors may influence
other aspects of HPV biology such as replication, partition, and
particle maturation, as pointed out for the nuclear
compartmentalization of simian virus 40 (18).
Is HPV-16 transcription induced during carcinogenesis by
integration into cellular chromosomes?
As the normal life cycle of
HPVs involves only episomal genomes, transcriptional induction during
integration into cellular chromosomes cannot be a natural function of
viral MARs. However, even in HeLa cells with their high concentration
of CDP/Cut (44), chromosomal integration of HPV-16 MARs, and
of the endogenous HPV-18 copies, overcomes the repression by CDP/Cut,
possibly by bringing the viral genome into nuclear territories
favorable to transcription. This suggests that transcriptional
induction may also occur when HPV genomes integrate into cellular DNA
in situ, with the result of higher expression of the E6 and E7
oncoproteins and increased oncogenic properties of the affected cell.
In malignant lesions, HPV-16 genomes are more frequently chromosomally
integrated than in episomal form (
16,
64). Integration
occurs most often between the E7 and E2 genes, leaving oncoprotein
expression intact but annihilating expression of E2 (
4,
58).
As the HPV-16 and HPV-18 E2 proteins are repressors of E6 and
E7
transcription (
17,
63), such a recombination derepresses
the
oncogenes and may contribute to a more aggressive cancer cell
phenotype. Although this concept is most likely valid, we propose
that
MAR-dependent transcriptional stimulation might precede the
release of
E2-dependent repression. In a lesion containing only
episomal copies,
some viral genomes, but not simultaneously all
of them, may integrate
into a chromosome of any individual cell.
While these integrated
genomes would cease to transcribe E2, the
E2 repressor is still made
from the remaining episomes, leaving
the cellular phenotype unaltered.
Only MAR-dependent transcriptional
stimulation of intrachromosomal
copies constitutes a dominant
genotype that may overcome E2 repression,
establish enhanced E6
and E7 expression, and confer a phenotype
favorable to
carcinogenesis.
| |
ACKNOWLEDGMENTS |
We thank S. H. Orkin and E. Neufeld for providing plasmids
pMT2 and pMT2-CDP and anti-CDP antiserum, and we thank J. Bode for
stimulating discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Molecular and Cell Biology, National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore. Phone:
65-778-8823. Fax: 65-779-1117. E-mail:
mcbhub{at}imcb.nus.edu.sg.
Present address: National Institutes of Health, Unit of Cell Cycle
Regulation, Bethesda, MD 20892-5431.
Present address: KuDOS Pharmaceuticals Ltd., Cambridge CB4
4GW, United Kingdom.
| |
REFERENCES |
| 1.
|
Ai, W.,
E. Toussaint, and A. Roman.
1999.
CCAAT displacement protein binds to and negatively regulates human papillomavirus type 6 E6, E7, and E1 promoters.
J. Virol.
73:4220-4229[Abstract/Free Full Text].
|
| 2.
|
Auborn, K. J., and B. M. Steinberg.
1991.
A key DNA-protein interaction determines the function of the 5'URR enhancer in human papillomavirus type 11.
Virology
181:132-138[CrossRef][Medline].
|
| 3.
|
Aufiero, B.,
E. J. Neufeld, and S. H. Orkin.
1994.
Sequence-specific DNA binding of individual cut repeat of the human CCAAT displacement/cut homeodomain protein.
Proc. Natl. Acad. Sci. USA
91:7757-7761[Abstract/Free Full Text].
|
| 4.
|
Baker, C. C.,
W. C. Phelps,
V. Lindgren,
M. J. Braun,
M. A. Gonda, and P. M. Howley.
1987.
Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines.
J. Virol.
61:962-971[Abstract/Free Full Text].
|
| 5.
|
Banan, M.,
I. C. Rojas,
W. H. Lee,
H. L. King,
J. V. Harriss,
R. Kobayashi,
C. F. Webb, and P. D. Gottlieb.
1997.
Interaction of the nuclear matrix associated region (MAR)-binding proteins, SATB1 and CDP/Cux, with a MAR element (L2a) in an upstream regulatory region of the mouse CD8a gene.
J. Biol. Chem.
272:18440-18452[Abstract/Free Full Text].
|
| 6.
|
Barberis, A.,
G. Superti-Furga, and M. Busslinger.
1987.
Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter.
Cell
50:347-359[CrossRef][Medline].
|
| 7.
|
Berezney, R., and D. S. Coffey.
1974.
Identification of a nuclear protein matrix.
Biochem. Biophys. Res. Commun.
60:1410-1470[CrossRef][Medline].
|
| 8.
|
Berezney, R.,
M. J. Mortillaro,
H. Ma,
X. Wei, and J. Samarabandu.
1995.
The nuclear matrix: a structural milieu for nuclear genomic function.
Int. Rev. Cytol.
162A:1-65.
|
| 9.
|
Bode, J.,
Y. Kohwi,
L. Dickinson,
T. Joh,
D. Klehr,
C. Mielke, and T. Kohwi-Shigematsu.
1992.
Biological significance of unwinding capability of nuclear matrix-associating DNAs.
Science
255:195-197[Abstract/Free Full Text].
|
| 10.
|
Bode,
J. M. Stengert-Iber,
V. May,
T. Schlake, and A. Dietz-Pfeilstetter.
1996.
Scaffold/matrix-attached regions: topological switches with multiple regulatory functions.
Crit. Rev. Eukaryot. Gene Expr.
6:115-138[Medline].
|
| 11.
|
Boulikas, T.
1995.
Chromatin domains and prediction of MAR sequences.
Int. Rev. Cytol.
162A:279-388.
|
| 12.
|
Chan, W. K.,
G. Klock, and H. U. Bernard.
1989.
Progesterone and glucocorticoid response elements occur in the long control regions of several human papillomaviruses involved in anogenital neoplasia.
J. Virol.
63:3261-3269[Abstract/Free Full Text].
|
| 13.
|
Chong, T.,
D. Apt,
B. Gloss,
M. Isa, and H. U. Bernard.
1991.
The enhancer of human papillomavirus-16: binding sites for the ubiquitous transcription factors Oct-1, NFA, TEF-2, NFI, and AP1 participate in the epithelial specific transcription.
J. Virol.
65:5933-5943[Abstract/Free Full Text].
|
| 14.
|
Cockerill, P. N.,
M. H. Yuen, and W. T. Garrard.
1987.
The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements.
J. Biol. Chem.
262:5394-5397[Abstract/Free Full Text].
|
| 15.
|
Cripe, T. P.,
T. H. Haugen,
J. P. Turk,
F. Tabatabai,
P. G. Schmid,
M. Dürst,
L. Gissmann,
A. Roman, and L. P. Turek.
1987.
Transcriptional regulation of the human papillomavirus-16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis.
EMBO J.
6:3745-3753[Medline].
|
| 16.
|
Daniel, B.,
G. Mukherjee,
L. Seshadri,
E. Vallikad, and S. Krishna.
1995.
Changes in the physical state and expression of human papillomavirus type 16 in the progression of cervical intraepithelial neoplasia lesions analysed by PCR.
J. Gen. Virol.
76:2589-2593[Abstract/Free Full Text].
|
| 17.
|
Demeret, C.,
C. Desaintes,
M. Yaniv, and F. Thierry.
1997.
Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes.
J. Virol.
71:9343-9349[Abstract].
|
| 18.
|
Deppert, W., and R. Schirmbeck.
1995.
The nuclear matrix and virus function.
Int. Rev. Cytol.
162A:485-537.
|
| 19.
|
Dickinson, L. A.,
T. Joh,
Y. Kohwi, and T. Kohwi-Shigematsu.
1992.
A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition.
Cell
70:631-645[CrossRef][Medline].
|
| 20.
|
Dickinson, L. A., and T. Kohwi-Shigematsu.
1995.
Nucleolin is a matrix attachment region DNA binding protein that specifically recognizes a region with high base-unpairing potential.
Mol. Cell. Biol.
15:456-465[Abstract].
|
| 21.
|
Dignam, J. D.,
R. M. Lebowitz, and R. G. Roeder.
1983.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:1475-1489[Abstract/Free Full Text].
|
| 22.
|
Dollard, S. C.,
T. R. Broker, and L. T. Chow.
1993.
Regulation of the human papillomavirus type 11 E6 promoter by viral and host transcription factors in primary human keratinocytes.
J. Virol.
67:1721-1726[Abstract/Free Full Text].
|
| 23.
|
Dong, G.,
T. R. Broker, and L. T. Chow.
1994.
Human papillomavirus type 11 E2 proteins repress the homologous E6 promoter by interfering with the binding of host transcription factors to adjacent elements.
J. Virol.
68:1115-1127[Abstract/Free Full Text].
|
| 24.
|
Forrester, W. C.,
C. van Genderen,
T. Jenuwein, and R. Grosschedl.
1994.
Dependence of enhancer-mediated transcription of the immunoglobulin µ gene on nuclear matrix attachment regions.
Science
265:1221-1225[Abstract/Free Full Text].
|
| 25.
|
Gasser, S. M., and U. K. Laemmli.
1986.
Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster.
Cell
46:521-530[CrossRef][Medline].
|
| 26.
|
Gloss, B.,
H. U. Bernard,
K. Seedorf, and G. Klock.
1987.
The upstream regulatory region of the human papillomavirus-16 contains an E2 protein independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones.
EMBO J.
6:3735-3743[Medline].
|
| 27.
|
Gloss, B.,
T. Chong, and H. U. Bernard.
1989.
Numerous nuclear proteins bind the long control region of human papillomavirus type 16: a subset of 6 of 23 DNase I-protected segments coincides with the location of the cell-type-specific enhancer.
J. Virol.
63:1142-1152[Abstract/Free Full Text].
|
| 28.
|
Harada, R.,
G. Berube,
O. J. Tamplin,
C. Denis-Larose, and A. Nepveu.
1995.
DNA-binding specificity of the cut repeats from the human cut-like protein.
Mol. Cell. Biol.
15:129-140[Abstract].
|
| 29.
|
Hoppe-Seyler, F., and K. Butz.
1993.
A novel cis-stimulatory element maps to the 5' portion of the human papillomavirus type 18 upstream regulatory region and is functionally dependent on a sequence-aberrant Sp1 binding site.
J. Gen. Virol.
74:281-286[Abstract/Free Full Text].
|
| 30.
|
Howley, P. M.
1996.
Papillomavirinae: the viruses and their replication, p. 2045-2076.
In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Lippincott-Raven, Philadelphia, Pa.
|
| 31.
|
International Agency for Research on Cancer.
1995.
IARC monographs on the evaluation of carcinogenic risks to humans, vol. 64. Human papillomaviruses.
International Agency for Research on Cancer, Lyon, France.
|
| 32.
|
Kanaya, T.,
S. Kyo, and L. A. Laimins.
1997.
The 5' region of the human papillomavirus type 31 upstream regulatory region acts as an enhancer which augments viral early expression through the action of YY1.
Virology
237:159-169[CrossRef][Medline].
|
| 33.
|
Kimura, H.,
Y. Tao,
R. G. Roeder, and P. R. Cook.
1999.
Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure.
Mol. Cell. Biol.
19:5383-5392[Abstract/Free Full Text].
|
| 34.
|
Klehr, D.,
K. Maass, and J. Bode.
1991.
Scaffold-attached regions from the human interferon domain can be used to enhance the stable expression of genes under the control of various promoters.
Biochemistry
30:1264-1270[CrossRef][Medline].
|
| 35.
|
Kohwi-Shigematsu, T.,
K. Maass, and J. Bode.
1997.
A thymocyte factor SATB1 suppresses transcription of stably integrated matrix-attachment region-lined reporter genes.
Biochemistry
36:12005-12010[CrossRef][Medline].
|
| 36.
|
Lamond, A. I., and W. C. Earnshaw.
1998.
Structure and function in the nucleus.
Science
280:547-553[Abstract/Free Full Text].
|
| 37.
|
Lee, D.,
H. Sohn,
G. V. Kalpana, and J. Choe.
1999.
Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA.
Nature
399:487-491[CrossRef][Medline].
|
| 37a.
|
Li, R., and M. R. Botchan.
1994.
Acidic transcription factors alleviate nucleosome-mediated repression of DNA replication of bovine papillomavirus type 1.
Proc. Natl. Acad. Sci. USA
91:7051-7015[Abstract/Free Full Text].
|
| 38.
|
Li, S.,
L. Moy,
N. Pittman,
G. Shue,
B. Aufiero,
E. J. Neufeld,
N. S. LeLeiko, and M. J. Walsh.
1999.
Transcriptional repression of the cystic fibrosis transmembrane conductance regulator gene, mediated by CCAAT displacement protein/cut homolog, is associated with histone deacetylation.
J. Biol. Chem.
274:7803-7815[Abstract/Free Full Text].
|
| 39.
|
Liu, J.,
A. Barnett,
E. J. Neufeld, and J. P. Dudley.
1999.
Homeoproteins CDP and SATB1 interact: potential for tissue-specific regulation.
Mol. Cell. Biol.
19:4918-4926[Abstract/Free Full Text].
|
| 40.
|
Liu, Y.,
J. Chen,
Q. Gao,
S. Dalal,
Y. Hong,
C. P. Mansur,
V. Band, and E. Androphy.
1999.
Multiple functions of human papillomavirus type 16 E6 contribute to the immortalization of mammary epithelial cells.
J. Virol.
73:7297-7307[Abstract/Free Full Text].
|
| 41.
|
Luderus, M. E. E.,
J. L. den Blaauwen,
O. J. B. de Smit,
D. A. Compton, and R. van Driel.
1994.
Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.
Mol. Cell. Biol.
14:6297-6305[Abstract/Free Full Text].
|
| 42.
|
Mailly, F.,
G. Berube,
R. Harada,
P. L. Mao,
S. Phillips, and A. Nepveu.
1996.
The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy.
Mol. Cell. Biol.
16:5346-5357[Abstract].
|
| 43.
|
Myers, G.,
H. U. Bernard,
H. Delius,
M. Favre,
J. Icenogle,
M. van Ranst, and C. Wheeler.
1994.
Human papillomaviruses. A compilation and analysis of nucleic acid and amino acid sequences.
Los Alamos National Laboratory, Los Alamos, N.Mex.
|
| 44.
|
Neufeld, E. J.,
D. G. Skalnick,
P. M. J. Lievens, and S. H. Orkin.
1992.
Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut.
Nat. Genet.
1:50-55[CrossRef][Medline].
|
| 45.
|
Nickerson, J. A.,
G. Krockmalnic,
K. M. Wan, and S. Penman.
1997.
The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus.
Proc. Natl. Acad. Sci. USA
94:4446-4450[Abstract/Free Full Text].
|
| 46.
|
O'Connor, M.,
S. Y. Chan, and H. U. Bernard.
1995.
Transcription factor binding sites in the long control regions of genital HPVs, p. 21-40.
In
G. Myers, H. U. Bernard, H. Delius, C. Baker, J. Icenogle, A. Halpern, and C. Wheeler (ed.), Human papillomaviruses 1995, part III-A. Compendium. Los Alamos National Laboratory, Los Alamos, N.Mex.
|
| 47.
|
O'Connor, M. J.,
W. Stünkel,
C. H. Koh,
H. Zimmermann, and H. U. Bernard.
2000.
The differentiation-specific factor CDP/Cut represses transcription and replication of human papillomaviruses through a conserved silencing element.
J. Virol.
74:401-410[Abstract/Free Full Text].
|
| 48.
|
O'Connor, M. J.,
W. Stünkel,
H. Zimmermann,
C. H. Koh, and H. U. Bernard.
1998.
A novel YY1-independent silencer represses the activity of the human papillomavirus type 16 enhancer.
J. Virol.
72:10083-10092[Abstract/Free Full Text].
|
| 49.
|
O'Connor, M. J.,
S. H. Tan,
C. H. Tan, and H. U. Bernard.
1996.
YY1 represses human papillomavirus type 16 transcription by quenching AP-1 activity.
J. Virol.
70:6529-6539[Abstract/Free Full Text].
|
| 50.
|
Parker, J. N.,
W. Zhao,
K. J. Askins,
T. R. Broker, and L. T. Chow.
1997.
Mutational analysis of differentiation-dependent human papillomavirus type-18 enhancer elements in epithelial raft cultures of neonatal foreskin keratinocytes.
Cell Growth Differ.
8:751-762[Abstract].
|
| 51.
|
Pater, M. M.,
G. A. Hughes,
D. E. Hyslop,
H. Nakshatri, and A. Pater.
1988.
Glucocorticoid-dependent oncogenic transformation by type 16 but not type 11 human papilloma virus DNA.
Nature
335:832-835[CrossRef][Medline].
|
| 52.
|
Pattison, S.,
D. G. Skalnik, and A. Roman.
1997.
CCAAT displacement protein, a regulator of differentiation-specific gene expression, binds a negative regulatory element within the 5' end of the human papillomavirus type 6 long control region.
J. Virol.
71:2013-2022[Abstract].
|
| 53.
|
Rapp, L., and J. J. Chen.
1998.
The papillomavirus E6 proteins.
Biochim. Biophys. Acta
1378:F1-F19[Medline].
|
| 54.
|
Romig, H.,
F. O. Fackelmayer,
A. Renz,
U. Ramsperger, and A. Richter.
1992.
Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment elements.
EMBO J.
11:3431-3440[Medline].
|
| 55.
|
Sailaja, G.,
R. M. Watts, and H. U. Bernard.
1999.
Many different papillomaviruses have low transcriptional activity in spite of strong epithelial specific enhancers.
J. Gen. Virol.
80:1715-1724[Abstract].
|
| 56.
|
Schiffman, M. H., and L. A. Brinton.
1995.
The epidemiology of cervical carcinogenesis.
Cancer
76:1888-1901[CrossRef][Medline].
|
| 57.
|
Schübeler, D.,
C. Mielke,
K. Maass, and J. Bode.
1996.
Scaffold/matrix-attached regions act upon transcription in a context-dependent manner.
Biochemistry
35:11160-11169[CrossRef][Medline].
|
| 58.
|
Schwarz, E.,
U. K. Freese,
L. Gissmann,
W. Mayer,
B. Roggenbuck,
A. Stremlau, and H. zur Hausen.
1985.
Structure and transcription of human papillomavirus sequences in cervical carcinoma cells.
Nature
314:111-114[CrossRef][Medline].
|
| 59.
|
Skalnik, D. G.,
E. C. Strauss, and S. H. Orkin.
1991.
CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter.
J. Biol. Chem.
266:16736-16744[Abstract/Free Full Text].
|
| 60.
|
Strouboulis, J., and A. P. Wolffe.
1996.
Functional compartmentalization of the nucleus.
J. Cell Sci.
109:1991-2000[Abstract].
|
| 61.
|
Stünkel, W., and H. U. Bernard.
1999.
The chromatin structure of the long control region of human papillomavirus type 16 represses viral oncoprotein expression.
J. Virol.
73:1918-1930[Abstract/Free Full Text].
|
| 62.
|
Tan, S. H.,
D. Bartsch,
E. Schwarz, and H. U. Bernard.
1998.
Nuclear matrix attachment regions of human papillomavirus type 16 point toward conservation of these genomic elements in all genital papillomaviruses.
J. Virol.
72:3610-3622[Abstract/Free Full Text].
|
| 63.
|
Tan, S. H.,
L. E. C. Leong,
P. Walker, and H. U. Bernard.
1994.
The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID.
J. Virol.
68:6411-6420[Abstract/Free Full Text].
|
| 64.
|
Vernon, S. D.,
E. R. Unger,
D. L. Miller,
D. R. Lee, and W. C. Reeves.
1997.
Association of human papillomavirus type 16 integration in the E2 gene with poor disease-free survival from cervical cancer.
Int. J. Cancer
74:50-56[CrossRef][Medline].
|
| 65.
|
Wang, H.,
K. Liu,
F. Yuan,
L. Berdichevsky,
L. B. Taichman, and K. Auborn.
1996.
C/EBP is a negative regulator of human papillomavirus type 11 in keratinocytes.
J. Virol.
70:4839-4844[Abstract].
|
| 66.
|
Wang, Z.,
A. Goldstein,
R. T. Zong,
D. Lin,
E. J. Neufeld,
R. H. Scheuermann, and P. W. Tucker.
1999.
Cux/CDP homeoprotein is a component of NF-µNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the Bright transcription enhancer.
Mol. Cell. Biol.
19:284-298[Abstract/Free Full Text].
|
| 67.
|
Wei, X.,
J. Samarabandu,
R. S. Devdhar,
A. J. Siegel,
R. Acharya, and R. Berezney.
1998.
Segregation of transcription and replication sites into higher order domains.
Science
281:1502-1506[Abstract/Free Full Text].
|
| 68.
|
Zhao, W.,
F. Noya,
W. Y. Chen,
T. M. Townes,
L. T. Chow, and T. R. Broker.
1999.
Trichostatin A up-regulates human papillomavirus type 11 upstream regulatory region-E6 promoter activity in undifferentiated primary human keratinocytes.
J. Virol.
73:5026-5033[Abstract/Free Full Text].
|
| 69.
|
Zong, R. T., and R. H. Scheuermann.
1995.
Mutually exclusive interaction of a novel matrix attachment region binding protein and the NF-µNR enhancer repressor.
J. Biol. Chem.
270:24010-24018[Abstract/Free Full Text].
|
| 70.
|
zur Hausen, H.
1996.
Papillomavirus infections a major cause of human cancers.
Biochim. Biophys. Acta
1288:F55-F78[Medline].
|
Journal of Virology, March 2000, p. 2489-2501, Vol. 74, No. 6
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
